Poly(ethylene glycol) amphiphile adsorption and liposome partition

Surface localized poly(ethylene glycol) (PEG) amphiphiles of type C_16:0-EO₁₅₁ and C_18:2-EO₁₅₁ were studied via ellipsometry at macroscopic, flat methylated silica (MeSi), phosphatidic acid (PA), and phosphatidylcholine (PC) surfaces. At these surfaces the amphiphiles adsorb similarly, in a non-cooperative manner, achieving a plateau (≈0.1 PEG chains/nm²) well below amphiphile critical micelle concentration (CMC). The resultant PEG-enriched layers were 10–15 nm thick, with a polymer concentration (≈0.07 g/cm³) greater than the PEG-enriched phase of many dextran, PEG aqueous two-phase systems. PEG-amphiphile adsorption (mg/m²) at hydrophobic and phospholipid flat surfaces correlated with changes in the partition (log K) of PC liposomes in such two-phase systems. PEG-amphiphile adsorption at macroscopic surfaces appears to represent a balance between hydrophobic attraction and repulsive intra-chain interactions which promote chain elongation normal to the surface.     

Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol)

Hybrid gels constructed from proteins and polymers have attracted a wide range of attention in the field of biomedicine and bioengineering. We report herein the enzymatic preparation of polymer–protein hybrid hydrogels composed of terminally bis-functionalized linear poly(ethylene glycol) (PEG) and streptavidin (SA). PEG was conjugated with tyramine to introduce terminal phenolic hydroxyl (Ph-OH) groups. A peptide tag containing a tyrosine residue (G₄Y-tag) was genetically introduced at the C-terminus of SA. The Ph-OH-modified PEG and G₄Y-tagged SA (SA-G₄Y) were treated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) to yield (PEG-Ph-OH)–(SA-G₄Y) hybrid gels. Biotinylated enhanced green fluorescent protein (biotin-EGFP) was selectively captured in the obtained hybrid gels, indicating that SA-G₄Y retained its biological function. The amount of biotin-EGFP immobilized in the hybrid gels depended on the concentration of SA-G₄Y. In addition, biotinylated bacterial alkaline phosphatase (biotin-BAP) was immobilized in the hybrid gel. The immobilized biotin-BAP exhibited more than 95% of the initial activity after 5 rounds of recycling. The results suggest the facile functionalization of the hybrid gel with a variety of biotinylated functional molecules.     

Analytical partitioning of poly(ethylene glycol)-modified proteins

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.     

Preparation and characterization of poly(ethylene glycol)-g-chitosan with water- and organosolubility

A new scheme was proposed for synthesizing poly(ethylene glycol)-g-chitosan (PEG-g-CS), where methoxy poly(ethylene glycol) iodide (MPEG-I) (M_n 2000) was used for N-substitution of triphenylmethyl chitosan (TPM-CS) in organic medium. The graft copolymers were obtained by subsequent removal of protecting groups with dichloroacetic acid. By varying PEG-I/TPM-CS feed ratio, the grafting levels (GL) of PEG can be adjusted. The chitosan derivatives were characterized by FTIR, ¹H NMR, ¹³C NMR and DSC. All the copolymers were soluble in water over wide pH range. Furthermore, organosolubility of the hybrids in DMF and DMSO was also achieved when the DS value more than 24%. The lysozyme degradation rate of the copolymers in aqueous neutral medium decreased with the increase of GL value.     

Partitioning behavior of group 1 and 2 cations in poly(ethylene glycol)-based aqueous biphasic systems

The partitioning behavior of several Group 1 and 2 cations was investigated in poly(ethylene glycol) (PEG)-based aqueous biphasic systems. All of these metal ions prefer the salt-rich phase over the PEG-rich phase with distribution ratios all well below one regardless of the system investigated. The relative salting-out ability of the individual cations can be directly correlated to their Gibbs free energy of hydration (ΔG_hyd). In addition, the relative magnitude of the distribution ratios for these metal ions can also be explained in terms of ΔG_hyd.     

Chitosan cross-linked with poly(ethylene glycol)dialdehyde via reductive amination as effective controlled release carriers for oral protein drug delivery

The covalently cross-linked chitosan-poly(ethylene glycol)₁₅₄₀ derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG₁₅₄₀-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2 h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12 h. The results proved that the release-and-hold behavior of the cross-linked CS–PEG₁₅₄₀H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.     

Preparation and spectroscopic characterization of methoxy poly(ethylene glycol)-grafted water-soluble chitosan

The object of this study was to test the solubility of a methoxy poly(ethylene glycol) (MPEG)-grafted chitosan copolymer in organic solvents and aqueous solution. Water-soluble chitosan with low molecular weight (LMWSC) was used in a PEG-graft copolymerization. The MPEG was conjugated to chitosan using 4-dicyclohexylcarbodimide (DCC), and N-hydroxysuccimide (NHS). Introduction of PEG was confirmed by (1)H and (13)C NMR spectroscopy and FT-IR spectroscopy. The degree of substitution (DS) of MPEG into chitosan was calculated from (1)H NMR data and also by estimating the molecular weight (MW) using gel permeation chromatography (GPC). The DS values obtained from (1)H NMR spectroscopy and GPC were similar, indicating that MPEG-grafted LMWSC was synthesized and properly characterized. Furthermore, the introduction of PEG into chitosan increases the solubility in aqueous solutions over a range of pH values (4.0-11.0) and organic solvents such as DMF, DMSO, ethanol, and acetone.     

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

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.     

Effects of poly(ethylene glycol) on liposomes and erythrocytes: Permeability changes and membrane fusion

Poly(ethylene glycol) 6000 induced a concentration-dependent, time-dependent decrease in the latency of the reaction between Arsenazo III sequestered in liposomes and extraliposomal Ca²⁺. This was mediated by a gross change in liposomal permeability, i.e. by a release of Arsenazo III from liposomes rather than simply by an entry of Ca²⁺. The loss of latency was strongly temperature-dependent, and it was markedly diminished on increasing the cholesterol content of the liposomes. It was apparently not due to an osmotic stress of the polymer. The high activation energy found (63 kJ · mol^?1) is thought to indicate that the loss of latency resulted from local discontinuities in the lipid bilayers, caused by dehydration, rather than from partial or total lysis. Related microscopy experiments indicated that the polymer also caused the liposomes to fuse, and it is suggested that membrane fusion may have occurred at the sites of dehydration-induced discontinuities in adjacent bilayers, in addition the polymer was found to enhance the permeability of hen erythrocytes to Ca²⁺ in a manner that was comparable to its effect on liposomal latency, and it is proposed that cell fusion induced by poly(ethylene glycol) may occur at the sites of similarly induced discontinuities in the phospholipid bilayers of two closely adjacent cells.     

Determination of low levels of poly(ethylene glycol) 400 in plasma and urine by capillary gas chromatography-selected ion-monitoring mass spectrometry after solid-phase extraction

A convenient and sensitive method for the quantitative determination of poly(ethylene glycol) 400 in plasma and urine with capillary gas chromatography-mass spectrometry has been developed. The sample preparation involves solid-phase extraction with subsequent derivatization with heptafluorobutyric anhydride, which proved to give the most stable derivative. The derivatization procedure was optimized using experimental design, and different solid-phase extraction columns were evaluated. The limit of quantitation was 1 μmol/l (0.4 μg/ml) for both plasma and urine.     

Capillary electrophoresis analysis of poly(ethylene glycol) and ligand-modified polylysine gene delivery vectors

Cationic polymers including polylysine (PLL) and polyethylenimine are being widely tested as gene delivery vectors in various gene therapy applications. In many cases, the polymers were further modified by hydrophilic polymer grafting or ligand conjugation, which had been shown to greatly affect the vector stability, delivery efficiency and specificity. The characterization of modified polycation is particularly critical for quality control and vector development. Here several different separation modes using capillary electrophoresis for the analytical characterization of the modified polymers are described. PLL molecules were grafted with poly(ethylene glycol) (PEG) chain or conjugated with epidermal growth factor and analyzed under various analytical conditions. Poly(N,N'-dimethylacrylamide)-coated capillary was used to analyze the modified PLL to reduce the interaction between the samples and the capillary wall. PLLs containing different numbers of conjugated ligands were well separated with the coating method but, for PLL-g-PEG, the separation was poor under the same conditions. A method using low buffer pH and hydroxypropylmethyl cellulose additive was developed. These methods are useful to characterize various polycations and important for the quality control and application of potential gene delivery vectors.     

Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG₃) and hexa(ethylene glycol) (EG₆) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.     

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange

(1) The water soluble polymer, poly(ethylene glycol), causes aggregation of sonicated vesicles of dimyristoylphosphatidylcholine in a manner consistent with a steric exclusion mechanism. (2) Poly(ethylene glycol) promotes the exchange of lipids between multilamellar vesicles of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine when the lipids are in the liquid-crystalline state. (3) ³¹P-NMR studies demonstrate that the bilayer configuration of smectic mesophases of dipalmitoylphosphatidylcholine is substantially maintained in the presence of poly(ethylene glycol).     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.     

Protein resistance of dextran and dextran-poly(ethylene glycol) copolymer films

The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1–1.2 mg m^?2) of three molecular weights (10,000, 66,900, 400,000 g mol^?1) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ～5 to 0.5 mg m^?2 with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (～2 mg m^?2) indicating ternary adsorption of the smaller protein within the dextran layer.