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.     

Separation of macromolecules by ultrafiltration: Removal of poly(ethylene glycol) from human albumin

Mixtures of albumin and poly(ethylene glycol) (PEG) were used to elucidate some of the factors which influence the separation of macromolecules by thin-channel ultrafiltration. Several membranes which readily passed PEG-4000 in the absence of protein were found to exhibit increased rejection of the synthetic polymer when albumin was added to the system. Based on a comparison of filtration flux and net sieving properties, the PM-30 membrane of Amicon was chosen for further characterization. The increased rejection of PEG-4000 in the presence of albumin was independent of albumin concentration between 1 and 100 mg/ml and persisted even after albumin was removed and the system flushed with water. Overnight incubation of the membrane with trypsin restored the original sieving properties, indicating that the ‘permanent’ effects were due to irreversible adsorption to the membrane. By measuring flux over a 10⁶-fold range of albumin concentration it was possible to resolve the effects of protein adsorption, a saturable process which occurs at low protein concentration (<0.01 mg/ml), from the effects of concentration polarization which occur at high protein concentration (>0.1 mg/ml). Only the former process has an effect on the net sieving properties in this system. In spite of the adverse effects of protein adsorption, it was still possible to obtain efficient removal of PEG-4000 from albumin. Exchange of approximately 5 vols. of solvent at room temperature resulted in a 10-fold reduction in the concentration of PEG in the sample, with no loss of albumin, and no formation of albumin dimers.     

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL)

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.     

The effects of poly(ethylene glycol) on the solution structure of human serum albumin

Protein physical and chemical properties can be altered by polymer interaction. The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many organic and polymer molecules. This study was designed to examine the interaction of HSA with poly(ethylene glycol) (PEG) in aqueous solution at physiological conditions. Fourier transform infrared, ultraviolet-visible, and CD spectroscopic methods were used to determine the polymer binding mode, the binding constant, and the effects of polymer complexation on protein secondary structure.The spectroscopic results showed that PEG is located along the polypeptide chains through H-bonding interactions with an overall affinity constant of K = 4.12 x 10(5) M(-1). The protein secondary structure showed no alterations at low PEG concentration (0.1 mM), whereas at high polymer content (1 mM), a reduction of alpha-helix from 59 (free HSA) to 53% and an increase of beta-turn from 11 (free HSA) to 22% occurred in the PEG-HSA complexes (infrared data). The CDSSTR program (CD data) also showed no major alterations of the protein secondary structure at low PEG concentrations (0.1 and 0.5 mM), while at high polymer content (1 mM), a major reduction of alpha-helix from 69 (free HSA) to 58% and an increase of beta-turn from 7 (free HSA) to 18% was observed.     

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.     

Effect of membrane cholesterol enrichment or depletion on the partition behavior of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases

It has previously been shown that by appropriate manipulation of polymer concentrations and ionic composition and concentration one can select whether charge-associated or lipid-related membrane surface properties are reflected by cell partition in dextran-poly(ethylene glycol) aqueous two-phase systems (Walter, H. (1977) in Methods of Cell Separation ((Catsimpoolas, N., ed.), Vol. 1, pp. 307–354, Plenum Press, New York). In the current experiments we have studied the partition behavior of human erythrocytes and found that not only lipid-related but also charge-associated membrane properties are altered as a consequence of cholesterol-enrichment or -depletion. Results further indicate that, just as cell partition in charged phase systems reflects membrane charge-associated properties not readily measured by means other than partition (Brooks, D.E., Seaman, G.V.F. and Walter, H. (1971) Nat. New Biol. 234, 61–62; Walter, H., Tung, R., Jackson, L.J. and Seaman, G.V.F. (1972) Biochem. Biophys. Res. Commun. 48, 565–571), cell partition in uncharged phases reflects membrane lipid-related properties also not readily measured by other means.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

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).     

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.     

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.     

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.     

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.     

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.     

One-pot synthesis in aqueous system for water-soluble chitosan-graft-poly(ethylene glycol) methyl ether

Chitosan is functionalized with poly(ethylene glycol) methyl ether (mPEG) at the amino and hydroxyl groups via a single step reaction in a homogeneous aqueous system. A chitosan aqueous solution obtained from the mixture of chitosan and hydroxybenzotriazole (HOBt) in water is a key factor in providing mild conditions to conjugate mPEG by using a carbodiimide conjugating agent. The reaction at ambient temperature for 24 h gives chitosan-g-mPEG with water solubility with mPEG content as high as 42%. This work demonstrates that a water-soluble chitosan-HOBt complex is an effective system for the preparation of chitosan derivatives via the aqueous system without the use of acids or organic solvents.     

Poly(ethylene glycol) quantitation by laser nephelometry

When poly(ethylene glycol) 3350 is estimated by the method of Skoog [(1979) Vox Sang. 37, 345-349], fine particles form. The particles are not attributable to residual protein but to a poly(ethylene glycol)/barium/iodine complex that can be quantitated by means of a laser nephelometer. The method is sensitive to at least 10 mg% poly(ethylene glycol) 3350 (4 micrograms in the cuvette) in 2500 mg% protein, and nephelometer response is approximately linear between 30 and 200 mg% of the polymer. The coefficient of variance is about 8%. Triton X-100, Pluronic F-68, Varonic 1000MS, and poly(ethylene glycol) of higher and lower molecular weight react well. Alkylated celluloses, dextrans, glycerol, glycine, and sodium dodecyl sulfate do not react significantly. Barium can be replaced with Mg, Ca, Ni, Fe, and other divalent cations in the reaction, but other than for Hg, light-scattering is most intense with Ba. The reaction goes to completion in about 5 min and is most intense when the barium is added before the iodine.     

Cross-linking density alters early metabolic activities in chondrocytes encapsulated in poly(ethylene glycol) hydrogels and cultured in the rotating wall vessel

In designing a tissue engineering strategy for cartilage repair, selection of both the bioreactor, and scaffold is important to the development of a mechanically functional tissue. The hydrodynamic environment associated with many bioreactors enhances nutrient transport, but also introduces fluid shear stress, which may influence cellular response. This study examined the combined effects of hydrogel cross-linking and the hydrodynamic environment on early chondrocyte response. Specifically, chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels having two different cross-linked structures, corresponding to a low and high cross-linking density. Both cross-linked gels yielded high water contents (92% and 79%, respectively) and mesh sizes of 150 and 60 A respectively. Cell-laden PEG hydrogels were cultured in rotating wall vessels (RWV) or under static cultures for up to 5 days. Rotating cultures yielded low fluid shear stresses (< or = 0.11 Pa) at the hydrogel periphery indicating a laminar hydrodynamic environment. Chondrocyte response was measured through total DNA content, total nitric oxide (NO) production, and matrix deposition for glycosaminoglycans (GAG). In static cultures, gel cross-linking had no effect on DNA content, NO production, or GAG production; although GAG production increased with culture time for both cross-linked gels. In rotating cultures, DNA content increased, NO production decreased, and overall GAG production decreased when compared to static controls for the low cross-linked gels. For the high cross-linked gels, the hydrodynamic environment had no effect on DNA content, but exhibited similar results to the low cross-linked gel for NO production, and matrix production. Our findings demonstrated that at early culture times, when there is limited matrix production, the hydrodynamic environment dramatically influences cell response in a manner dependent on the gel cross-linking, which may impact long-term tissue development.     

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat‐induced deamidation and β‐elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alchol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S‐carboxy‐methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers––PEG and PVP––and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution. Biotechnol. Bioeng. 2012; 109: 2543–2552. © 2012 Wiley Periodicals, Inc.     

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.