Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

Photografted poly(ethylene glycol) matrix for affinity interaction studies

A poly(ethylene glycol) (PEG)-based matrix for studies of affinity interactions is developed and demonstrated. The PEG matrix, less than 0.1 microm thick, is graft copolymerized onto a cycloolefin polymer from a mixture of PEG methacrylates using a free radical reaction initiated by UV light at 254 nm. The grafting process is monitored in real time, and characteristics such as thickness, homogeneity, relative composition, photostability, and performance in terms of protein resistance in complex biofluids and sensor qualities are investigated with null ellipsometry, infrared spectroscopy, and surface plasmon resonance. The matrix is subsequently modified to contain carboxyl groups, thereby making it possible to immobilize ligands in a controlled and functional manner. Human serum albumin and fibrinogen are immobilized and successfully detected by antibody recognition using surface plasmon resonance. The results are encouraging and suggest that the PEG matrix is suitable for biochip and biosensor applications in demanding biofluids.     

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.     

Effect of solute hydrogen bonding capacity on osmotic stability of lysosomes

The effect of solute hydrogen bonding capacity on the osmotic stability of lysosomes was examined through measurement of free enzyme activity of lysosomes after their incubation in sucrose and poly(ethylene glycol) (PEG) (1500–6000 Da molecular mass) media. Free enzyme activity of the lysosomes was less in the PEG medium than that in the sucrose medium under the same hypotonic condition. The lysosomal enzyme latency loss decreased with increasing hydrogen bonding capacity of the solute. In addition, the lysosomes lost less latency at lower incubation temperature. The results indicate that solute hydrogen bonding capacity plays an important role in the osmotic protection of an incubation medium to lysosomes.     

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

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.     

Contact angle, WAXS, and SAXS analysis of poly(beta-hydroxybutyrate) and poly(ethylene glycol) block copolymers obtained via Azotobacter vinelandii UWD

This study investigated and correlated physical properties and cell interactions of copolymers obtained by a poly(ethylene glycol) (PEG)-modulated fermentation of Azotobacter vinelandii UWD. PEGs with molecular weights of 400 and 3400 Da and di(ethylene glycol) (DEG) were used to modulate the bacterial synthesis of poly(beta-hydroxybutyrate) (PHB). The PHB crystallinity was determined by wide-angle X-ray scattering (WAXS). Small-angle X-ray scattering (SAXS) showed that lamellar distances decreased between the PHB and the PHB modulated with PEG or DEG. Furthermore, the contact angle of water on the PHB/PEG polymer surfaces decreased when compared to that of PHB. The significant decrease of the contact angle and corresponding increase in surface tension, as well as significant decrease in cell adhesion, suggest the presence of hydrophilic PEG and DEG within the hydrophobic surface.     

Increased activity and stability of poly(ethylene glycol)-modified trypsin.

The reaction of trypsin with activated monomethoxypoly(ethylene glycol) with various molecular masses led to the development of a series of poly(ethylene glycol)-modified trypsins (PEG-trypsins). On determining the catalytic properties of PEG-trypsin using N-benzoyl-L-arginine p-nitroanilide as a substrate, a three- to fourfold increase in the maximal velocity of hydrolysis was found to occur, whatever the size of the PEG moiety used. PEG-trypsin with higher molecular mass moieties showed lower Michaelis constant values. The activation of trypsin was neither reversed by nucleophiles such as hydroxylamine, nor prevented when modification was carried out in the presence of benzamidine or in the presence of the polypeptidic soybean trypsin inhibitor. Chemical modification of about 80% of the free amino groups with PEG chains significantly improved the resistance to heat and detergents. This might result from the formation of a highly hydrogen-bonded structure around the enzyme.     

Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate-co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA-co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(100) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA-co-PEGMEMA) microdomains were activated directly by 1,1'-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.     

Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) networks for enhanced blood compatibility

Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) (PEG-g-PHU) networks were prepared by irradiating homogeneous solutions of poly(3-hydroxyundecenoate) (PHU) and the monoacrylate of poly(ethylene glycol) (PEG) with UV light. The resulting polymer networks were characterized by measuring the water contact angle, water uptake, and mechanical properties and by performing attenuated total reflectance infrared spectroscopy and scanning electron microscopy. These measurements showed that the PEG chains were present in polymer networks. Adsorption of blood proteins and platelets on cross-linked PHU (CLPHU) and PEG-g-PHU were examined using poly(L-lactide) (PLLA) surfaces as control. Blood proteins and platelets had significantly lower tendency of adhesion to surfaces composed of CLPHU and PEG-g-PHU networks than to PLLA. Blood compatibility of polymer networks increased as the fraction of grafted PEG increased. The results of this study suggest that PEG-g-PHU networks might be useful for blood-compatible biomedical applications.     

Interaction forces and morphology of a protein-resistant poly(ethylene glycol) layer

The molecular interactions on a protein-resistant surface coated with low-molecular-weight poly(ethylene glycol) (PEG) copolymer brushes are investigated using the extended surface forces apparatus. The observed interaction force is predominantly repulsive and nearly elastic. The chains are extended with respect to the Flory radius, which is in agreement with qualitative predictions of scaling theory. Comparison with theory allows the determination of relevant quantities such as brush length and adsorbed mass. Based on these results, we propose a molecular model for the adsorbed copolymer morphology. Surface-force isotherms measured at high resolution allow distinctive structural forces to be detected, suggesting the existence of a weak equilibrium network between poly(ethylene glycol) and water--a finding in accordance with the remarkable solution properties of PEG. The occurrence of a fine structure is interpreted as a water-induced restriction of the polymer's conformational space. This restriction is highly relevant for the phenomenon of PEG protein resistance. Protein adsorption requires conformational transitions, both in the protein as well as in the PEG layer, which are energetically and kinetically unfavorable.     

PNA conjugated to high-molecular weight poly(ethylene glycol): synthesis and properties

The conjugation of a bioactive, fluorescent PNA sequence to high-molecular weight poly(ethylene glycol) (PEG) is described and the properties of the PEG-PNA conjugate are evaluated.     

Structural effects on the biodistribution and positron emission tomography (PET) imaging of well-defined (64)Cu-labeled nanoparticles comprised of amphiphilic block graft copolymers

The synthesis of poly(methyl methacrylate-co-methacryloxysuccinimide-graft-poly(ethylene glycol)) (PMMA-co-PMASI-g-PEG) via living free radical polymerization provides a convenient route to well-defined amphiphilic graft copolymers having a controllable number of reactive functional groups, variable length PEG grafts, and low polydispersity. These copolymers were shown to form PMMA-core/PEG-shell nanoparticles upon hydrophobic collapse in water, with the hydrodynamic size being defined by the molecular weight of the backbone and the PEG grafts. Functionalization of these polymeric nanoparticles with a 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) ligand capable of chelating radioactive 64Cu nuclei enabled the biodistribution and in vivo positron emission tomography of these materials to be studied and directly correlated to the initial structure. Results indicate that nanoparticles with increasing PEG chain lengths show increased blood circulation and low accumulation in excretory organs, suggesting the possible use of these materials as stealth carriers for medical imaging and systemic administration.     

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to ¹H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.     

Compatibilization effect of poly(epsilon-caprolactone)-b-poly(ethylene glycol) block copolymers and phase morphology analysis in immiscible poly(lactide)/poly(epsilon-caprolactone) blends

The miscibility and phase behavior of two stereoisomer forms of poly(lactide) (PLA: poly (L-lactide) (PLLA) and poly(DL-lactide) (PDLLA)) blends with poly(epsilon-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) and PCL-b-monomethoxy-PEG (PCL-b-MPEG) block copolymers have been investigated by differential scanning calorimetry (DSC). The DSC thermal behavior of both the blend systems revealed that PLA is miscible with the PEG segment phase of PCL-b-(M)PEG but is still immiscible with its PCL segment phase although PCL was block-copolymerized with PEG. On the basis of these results, PCL-b-PEG was added as a compatibilizer to PLA/PCL binary blends. The improvement in mechanical properties of PLA/PCL blends was achieved as anticipated upon the addition of PCL-b-PEG. In addition, atomic force microscopy (AFM) measurements have been performed in order to study the compositional synergism to be observed in mechanical tests. AFM observations of the morphological dependency on blend composition indicate that PLA/PCL blends are immiscible but compatible to some extent and that synergism of compatibilizing may be maximized in the compositional blend ratio before apparent phase separation and coarsening.     

Phase separation in the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution driven by a specific interaction between the glucose side chain and poly(ethylene oxide)

We found that the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution underwent phase separation at around 3-4 degrees C, and this temperature was independent of both polymer concentration and the difference in poly(ethylene oxide) molecular weight (Mw 6000 and 70,000). The phase-separation took place at the same temperature at which the optical rotation changed. Since the optical rotation change is ascribed to the difference in the nature of hydrogen bonding between the schizophyllan side chain and water, the phase separation is also considered to be due to an interaction between poly(ethylene oxide) and schizophyllan. The phase-separation temperature increased on changing H2O to D2O in accordance with a change in the optical rotation, confirming the specific interaction essential for the phase separation.     

Morphological control of poly(ethylene glycol)-block-poly(epsilon-caprolactone) copolymer aggregates in aqueous solution

In aqueous solution, it was found that the amphiphilic copolymer poly(ethylene glycol)-b-poly(caprolactone) (PEG(5000)-b-PCL(4100)) formed different morphologies, including long rod-like, short rod-like, or spherical aggregates, when the copolymer concentration was increased. Nearly identical morphologies were observed with the addition of increasing amounts of PEG(2000)-distearoylphosphoethanolamine (PEG(2000)-DSPE) to the copolymer. The morphologies of the aggregates in solution were confirmed by negative stain transmission electron microscopy (TEM) and cryogenic-TEM (cryo-TEM). The critical micelle concentrations of the PEG(5000)-b-PCL(4100) copolymer, PEG(2000)-DSPE and a mixture of the two materials (PEG(5000)-b-PCL 4100/PEG(2000)-DSPE) were evaluated to determine the thermodynamic stability of the aggregates. Differential scanning calorimetry was performed to gain insight into the degree of mixing of PEG(5000)-b-PCL(4100) and PEG(2000)-DSPE. Overall, combining PEG(5000)-b-PCL(4100) and PEG(2000)-DSPE produced a single population of mixed micelles with rod-like or spherical morphologies depending on the material composition and concentration.     

Variation of penicillin acylase partition coefficient with phase volume ratio in poly(ethylene glycol)–sodium citrate aqueous two-phase systems

The influence of phase volume ratio on partition and purification of penicillin acylase from Escherichia coli on poly(ethylene glycol)–sodium citrate aqueous two-phase systems was studied. In PEG 1000 systems both partition coefficients of the enzyme and total protein increased with decreasing phase volume ratio. However, in PEG 3350 containing NaCl, penicillin acylase follows a reverse trend, while total protein behaves in the same way. Implications for protein purification designs are discussed.     

Study of the synthesis, crystallization, and morphology of poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers

Poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers PEG-PCL were synthesized by ring-opening polymerization of epsilon-caprolactone using monomethoxy poly(ethylene glycol) as the macroinitiator and calcium ammoniate as the catalyst. Obvious mutual influence between PEG and PCL crystallization was studied by altering the relative block length. Fixing the length of the PEG block (Mn = 5000) and increasing the length of the PCL block, the crystallization temperature of the PCL block rose gradually from 1 to about 35 degrees C while that of the PEG block dropped from 36 to -6.6 degrees C. Meanwhile, the melting temperature of the PCL block went up from 30 to 60 degrees C, while that of the PEG block declined from 60 to 41 degrees C. If the PCL block was longer than the PEG block, the former would crystallize first when cooling from a molten state and led to obviously imperfect crystallization of PEG and vice versa. And they both crystallized at the same temperature, if their weight fractions were equal. We found that the PEG block could still crystallize at -6.6 degrees C even when its weight fraction is only 14%. A unique morphology of concentric spherulites was observed for PEG5000-PCL5000. According to their morphology and real-time growth rates, it is concluded that the central and outer sections in the concentric spherulites were PCL and PEG, respectively, and during the formation of the concentric spherulite, the PEG crystallized quickly from the free space of the PCL crystal at the earlier stage, followed by outgrowing from the PCL spherulites in the direction of right angles to the circle boundaries until the entire area was occupied.