Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles

The critical micelle concentrations of 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 microM, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG-DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.     

Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes.

Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.     

Hydration of polyethylene glycol-grafted liposomes.

This study aimed to characterize the effect of polyethylene glycol of 2000 molecular weight (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodynamic stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodynamic and hydrational characterization. Using a differential scanning calorimetry technique we showed that each molecule of PEG2000 binds 136 +/- 4 molecules of water. For PEG2000 covalently attached to the lipid molecules organized in micelles, the water binding increases to 210 +/- 6 water molecules. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concentrations over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calculated from ultrasound velocity and density) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concentration up to approximately 7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodynamic stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concentrations, compressibility and partial volume of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five additional water molecules per lipid molecule for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.     

Osmoelastic coupling in biological structures: decrease in membrane fluidity and osmophobic association of phospholipid vesicles in response to osmotic stress

Poly(ethylene glycol)- (PEG-) induced change in membrane fluidity and aggregation of phospholipid vesicles were studied. A threshold concentration of PEG was required to induce the aggregation. This concentration increased with a decrease in the molecular weight of PEG, e.g., from 5% (w/w) with PEG 6000 (PEG with an average molecular weight of 7500) to more than 30% (w/w) with PEG 200. The aggregation was reversible upon dilution of PEG if the initial PEG concentration was smaller than a certain value, e.g., 22% (w/w) for PEG 6000. Addition of PEG caused a decrease in membrane fluidity of the vesicles detected by fluorescence anisotropy of diphenylhexatriene and by electron spin resonance of a spin-labeled fatty acid. The anisotropy change of diphenylhexatriene fluidity change had an inflection point at approximately 5% (w/w) of PEG 6000, which might suggest that the aggregation would make the decrease of membrane fluidity smaller. Transfer of lipid molecules between phospholipid vesicles was enhanced by the PEG-induced aggregation. The enhancement occurred not only upon direct addition of PEG to the suspending medium, but also upon dialysis of the vesicle suspension against a high concentration of PEG. All these features are consistent with osmoelastic coupling in the phospholipid membranes and the subsequent osmophobic association of the vesicles. The imbalance of osmolarity between the region adjacent to the vesicle surface (exclusion layer) and the bulk aqueous phase, which results from the preferential exclusion of PEG from the exclusion layer in the case of direct addition of PEG, exerts an osmotic stress on the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusogenic activity of PEGylated pH-sensitive liposomes

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG???? was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG???? was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG???? or sterol-PEG???? into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG???? in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.     

Complex formation of bovine serum albumin with a poly(ethylene glycol) lipid conjugate

In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes.     

Comparative electrophysiological study of reconstituted giant vesicle preparations of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were reconstituted into two types of giant vesicles: (1) Giant proteoliposomes prepared by freeze-thawing of a mixture of SR vesicles and sonicated phospholipid vesicles without the use of detergent. (2) Giant SR vesicles prepared by fusion of SR vesicles using poly(ethylene glycol) (PEG) as a fusogen and without the addition of exogenous lipid. These giant vesicles were patch-clamped and properties of the single voltage-dependent potassium channel in the excised patch were studied. Single-channel conductance in a symmetrical solution of 0.1 M KCl and 1 mM CaCl2 was 140.0 +/- 10 pS (n = 5) for freeze-thawed vesicles and 136.4 +/- 15 pS (n = 7) for PEG vesicles. Both types of vesicles exhibited a sub-conductance state having 55% of the fully open state conductance. The voltage-dependence of open-channel probability could be expressed in terms of thermodynamic parameters of delta Gi = 0.95 kcal/mol and z = -0.77 for freeze-thawed vesicles and delta Gi = 0.92 kcal/mol and z = -0.87 for PEG vesicles. These values correlated well with previous data obtained by fusion of native SR vesicles with a planar lipid membrane. Channel orientation was found to be conserved in both types of vesicles used in the present study.     

Comparative studies of irinotecan-loaded polyethylene glycol-modified liposomes prepared using different PEG-modification methods

Recently, a polyethylene glycol (PEG)-modification method for liposomes prepared using pH-gradient method has been proposed. The differences in the pharmacokinetics and the impact on the antitumor effect were examined; however the impact of PEG-lipid molar weight has not been investigated yet. The main purpose of this study is to evaluate the impact of PEG-lipid molar weight against the differences in the pharmacokinetics, the drug-release profile, and the antitumor effect between the proposed PEG-modification method, called the post-modification method, and the conventional PEG-modification method, called the pre-modification method. Various comparative studies were performed using irinotecan as a general model drug. The results showed that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, prolonged circulation time was observed in the post-modification method. The sustained drug-release was observed in the post-modification method by the results of the drug-releasing test in plasma. Moreover, a higher antitumor effect was observed in the post-modification method. It was also confirmed that the same behaviors were observed in all comparative studies even though the PEG molecular weight was lower. In conclusion, the post-modification method has the potential to be a valuable PEG-modification method that can achieve higher preservation stability of PEG-lipid, prolonged circulation time, and higher antitumor effect with only half the amount of PEG-lipid as compared to the pre-modification method. Furthermore, it was demonstrated that PEG(5000)-lipid would be more desirable than PEG(2000)-lipid since it requires much smaller amount of PEG-lipid to demonstrate the same performances.     

Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes

Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo experiments.     

Characterization of the PEG layer of sterically stabilized liposomes: a SAXS study

Synchrotron small-angle X-ray scattering analysis of the bilayer structure of a pharmacologically relevant sterically stabilized liposome system is presented. Describing the electron density profile of the bilayer with the superposition of Gaussian functions, the contribution of the poly(ethylene glycol) (PEG) layers to the total electron density was identified. The changes in the thickness of the PEG layer as well as the distribution of the PEG chains among the outer and inner leaflets of the bilayers were followed by changing the molar ratio of the PEG-lipid and the molar weight of the PEG molecule.     

Phase behavior and aggregate structure in mixtures of dioleoylphosphatidylethanolamine and poly(ethylene glycol)-lipids

Cryo-transmission electron microscopy has been used to investigate the phase behavior and aggregate structure in dilute aqueous mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-phospholipids (PEG-lipids). It is shown that PEG-lipids (micelle-forming lipids) induce a lamellar phase in mixtures with DOPE (inverted hexagonal forming lipid). The amount of PEG-lipid that is needed to induce a pure dispersed lamellar phase, at physiological conditions, depends on the size of the PEG headgroup. In the transition region between the inverted hexagonal phase and the lamellar phase, particles with dense inner textures are formed. It is proposed that these aggregates constitute dispersed cubic phase particles. Above bilayer saturating concentration of PEG-lipid, small disks and spherical micelles are formed. The stability of DOPE/PEG-lipid liposomes, prepared at high pH, against a rapid drop of the pH was also investigated. It is shown that the density of PEG-lipid in the membrane, sufficient to prevent liposome aggregation and subsequent phase transition, depends on the size of the PEG headgroup. Below a certain density of PEG-lipid, aggregation and phase transition occurs, but the processes involved proceed relatively slow, over the time scale of weeks. This allows detailed studies of the aggregate structure during membrane fusion.     

Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol).

Liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are being developed for in vivo drug delivery. In this paper we determine the structure and phase behavior of fully hydrated distearoylphosphatidylcholine (DSPC) suspensions containing PEG-lipids composed of distearoylphosphatidylethanolamine with attached PEGs of molecular weights ranging from 350 to 5000. For DSPC:PEG-lipid suspensions containing 0-60 mol % PEG-lipid, differential scanning calorimetry shows main endothermic transitions ranging from 55 to 64 degrees C, depending on the size of the PEG and concentration of PEG-lipid. The enthalpy of this main transition remains constant for all PEG-350 concentrations but decreases with increasing amounts of PEG-750, PEG-2000, or PEG-5000, ultimately disappearing at PEG-lipid concentrations greater than about 60 mol %. Low-angle and wide-angle x-ray diffraction show that tilted gel (L beta') phase bilayers are formed for all PEG-lipid molecular weights at concentrations of about 10 mol % or less, with the distance between bilayers depending on PEG molecular weight and PEG-lipid concentration. At PEG-lipid concentrations greater than 10 mol %, the lipid structure depends on the size of the PEG moiety. X-ray diffraction analysis shows that untilted interdigitated (L beta I) gel phase bilayers form with the incorporation of 40-100 mol % PEG-350 or 20-70 mol % PEG-750, and untilted gel (L beta) phase bilayers are formed in the presence of about 20-60 mol % PEG-2000 and PEG-5000. Light microscopy, turbidity measurements, x-ray diffraction, and 1H-NMR indicate that a pure micellar phase forms in the presence of greater than about 60% PEG-750, PEG-2000, or PEG-5000.     

Effects of PEG-lipids on permeability of phosphatidylcholine/cholesterol liposomes in buffer and in human serum

The permeability of liposomal membranes was studied as a function of the amount of incorporated PEG-lipid. The fluorescent dyes ethidium, propidium and 5(6)-carboxy fluorescein were used as markers for measurements of spontaneous leakage. The results show that addition of up to 8 mol% of PEG(2000)-DSPE into liposomal membranes of DSPC/Cho and EPC/Cho reduces the permeability of carboxyfluorescein in buffer solution. In contrast, the leakage of the more amphiphilic dye ethidium was not to any measurable extent affected by PEG-lipid inclusion. Another important difference was that ethidum leakage showed a clear dependence on temperature whereas leakage of carboxyfluorescein from pegylated liposomes did not. We conclude that the mechanisms by which the two dyes permeate the liposomal bilayer are qualitatively different. Both ethidium and carboxyfluorescein did interact with human serum components in a way that made measurements in serum unreliable. The more hydrophilic ethidium analogue propidium was shown not to interact with human serum components to any detectable extent. This made propidium suitable for permeability determinations in human serum. It was found that liposomes composed of pure EPC or EPC with 5 mol% DSPE-PEG, displayed a dramatic increase in permeability when subjected to a medium composed of 20% human serum in buffer. Addition of 40 mol% cholesterol to the EPC bilayers reduced the observed release rate in human serum substantially, whereas no stabilizing effect was observed upon PEG-lipid inclusion.     

Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol).

The interactive properties of liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are of interest because such liposomes are being developed as drug delivery vehicles and also are ideal model systems for measuring the properties of surface-grafted polymers. For bilayers containing PEG-lipids with PEG molecular weights of 350, 750, 2000, and 5000, pressure-distance relations have been measured by X-ray diffraction analysis of liposomes subjected to known applied osmotic pressures. The distance between apposing bilayers decreased monotonically with increasing applied pressure for each concentration of a given PEG-lipid. Although for bilayers containing PEG-350 and PEG-750 the contribution of electrostatic repulsion to interbilayer interactions was significant, for bilayers containing PEG-2000 and PEG-5000 the major repulsive pressure between bilayers was a steric pressure due to the attached PEG. The range and magnitude of this steric pressure increased both with increasing PEG-lipid concentration and PEG size, and the extension length of the PEG from the bilayer surface at maximum PEG-lipid concentration depended strongly on the size of the PEG, being less than 35 A for PEG-750, and about 65 A for PEG-2000 and 115 A for PEG-5000. The measured pressure-distance relations have been modeled in terms of current theories (deGennes, 1987; Milner et al., 1988b) for the steric pressure produced by surface-grafted polymers, as modified by us to take into account the effects of polymer polydispersity and the possibility that, at low grafting densities, polymers from apposing bilayers surfaces can interpenetrate or interdigitate. No one theoretical scheme is sufficient to account for all the experimental results. However, for a given pressure regime, PEG-lipid size, and PEG-lipid surface density, the appropriately modified theoretical treatment gives a reasonable fit to the pressure-distance data.     

Identification of the sites of deoxyhaemoglobin PEGylation

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further treated with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate 'blood substitute' in which human Hb (haemoglobin) is conjugated with long chains of PEG [poly(ethylene glycol)]. To identify the thiolation sites by MS, we have carried out the reaction using deoxyHb bound to inositol hexaphosphate to protect some of the residues crucial for function and NEM (N-ethylmaleimide) to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 PEG chains per tetramer, the thiolated residues were Lys7, Lys11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys60 of the a-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the b-chain. The a-amino groups of a- and b-chains were not modified and the reaction of the Cysb93 residues with NEM was minor or absent. After the modification with thiolane and NEM of up to five to eight lysine residues per tetramer, the products retained a large proportion of the properties of native Hb, such as low oxygen affinity, co-operativity, effect of the modulators and stability to autoxidation. Under identical anaerobic conditions, the conjugation of the thiolated Hb tetramer with five or six chains of the maleimido derivative of 6 kDa PEG yielded products with diminished co-operativity, Hill coefficient h=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to autoxidation. Co-operativity was apparently independent of the topological distribution of the PEGylated sites as obtained by treating partly the thiolated protein with NEM prior to PEGylation [poly(ethylene glycol)ation].     

Poly(ethylene glycol)-conjugated human serum albumin including iron porphyrins: surface modification improves the O2-transporting ability

Artificial O2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene glycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the rheological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O2-carrying plasma expander as a red cell substitute.     

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.     

Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.     

Proton magnetic resonance relaxation studies on the structure of mixed micelles of Triton X-100 and dimyristoylphosphatidylcholine.

Proton magnetic resonance and gel chromatographic studies on mixtures of phospholipid and the nonionic surfactant Triton X-200 have shown that at temperatures above the thermotropic phase transition of the phospholipid and below the cloud point of Triton, mixed micelles are present at molar ratios above about 2:1 Triton/phospholipid. Proton T1 and T2 (from line widths) relaxation times are reported for protons in Triton micelles and in mixed micelles of Triton and dimyristoylphosphatidylcholine at a molar ratio of 3:1 Triton/phospholipid. The T1 values and their temperature dependence and the activation energies of the various Triton proton groups appear to reflect internal motions of the Triton molecules in the micelle. Measurements of the T1/T2 ratio and frequency dependence (55-220 MHz) suggest that the hydrophobic tert-butyl group in Triton is observed under extreme narrowing conditions. The T1 and T2 values of Triton are unchanged in the presence of phosphatidylcholine. The T1 values of various protons of dimyristoylphosphatidylcholine in mixed micelles are similar to those reported for the phospholipid in sonicated vesicles, which are used as membrane models, and presumably the same coupled trans-gauche motions dominate. The T2 values for the terminal methyl and choline methyl protons in the phospholipid are longer than those reported for these groups in vesicles. Hence, the motion of the phospholipid in the mixed micelles appears to be less restricted than in vesicles. T1 measurements in H20/D20 mixtures are consistent with the idea that water does not penetrate the hydrophobic core of the mixed micelles, while water does solvate the polar oxyethylene and choline methyl groups. Titration with Mn2+ confirms that the oxyethylene and choline methyl groups are on the exterior of the mixed micelle while the hydrophobic groups are located in the micellar interior.     

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.