Functionalization of poly(ethylene glycol) and monomethoxy-poly(ethylene glycol)

Simple methods are described for the substitution of poly(ethylene glycol) and monomethoxy-poly(ethylene glycol) substitution. Affinity ligands, coenzymes, or enzymes can be covalently attached to the substitution product or they can be used as liquid ionexchangers.     

Branched poly(ethylene glycol) linkers

Novel types of methoxy poly(ethylene glycol) (PEG) linkers (U-PEG linkers) have been synthesized. These PEG linkers are linear polymers that attach to bioactive agents via a functional group, derived from a 2° alcohol, located in the center of the polymer chain versus the traditional terminal attachment site. These new types of linkers can be prepared with different functional groups (e.g. active ester, succinimidyl carbonate, and carbazate) for selected point of attachment, including ethylene oxide oligomers to provide “stems” when steric factors need to be addressed. Conversion of p-nitrophenyl carbonates to the more desirable succinimidyl carbonates has also been accomplished by a novel nucleophilic displacement procedure. Modification of proteins with these reagents is easily accomplished and is illustrated by the conjugation of a U-PEG linker with L -asparaginase.     

Thermoresponsive degradable poly(ethylene glycol) analogues

Thermoresponsive polymers have many biomedical applications, but their nondegradability limits their in vivo applications. Herein, we report a new type of degradable thermoresponsive polymers-degradable poly (ethylene glycol) analogues (DPEGs) having lower critical solution temperatures (LCSTs) ranging 10-50 degrees C. DPEGs were synthesized by condensation polymerization of PEG-di(meth)acrylates (PEGDA or PEGDMA) with dithiols. Their LCSTs could be easily tuned by the PEG-chain length and the types of the double bond in the PEG monomers and dithiols. Long PEG chain and the presence of hydrophilic groups in the dithiol monomer increased the LCST of the resulting DPEG. Crosslinking DPEG chains produced thermoresponsive hydrogels. The hydrogels prepared by the end-capping method maintained the thermoresponsive properties of the linear DPEG. The degradable thermoresponsive DPEGs and their hydrogels have great potentials for in vivo biomedical applications.     

Complex formation between poly(vinyl ether) of ethylene glycol and poly(acrylic acid) in aqueous and organic solutions

Interpolymer reactions between poly(acrylic acid) and poly(vinyl ether) of ethylene glycol were studied by viscometric and spectroturbidimetric methods in aqueous and organic solutions of different nature. It is shown that the formation of interpolymer complexes strongly depends on the strength of the polymer-solvent interaction. A decrease of the thermodynamical quality of the solvents should be favorable for the complexation process.     

Synthesis and characterization of poly(L-alanine)-block-poly(ethylene glycol) monomethyl ether as amphiphilic biodegradable co-polymers

Di-block co-polymers of poly(L-alanine) with poly(ethylene glycol) monomethyl ether (MPEG) were synthesized as amphiphilic biodegradable co-polymers. The ring-opening polymerization of N-carboxy-L-alanine anhydride (NCA) in dichloromethane was initiated by amino-terminated poly(ethylene glycol) monomethyl ether (MPEG-NH2, M(n) = 2000) to afford poly(L-alanine)-block-MPEG. The weight ratio of two blocks in the co-polymers could be altered by adjusting the feeding ratio of NCA to MPEG-NH2. Their chemical structures were characterized on the basis of infrared spectrometry and nuclear magnetic resonance. According to circular dichroism measurement, the poly(L-alanine) chain on the co-polymers in an aqueous medium had a alpha-helix conformation. Two melting points from MPEG block and poly(L-alanine), respectively, could be observed in differential scanning calorimetry curves of the co-polymers, suggesting that a micro-domain phase separation appeared in their bulky states. The co-polymers could take up some water and the capacity was dependent on the ratio of poly(L-alanine) block to MPEG. Such co-polymers might be useful in drug-delivery systems and other biomedical applications.     

Synthesis and characterization of poly(L-alanine)-block-poly(ethylene glycol) monomethyl ether as amphiphilic biodegradable co-polymers

Di-block co-polymers of poly(L-alanine) with poly(ethylene glycol) monomethyl ether (MPEG) were synthesized as amphiphilic biodegradable co-polymers. The ring-opening polymerization of N-carboxy-L-alanine anhydride (NCA) in dichloromethane was initiated by amino-terminated poly(ethylene glycol) monomethyl ether (MPEG-NH₂, M _n = 2000) to afford poly(L-alanine)-block-MPEG. The weight ratio of two blocks in the co-polymers could be altered by adjusting the feeding ratio of NCA to MPEG-NH₂. Their chemical structures were characterized on the basis of infrared spectrometry and nuclear magnetic resonance. According to circular dichroism measurement, the poly(L-alanine) chain on the co-polymers in an aqueous medium had a α-helix conformation. Two melting points from MPEG block and poly(L-alanine), respectively, could be observed in differential scanning calorimetry curves of the co-polymers, suggesting that a micro-domain phase separation appeared in their bulky states. The co-polymers could take up some water and the capacity was dependent on the ratio of poly(L-alanine) block to MPEG. Such co-polymers might be useful in drug-delivery systems and other biomedical applications.     

Poly(ether/ester)s based on poly(butylene terephthalate) and poly(ethylene glycol), 2. Effect of polyether segment length

Structure-properties relationships of poly(ether/ester)s (PEEs) 1 based on poly(butylene terephthalate) (PBT) and poly(ethylene glycol) (PEG) are studied. By varying the length of the soft segments (PEG) of molecular weights 600, 1000 and 2000) two series of PEEs are obtained, (i) with constant mole ratio PBT:PEG (the PBT block length being approximately the same) and (ii) with constant weight ratio PBT:PEG (different PBT segment length). The block structure is proved by ¹H nuclear magnetic resonance and differential scanning calorimetry studies. A three-phase morphology is established, i.e. two amorphous phases (polyether and polyester) and a crystalline phase (PBT), each one distinguished by its own transition temperature. At low temperatures (between ?60 and 20°C) a fourth phase (crystalline PEG) appears. Small angle X-ray scattering intensity and long spacing increase abruptly for the compositions with PEG 2000. The mechanical characteristics of the studied PEEs depend on the weight ratio of the two types of blocks.     

In vivo and in vitro degradation of poly(ether ester) block copolymers based on poly(ethylene glycol) and poly(butylene terephthalate)

Two in vivo degradation studies were performed on segmented poly(ether ester)s based on polyethylene glycol (PEG) and poly(butylene terephthalate) (PBT) (PEOT/PBT). In a first series of experiments, the in vivo degradation of melt-pressed discs of different copolymer compositions were followed up for 24 weeks after subcutaneous implantation in rats. The second series of experiments aimed to simulate long-term in vivo degradation. For this, PEOT/PBT samples were pre-degraded in phosphate buffer saline (PBS) at 100 degrees C and subsequently implanted. In both series, explanted materials were characterized by intrinsic viscosity measurements, mass loss, proton nuclear magnetic resonance spectroscopy (1H-NMR) and differential scanning calorimetry (DSC). In both studies the copolymer with the higher PEO content degraded the fastest, although all materials degraded relatively slowly. To determine the nature of the degradation products formed during hydrolysis of the copolymers, 1000 PEOT71PBT29 (a copolymer based on PEG with a molecular weight of 1000 g/mol and 71 wt% of PEO-containing soft segments) was degraded in vitro at 100 degrees C in phosphate buffer saline (PBS) during 14 days. The degradation products present in PBS were analyzed by 1H-NMR and high performance liquid chromatography/mass spectroscopy (HPLC/MS). These degradation products consisted of a fraction with high contents of PEO that was soluble in PBS and a PEOT/PBT fraction that was insoluble at room temperature. From the different in vitro and in vivo degradation experiments performed, it can be concluded that PEOT/PBT degradation is a slow process and generates insoluble polymeric residues with high PBT contents.     

Preparation of poly(ethylene glycol) grafted poly(3-hydroxyalkanoate) networks

Poly(3-hydroxyalkanoate)-g-poly(ethylene glycol) crosslinked graft copolymers are described. Poly(3-hydroxyalkanoate)s containing double bonds in the side chain (PHA-DB) were obtained by co-feeding Pseudomonas oleovorans with a mixture of nonanoic acid and anchovy (hamci) oily acid (in weight ratios of 50/50 and 70/30). PHA-DB was thermally grafted with a polyazoester synthesized by the reaction of poly(ethylene glycol) with MW of 400 (PEG-400) and 4,4′-azobis(4-cyanopentanoyl chloride). Sol-gel analysis and spectrometric and thermal characterization of the networks are reported.     

Biodegradable and thermoreversible hydrogels of poly(ethylene glycol)-poly(epsilon-caprolactone-co-glycolide)-poly(ethylene glycol) aqueous solutions

The aqueous solutions of triblock copolymers of poly(ethylene glycol)-poly(epsilon-caprolactone-co-glycolide)-poly(ethylene glycol) [PEG-P(CL-GA)-PEG] undergoing sol-gel transition as the temperature increases from 20 to 60 degrees C were successfully prepared. The thermogelling block copolymers were synthesized by subtle control of the hydrophilic/hydrophobic balance and the chain microstructures. The amphiphilic block copolymer formed micelles in aqueous solution, and the micelle aggregated as the temperature increased. The sol-gel transition of the copolymer aqueous solutions was studied focusing on the structure-property relationship. GA was incorporated into the polymer chain to prevent crystallization of PCL component and increase the polymer degradation. It is expected to be a promising long-term delivery system for pH-sensitive drugs, proteins, and genes.     

Poly(ether/ester)s based on poly(tetramethylene terephthalate) and poly(ethylene glycol), 4. Modification with 2-butyne-1,4-diol

A series of poly(ether/ester)s derived from dimethyl terephthalate, 1,4-butanediol, 2-butyne-1,4-diol (2-BD-1,4) and α-hydro-ω-hydroxypoly(oxyethylene) (molecular weight 1000) is synthesized. The mole ratio of the starting components is selected to result in copolymers with constant hard : soft segment weight ratio (59:41). The amount of 2-BD-1,4 is varied from 0 to 20 wt.-% referred to the total amount of the short-chain diols used. The incorporation of 2-BD-1,4 in the macrochains is proved by ¹H NMR measurements. A small increase of the oxygen index is found with increasing the amount of 2-BD-1,4. It is established that the poly(ether/ester) containing 10 wt.-% of 2-BD-1,4 behaves as known poly(ether/ester)s. Data from differential scanning calorimetry suggest a three-phase structure, two amorphous and one crystalline one. Small-angle X-ray studies of the annealed samples reveal a strong tendency to phase separation with increasing the annealing temperature. The latter is in agreement with density measurements.     

Synthesis and polymerization of poly(ethylene glycol) fumarates

Fumaric esters of poly(ethylene glycol) ( 1a–d ) were prepared as macromonomers. The halfesters 1a and 1c were obtained by interaction of maleic anhydride with the monoethers of poly(ethylene glycol) ( 2a and 2b ) in the presence of 4-dimethylaminopyridine, and the diesters 1b and 1d by esterification of methyl hydrogen fumarate ( 13 ) with the monoethers of poly(ethylene glycol) ( 2a and 2b ). The macromonomers were found to homopolymerize and to copolymerize with styrene and methyl methacrylate following a radical polymerization mechanism.     

Effects of sterilization on poly(ethylene glycol) hydrogels

The past few decades have witnessed a dramatic increase in the development of polymeric biomaterials. These biomaterials have to undergo a sterilization procedure before implantation. However, many sterilization procedures have been shown to profoundly affect polymer properties. Poly(ethylene glycol) hydrogels have gained increasing importance in the controlled delivery of therapeutics and in tissue engineering. We evaluated the effect of ethylene oxide (EtO), hydrogen peroxide (H(2)O(2)), and gamma sterilization of poly(ethylene glycol) hydrogels on properties relevant to controlled drug delivery and tissue engineering. We observed that the release of cyclosporine (CyA) (an immunosuppressive drug that is effective in combating tissue rejection following organ transplantation) was significantly affected by the type of sterilization. However, that was not the case with rhodamine B, a dye. Hence, the drug release characteristics were observed to be dependent not only on the sterilization procedure but also on the type of agent that needs to be delivered. In addition, differences in the swelling ratios for the sterilized and unsterilized hydrogels were statistically significant for 1:1 crosslinked hydrogels derived from the 8000 MW polymer. Significant differences were also observed for gamma sterilization for 1:1 crosslinked hydrogels derived from the 3350 MW polymer and also the 2:1 crosslinked hydrogels derived from the 8000 MW polymer. Atomic force microscopy (AFM) studies revealed that the roughness parameter for the unsterilized and EtO-sterilized PEG hydrogels remained similar. However, a statistically significant reduction of the roughness parameter was observed for the H(2)O(2) and gamma-sterilized samples. Electron spin resonance (ESR) studies on the unsterilized and the sterilized samples revealed the presence of the peroxy and the triphenyl methyl carbon radical in the samples. The gamma and the H(2)O(2)-sterilized samples were observed to have a much higher concentration of the radical pecies when compared with the EtO and the unsterilized samples.     

Synthesis and water-swelling of thermo-responsive poly(ester urethane)s containing poly(epsilon-caprolactone), poly(ethylene glycol) and poly(propylene glycol)

Thermo-responsive multiblock poly(ester urethane)s comprising poly(epsilon-caprolactone) (PCL), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments were synthesized. The copolymers were characterized by GPC, NMR, FTIR, XRD, DSC and TGA. Water-swelling analysis carried out at different temperatures revealed that the bulk hydrophilicity of the copolymers could be controlled either by adjusting the composition of the copolymer or by changing the temperature of the environment. These thermo-responsive copolymer films formed highly swollen hydrogel-like materials when soaked in cold water and shrank when soaked in warm water. The changes are reversible. The mechanical properties of the copolymer films were assessed by tensile strength measurement. These copolymers were ductile when compared to PCL homopolymers. Young's modulus and the stress at break increased with increasing PCL content, whereas the strain at break increased with increasing PEG content. The results of the cytotoxicity tests based on the ISO 10993-5 protocol demonstrated that the copolymers were non-cytotoxic and could be potentially used in biomedical applications.     

Degradative properties and cytocompatibility of a mixed-mode hydrogel containing oligo[poly(ethylene glycol)fumarate] and poly(ethylene glycol)dithiol

Our laboratory is currently exploring synthetic oligo(poly(ethylene glycol)fumarate) (OPF)-based biomaterials as a means to deliver fibroblasts to promote regeneration of central/partial defects in tendons and ligaments. In order to further modulate the swelling and degradative characteristics of OPF-based hydrogels, OPF crosslinking via a radically initiated, mixed-mode reaction involving poly(ethylene glycol) (PEG)-diacrylate and PEG-dithiol was investigated. Results demonstrate that mixed-mode hydrogels containing OPF can be formed and that the presence of 20 wt.% PEG-dithiol increases swelling and decreases degradation time vs. 10 wt.% PEG-dithiol and non-thiol-containing hydrogels (20% thiol fold swelling 28.7+/-0.8; 10% thiol fold swelling 11.6+/-1.4; non-thiol 8.7+/-0.2; 20% thiol-containing hydrogels degrade within 15 days in vitro). After encapsulation, tendon/ligament fibroblasts remained largely viable over 8 days of static culture. While the presence of PEG-dithiol did not significantly affect cellularity or collagen production within the constructs over this time period, image analysis revealed that the 20% PEG-dithiol gels did appear to promote cell clustering, with greater values for aggregate area observed by day 8. These experiments suggest that mixed-mode OPF-based hydrogels may provide an interesting alternative as a cell carrier for engineering a variety of soft orthopedic tissues, particularly for applications when it is important to encourage cell-cell contact.     

Surface modification using silanated poly(ethylene glycol)s

Surface-grafted poly(ethylene glycol) (PEG) molecules are known to prevent protein adsorption to the surface. The protein-repulsive property of PEG molecules are maximized by covalent grafting. We have synthesized silanated monomethoxy-PEG (m-PEG) for covalent grafting of PEG to surfaces with oxide layers. Two different trialkoxysilylated PEGs were synthesized and characterized. The first trialkoxysilylated PEG was prepared by direct coupling of m-PEG with 3-isocyanatopropyltriethoxysilane through a urethane bond (silanated PEG I). The other silanated PEG (silanated PEG II) containing a long hydrophobic domain between PEG and a silane domain was prepared by reacting m-PEG with 1,6-diisocyanatohexane and 10-undecen-1-ol in sequence before silylation with 3-mercaptopropyl trimethoxysilane. Silanated PEGs I and II were grafted onto glass, a model surface used in our study. The PEG-grafted glass surfaces were characterized by contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Although contact angle did not change much as the bulk concentration of silanated PEG used for grafting increased from 0.1 to 20 mg/ml for both PEGs I and II, the surface atomic concentrations from XPS measurements showed successful PEG grafting. Surface PEG grafting increased concentration of surface carbon but decreased silicone concentration. The high resolution C1s spectra showed higher ether carbon with lower hydrocarbon compositions for the PEG-grafted surfaces compared to the control surface. AFM images showed that more PEG molecules were grafted onto the surface as the bulk concentration used for grafting was increased. AFM images of the dried surfaces showed that the surfaces were not completely covered by PEG molecules. After hydration, however, the surface appears to be covered completely probably due to the hydration of the grafted PEG chains. Glass surfaces modified with silanated PEGs reduced fibrinogen adsorption by more than 95% as compared with the control surface. Silanated PEGs provides a simple method for PEG grafting to the surface containing oxide layers.     

Polyethylenimine-grafted copolymer of poly(l-lysine) and poly(ethylene glycol) for gene delivery

A major challenge in gene therapy is the development of effective gene delivery vectors with low toxicity. In the present study, linear poly(ethylenimine) (lPEI) with low molecular weight was grafted onto the block copolymer (PPL) of poly(l-lysine) (PLL) and poly(ethylene glycol)(PEG), yielding a ternary copolymer PEG-b-PLL-g-lPEI (PPI) for gene delivery. In such molecular design, PLL, lPEI and PEG blocks were expected to render the vector biodegradability, proton buffering capacity, low cationic toxicity and potentially long circulation in vivo, respectively. Given proper control of molecular composition, the copolymers demonstrated lower cytotoxicity, proton buffering capacity, ability to condense pDNA and mediate effective gene transfection in various cell lines. With folate as an exemplary targeting ligand, the FA-PPI/pDNA complex showed much higher transgene activity than its nontargeting counterpart for both reporter and therapeutic genes in folate receptor(FR)-positive cells. FA-PPI mediated effective transfection of the TNF-related apoptosis-inducing ligand gene (TRAIL) in human hepatoma Bel 7402 cells, leading to cell apoptosis and great suppression of cell viability. Our results indicate that the copolymers might be a promising vector combining low cytotoxicity, biodegradability, and high gene transfection efficiency.     

Chainlength dependence of thermodynamic properties of poly(ethylene glycol)

Investigations of ten poly(ethylene glycol) samples with molecular weights of 62 to 20 000 showed that inverse gas chromatography is a sensitive tool to determine molecular weight dependent variations of thermodynamic parameters like specific retention volume, activity coefficient, and Flory-Huggins interaction parameter. As mobile phase 27 probe molecules with various functional groups could be characterized with respect to their solubility behaviour for the oligomers and polymers due to the ability to form hydrogen bonds. By application of the factor analysis technique two factors could be extracted from the data matrix of retention values. The best fit of the corresponding simple structure was achieved with a combination of group concentrations of the terminal OH-group and the repeating ether unit of the poly(ethylene glycol)s.     

The phase behaviour of a poly(ether sulfone) with poly(ethylene oxide)

Blends of poly(ethylene oxide) with a poly(ether sulfone) were prepared by casting from a common solvent and were found to be miscible and show a single, composition dependent, glass transition temperature. Mixtures in both cyclohexanone and N,N-dimethylformamide phase, separated on heating and thus conditions need to be carefully chosen to obtain homogeneous blends. At higher PEO contents, PEO crystallised from the blends at lower temperatures. The melting point depression, as determined by trubidity measurements, was used to calculate an interaction parameter which was negative, as expected for miscible polymers. The blends also phase separated on heating, and the cloud point curve could be measured by turbidity measurements and confirmed by both visible and electron microscopy. The cloud point curve was very skew with a minimum at around 10 wt.-% PES content. This was not a strong function of the molecular weight and the skew nature was thus presumably due to differences in the state parameters of the pure components. The blends showed a very high mobility with sharp and reproducible could points which might make them ideal for future miscibility studies.     

Cartilage-like mechanical properties of poly (ethylene glycol)-diacrylate hydrogels

Hydrogels prepared from poly-(ethylene glycol) (PEG) have been used in a variety of studies of cartilage tissue engineering. Such hydrogels may also be useful as a tunable mechanical material for cartilage repair. Previous studies have characterized the chemical and mechanical properties of PEG-based hydrogels, as modulated by precursor molecular weight and concentration. Cartilage mechanical properties vary substantially, with maturation, with depth from the articular surface, in health and disease, and in compression and tension. We hypothesized that PEG hydrogels could mimic a broad range of the compressive and tensile mechanical properties of articular cartilage. The objective of this study was to characterize the mechanical properties of PEG hydrogels over a broad range and with reference to articular cartilage. In particular, we assessed the effects of PEG precursor molecular weight (508?Da, 3.4?kDa, 6?kDa, and 10?kDa) and concentration (10-40%) on swelling property, equilibrium confined compressive modulus (H(A0)), compressive dynamic stiffness, and hydraulic permeability (k(p0)) of PEG hydrogels in static/dynamic confined compression tests, and equilibrium tensile modulus (E(ten)) in tension tests. As molecular weight of PEG decreased and concentration increased, hydrogels exhibited a decrease in swelling ratio (31.5-2.2), an increase in H(A0) (0.01-2.46?MPa) and E(ten) (0.02-3.5?MPa), an increase in dynamic compressive stiffness (0.055-42.9?MPa), and a decrease in k(p0) (1.2?×?10(-15) to 8.5?×?10(-15)?m(2)/(Pa?s)). The frequency-dependence of dynamic compressive stiffness amplitude and phase, as well as the strain-dependence of permeability, were typical of the time- and strain-dependent mechanical behavior of articular cartilage. H(A0) and E(ten) were positively correlated with the final PEG concentration, accounting for swelling. These results indicate that PEG hydrogels can be prepared to mimic many of the static and dynamic mechanical properties of articular cartilage.