Effects of poly(ethylene glycol) (PEG) concentration on the permeability of PEG-grafted liposomes

Poly(ethylene glycol)-grafted liposomes (PEG-liposomes) were prepared from dipalmitoylphosphatidylcholine (DPPC) with various amounts of distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (DSPE-PEG) with PEG molecular weights of 1000, 2000, 3000 and 5000. The effects of DSPE-PEG concentration on the permeability of PEG-liposomes were investigated using carboxyfluorescein (CF). In the gel state, the CF leakage from PEG-liposomes was decreased with increasing mole fractions of DSPE-PEG for all PEG molecular weights. In the liquid-crystalline state, the CF leakage from PEG-liposomes containing DSPE-PEG1000 gradually increased with increasing mole fractions of DSPE-PEG, while that of PEG-liposomes whose molecular weight in PEG units was above 2000 rapidly decreased by the addition of DSPE-PEG. Furthermore, no effect of PEG molecular weight on CF leakage was observed. The relationship between the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) (or 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH)) and the mole fraction of DSPE-PEG for PEG-liposomes was also investigated. No significant changes in fluorescence polarization of DPH for liposomal bilayer membranes was observed in the gel and liquid-crystalline states due to the addition of DSPE-PEG, while that of TMA-DPH was decreased compared with that of liposomes without DSPE-PEG in both states.     

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.     

Calorimetry and cryo-transmission electron microscopic studies of PEG2000-grafted liposomes

The phase behavior of poly(ethylene glycol) grafted liposomes (PEG-liposomes) was investigated by differential scanning calorimetry (DSC), dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM). PEG-liposomes were prepared from mixtures of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamine with a covalently attached PEG molecular weight of 2000 (DSPE-PEG2000). From the results of DLS measurements, the coexistence of PEG-liposomes and small molecular assemblies were confirmed at mole fractions of DSPE-PEG2000 above about 0.1. Moreover, it was confirmed that small molecular assemblies were disk micelles by cryo-TEM. However, the phase transition enthalpies of PEG-liposomes were hardly changed according to the DSC measurement, though the mole fraction of the PEG lipid increased. From these results, it was suggested that the phase transition enthalpies hardly changed despite mixed micelles being formed because the bilayer structure of the disk micelle maintains high cooperativity between the DPPC molecules.     

Poly(ethylene glycol)-conjugated phospholipids in aqueous micellar solutions: hydration, static structure, and interparticle interactions

By means of dielectric relaxation spectroscopy (DRS) and small-angle X-ray scattering (SAXS), we have investigated hydration behavior, solvent dynamics, and static structures of aqueous solutions of poly(ethylene glycol)-conjugated distearoyl phosphatidylethanolamine (DSPE-PEG) (molecular weight of PEG: M(PEG)= 2000, 5000, and 12,000 Da). A quantitative analysis of the bulk-water relaxation amplitude revealed the effective hydration number of a DSPE-PEG molecule per ethylene oxide monomer unit to be approximately 5.0-5.5, virtually independent of M(PEG). The overall hydration number of a DSPE-PEG molecule is ca. 20% higher than that of the corresponding normal PEG (without DSPE). This is attributed to both hydration of a charged head group of phosphoric acid in DSPE and a packing effect of PEG chains into micellar structures. The pair-distance distribution functions, p(r), extracted from the GIFT analysis of SAXS intensities show that the DSPE-PEGs form spherical-like micelles in water having the maximum diameter of approximately 16, 22, and 31 nm, respectively, for M(PEG) = 2000, 5000, and 12,000 Da and nearly identical aggregation numbers of 72 (+/-10%). The DSPE-PEG micelles behave as charged colloids whose interparticle interaction potential can be approximated by the screened Coulomb potential model. The extracted pair correlation function g(r) demonstrates that both electrostatic repulsion induced by the charged head group and excluded volume effects of the fully hydrated PEG layer contribute to repulsive interactions among the PEG-lipid micelles. This should be a key factor for the function of PEG lipids as a stabilizer of liposomes.     

Structure of mixed micelles formed in PEG-lipid/lipid dispersions

Polyethylene glycol (PEG)-conjugated lipids are commonly employed for steric stabilization of liposomes. When added in high concentrations PEG-lipids induce formation of mixed micelles, and depending on the lipid composition of the sample, these may adapt either a discoidal or a long threadlike shape. The factors governing the type of micellar aggregate formed have so far not been investigated in detail. In this study we have systematically varied the lipid composition in lipid/PEG-lipid mixtures and characterized the aggregate structure by means of cryo-transmission electron microscopy (cryo-TEM). The effects caused by adding sterols, phosphatidylethanolamines, and phospholipids with saturated acyl chains to egg phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (EPC/DSPE-PEG2000) mixtures with a fixed amount (25 mol %) of DSPE-PEG2000 was studied. Further, the aggregate structure in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPC/DMPE-PEG2000) samples above and below the gel to liquid crystalline phase transition temperature (TC) was investigated. Our results revealed that lipid components, as well as environmental conditions, that reduce the lipid spontaneous curvature and increase the monolayer bending modulus tend to promote formation of discoidal micelles. At temperatures below the gel-to-liquid crystalline phase transition temperature reduced lipid/PEG-lipid miscibility, furthermore, likely contribute to the observed formation of discoidal rather than threadlike micelles.     

Effect of a PEG lipid (DSPE-PEG2000) and freeze-thawing process on phospholipid vesicle size and lamellarity

Liposomes containing distearoylphosphatidylethanolamine with covalently linked polyethylene glycol of molecular weight 2,000 (DSPE-PEG2000) covering a range of 0–30 mol% were prepared by a mechanical dispersion or detergent-removal method. The effects of DSPE-PEG2000 on particle sizes and lamellarity of liposomes were investigated. The average diameters of vesicles prepared from both methods decreased when the concentration of DSPE-PEG2000 was increased. The decrease in vesicle size with increase in DSPE-PEG2000 was ascribed to the steric hindrance of strongly hydrated PEG. The significant decrease in the sizes of DSPE-PEG2000-containing EggPC vesicles prepared by the detergent-removal method could be explained by the postvesiculation size growth in the process of micelle–vesicle transition. For DMPC vesicles prepared by the detergent-removal method, electron micrographs showed that inclusion of DSPE-PEG2000 promoted vesicle formation. Based on the results of investigation of calcein entrapment efficiency, we concluded that the lamellarity of liposomes is reduced as PEG lipid concentration is increased. Fragmentation of multilamellar vesicles into smaller unilamellar vesicles occurred more readily when the liposome suspension was subjected to repetitive freeze-thawing. After five cycles of freezing and thawing, vesicles containing more than 0.5 mol% DSPE-PEG2000 were fragmented into unilamellar vesicles with diameters smaller than 300 nm.     

Physicochemical properties of PEG-grafted liposomes

Egg phosphatidylcholine (EggPC) or dimyristoylphosphatidylcholine (DMPC) liposomes containing polyethylene glycol (PEG)-lipids covering a range of 0-30 mol% have been prepared by Extrusion method. The physicochemical properties including size evolution and calcein permeation were evaluated to investigate the effect of PEG-lipids on bilayer structure. The results from quasielasetic light scattering (QELS), freeze-fracture microscopy, and gel exclusion chromatography revealed that presence of low concentration of PEG-lipid results in decreasing of vesicle size and further increase in the PEG-lipid concentrations lead to a transition from the lamellar membranes to micelles. The permeability for calcein increased with increase in concentration of distearoylphosphatidylethanolamine (DSPE)-PEG. On the other hand, the permeability decreased with low amount of cholesterol-PEG (blow 20% cholesterol-PEG) and increased with high amount of it. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the composition of the bilayer. The concentration of DSPE-PEG2000 incorporated into vesicles without damaging vesicle structures were <20 mol% for EggPC and <10% for DMPC.     

Effects of Amphiphilic Copolymers Bearing Short Blocks of Lipid-Mimetic Units on the Membrane Properties and Morphology of DSPC Liposomes

Amphiphilic, nonionic diblock copolymers based on poly(ethylene glycol) (PEG 2000–5000), comprising short blocks of lipid-mimetic units, where tested for their ability to afford steric stabilization of distearoylphosphathydilcholine:cholesterol liposomes. The copolymers bear 1–4 lipid-mimetic anchors per copolymer chain. Effects on liposomes size depend on copolymer type and content. Cryo-TEM reveals well-separated, intact, predominantly spherical liposomes at copolymer contents up to 5 mol%. A “flat” liposomes fraction occurs upon incorporation of above 7.5 mol% of copolymers bearing 2 or 4 lipid anchors. 5,6-carboxyfluorescein assay indicates lower leakage of stabilized vs. plain liposomes up to concentration 7.5 mol%. Leakage from liposomes with higher copolymer concentration is insignificantly greater.     

Effect of molecular weight in amphipathic polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes

The effect in mice of the molecular weight of polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes (LUVs) was examined using four different distearoyl N-(monomethoxy polyethyleneglycol succinyl) phosphatidylethanolamines (DSPE-PEGs). The molecular weights tested were 1000, 2000, 5000 and 12000. Incorporation of 6 mol% of DSPE-PEG in LUV composed of distearoylphosphatidylcholine (DSPC) / cholesterol (CH) (1:1 in molar ratio) increased the blood circulation half-life significantly more than those without DSPE-PEG derivatives. DSPE-PEGs with molecular weights of 1000 and 2000 prolonged the circulation time of liposomes more than other DSPE-PEGs with higher molecular weights, such as 5000 and 12000. Their effects are also higher than ganglioside GM1, a well described glycolipid with this effect. DSPC/CH LUV-incorporated DSPE-PEG with a molecular weight of 2000 displayed a high concentration in the blood, approximately 40% of the dose, 6 h after the injection.     

Freeze-fracture electron microscopic and calorimetric studies on microscopic states of surface-modified liposomes with poly(ethylene glycol) chains

The differential scanning calorimetry (DSC) and the freeze-fracture electron microscopy of dipalmitoyl phosphatidylcholine (DPPC) liposomes containing distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (PEG-DSPE) were carried out. The DSC peak of DPPC liposomes containing PEG-DSPE had a shoulder. The main phase transition temperature of DPPC bilayer membranes containing PEG-DSPE whose molecular weight of PEG is less than 3000 was slightly shifted to a higher temperature, while that containing PEG-DSPE whose molecular weight of PEG is more than 5000 was slightly shifted to a lower temperature. The electron micrographs of freeze-fracture replicas of DPPC liposomes containing PEG-DSPE quenched from 37±2°C exhibited banded and planar textures, suggesting the lateral phase separation in the bilayer membranes.     

Properties of polyethylene glycol supported tetraarylporphyrin in aqueous solution and its interaction with liposomal membranes

5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin was functionalized by covalent attachment of poly(ethylene glycol) (PEG) chains of various molecular weights, 350, 2000, and 5000 Da. The properties of PEG-functionalized tetraarylporphyrins in aqueous solution and their interactions with liposomes have been studied. Electronic absorption spectroscopy, dynamic light scattering, atomic force microscopy, and fluorescence quenching were used to monitor aggregation of porphyrin chromophores and behavior of the attached PEG chains in the aqueous solution. The tendency for aggregation of porphyrin chromophores in aqueous solution and the efficiency of fluorescence quenching by KI decrease with increasing length of PEG chain linked to the porphyrin ring. The experimental results indicate that polymer clusters are present in aqueous solution of all pegylated porphyrins. The interactions between the pegylated porphyrins and phosphatidylcholine liposomes in the aqueous solution were studied using the fluorescence methods. The apparent binding constants of porphyrin chromophores to liposomes were determined. The degree of binding was found to be dependent on the molecular weight of the attached polymer.     

Synthesis and characterization of hydrophilic thermotropic block copolyetheresters

The block copolyetheresters with a hard segment of poly (hexamethylene p,p′-bibenzoate) and a soft segment of poly (ethylene oxide) were prepared by melt polycondensation of dimethyl-p,p′-bibenzoate, 1,6-hexanediol, and polyethylene glycol (PEG) with molecular weights of 400, 1000, 2000, or 4000. These block copolyetheresters were characterized by intrinsic viscosity, GPC, FT-IR, ¹H-NMR, and water absorption. The thermotropic liquid crystalline properties were investigated by DSC, polarized microscope, and x-ray diffraction. The block copolyetheresters exhibit smectic liquid crystallinity due to the polyester segment. The transitions are dependent on the molar content and the molecular weight of PEG used. The block copolyetheresters show high water absorption due to the hydrophilic nature of the poly (ethylene oxide) segment. The water absorption increases with increasing PEG content. As the molecular weight of PEG increases, the water absorption increases significantly. The results indicate that the water absorption of the poly (ethylene oxide) segment in the block copolymers is affected by the presence of polyester segments. © 1995 John Wiley & Sons, Inc.     

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.     

Mixing behavior of a poly(ethylene glycol)-grafted phospholipid in monolayers at the air/water interface

Mixed phospholipid monolayers hosting a poly(ethylene glycol) (PEG)-grafted distearoylphosphatidylethanolamine with a PEG molecular weight of 5000 (DSPE-PEG5000) spread at the air/water interface were used as model systems to study the effect of PEG-phospholipids on the lateral structure of PEG-grafted membrane-mimetic surfaces. DSPE-PEG5000 has been found to mix readily with distearoylphosphoethanolamine-succinyl (DSPE-succynil), a phospholipid whose structure resembles closely that of the phospholipid part of the DSPE-PEG5000 molecule. However, properties of mixed monolayers such as morphology and stability varied significantly with DSPE-PEG5000 content. In particular, our surface pressure, epifluorescence microscopy (EFM), and Brewster angle microscopy (BAM) studies have shown that mixtures containing 1-9 mol % of DSPE-PEG5000 form stable condensed monolayers with no sign of microscopic phase separation at surface pressures above approximately 25 mN/m. Yet, at 1 mol % of DSPE-PEG5000 in mixed monolayers, the two components have been found to behave nearly immiscibly at surface pressures below approximately 25 mN/m. For monolayers containing 18-75 mol % of DSPE-PEG5000, a high-pressure transition has been observed in the low-compressibility region of their isotherms, which has been identified on the basis of continuous BAM imaging of monolayer morphology, as reminiscent of the collapse nucleation in a pure DSPE-PEG5000 monolayer. Thus, the comparative analysis of our surface pressure, EFM, and BAM data has revealed that there exists a rather narrow range of mixture compositions with DSPE-PEG5000 content between 3 and 9 mol %, where somewhat homogeneous distribution of DSPE-PEG5000 molecules and high pressure stability can be achieved. This finding can be useful to "navigating" through possible mixture compositions while developing guidelines to the rational design of membrane-mimetic surfaces with highly controlled bio-nonfouling properties.     

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.     

PLLA-PEG共聚物的非等温结晶行为

采用熔融共聚法制备PLLA-PEG嵌段共聚物, 用WAXD和DSC方法研究其结晶行为, 并用Avrami方程的Jeziorny修正分析了非等温结晶动力学行为. 结果表明, PLLA结晶明显, 而PEG结晶难以观察到, PEG的柔性能促进PLLA结晶. PEG分子量的增加和投料量的增加都能使得结晶温度升高, 结晶度增大, 结晶速度加快.     

Nanoparticle Capping Agent Controlled Electron‐Transfer Dynamics in Ionic Liquids

Herein, we report a change in the mechanism of the oxidation of silver nanoparticles (Ag NPs) with the molecular weight of a poly(ethylene) glycol (PEG) capping agent. Characterisation of the modified nanoparticles is undertaken using dynamic light scattering and UV/Vis spectroscopy. Electrochemical analyses reveal that the oxidation of 6000 molecular weight (MW) PEG is consistent with a polymer‐gated mechanism, whilst for 2000 MW PEG the polymer does not hinder the oxidation. The 10,000 MW PEG Ag NPs are rendered almost electrochemically inactive. This study demonstrates the ability to alter and better understand the electron‐transfer mechanism in a room temperature ionic liquid (RTIL) by systematically altering the capping agent.     

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.     

Production and characterization of poly(3-hydroxybutyrateco-3-hydroxyhexanoate)-poly(ethylene glycol) hybird copolymer with adjustable molecular weight

A novel natural-synthetic hybrid block copolymer was synthesized by Aeromonas hydrophila 4AK4 in poly(ethylene glycol)(PEG,M_n=200) modified fermentation.This hybrid biomaterial consists of the natural hydrophobic polymer poly(3-hydroxybutyrat-co-3-hydroxyhexanoate)(PHBHHx) end-capped with hydrophilic PEG,which has the increased flexibility as well as the improved thermal stability.Addition of diethylene glycol(DEG) and ethylene glycol could not result in the accumulation of hybrid block copolymer.DEG and ethylene glycol,together with PEG-200,could cause a reduction of molar mass of PHBHHx,resulting in a series of low molecular weight polymer and the reduction of the polymer yield as well as the cellular productivity.In vitro degradation of PHBHHx and PHBHHx-PEG with different molecular weight showed that the decrease of molecular weight accelerated the degradation of copolymers,but PEG modification has little effect on its degradation rate.The results in this study provided a convenient and direct method to produce a series of PHBHHx and PHBHHx-PEG materials with adjustable molecular weight and broad molecular weight distribution which will be very useful for the biomedical applications.     

Preparation and characterization of macroporous poly(N‐isopropylacrylamide) hydrogels for the controlled release of proteins

Macroporous, temperature‐sensitive poly(N‐isopropylacrylamide) (PNIPAAm) hydrogels were synthesized with poly(ethylene glycol)s (PEGs; molecular weight = 2000–6000) as the pore‐forming agents. The influence of the molecular weight and PEG content on the responsive kinetics of these macroporous hydrogels was investigated. The PEG‐modified PNIPAAm hydrogels were characterized by the swelling ratio, deswelling–reswelling kinetics, Fourier transform infrared, and differential scanning calorimetry. The morphology of these hydrogels was analyzed with scanning electron microscopy. The prepared macroporous hydrogels exhibited some unique properties in comparison with the gels with low molecular weight PEGs (molecular weight < 2000) as the pore‐forming agents. In addition, a preliminary study on the controlled release of bovine serum albumin from these macroporous hydrogels was carried out. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 152–159, 2003