Poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] and poly(ethylene glycol) as candidate biomaterials: characterization and mechanical property study

Poly(ester urethane)s with poly[(R)-3-hydroxybutyrate] (PHB) as the hard and hydrophobic segment and poly(ethylene glycol) (PEG) as the soft and hydrophilic segment were synthesized from telechelic hydroxylated PHB (PHB-diol) and PEG using 1,6-hexamethylene diisocyanate as a nontoxic coupling reagent. Their chemical structures and molecular characteristics were studied by gel permeation chromatography, 1H NMR, and Fourier transform infrared spectroscopy. Results of differential scanning calorimetry and X-ray diffraction indicated that the PHB segment and PEG segment in the poly(ester urethane)s formed separate crystalline phases with lower crystallinity and a lower melting point than those of their corresponding precursors, except no PHB crystalline phase was observed in those with a relatively low PHB fraction. Thermogravimetric analysis showed that the poly(ester urethane)s had better thermal stability than their precursors. The segment compositions were calculated from the two-step thermal decomposition profiles, which were in good agreement with those obtained from 1H NMR. Water contact angle measurement and water swelling analysis revealed that both surface hydrophilicity and bulk hydrophilicity of the poly(ester urethane)s were enhanced by incorporating the PEG segment into PHB polymer chains. The mechanical properties of the poly(ester urethane)s were also assessed by tensile strength measurement. It was found that the poly(ester urethane)s were ductile, while natural source PHB is brittle. Young's modulus and the stress at break increased with increasing PHB segment length or PEG segment length, whereas the strain at break increased with increasing PEG segment length or decreasing PHB segment length.     

Polycaprolactone-poly(ethylene glycol) multiblock copolymers as potential substitutes for di(ethylhexyl) phthalate in flexible poly(vinyl chloride) formulations

New high-molecular-weight hydrophobic/hydrophilic segmented copolymers of poly(ester ether carbonate) structure, containing poly(epsilon-caprolactone) (PCL) and poly(ethylene glycol) (PEG) segments in their main chain, were synthesized and characterized. These copolymers were obtained by a two-step chain-extension reaction carried out in the presence of alpha,omega-dihydroxy-oligoPCL of molecular weight 1250 and PEG samples of molecular weight 150, 400, 600, 1000, and 2000. The molecular structures of all synthesized materials were characterized by means of (1)H NMR and (13)C NMR spectroscopy, their molecular weights were determined by means of size exclusion chromatography, and their thermal properties were obtained by means of differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The poly(ester ether carbonate)s of this study are partly or totally miscible at least up to 50 wt % with poly(vinyl chloride) (PVC) and could be used to produce flexible PVC formulations. The miscibility between PVC and the poly(ester ether carbonate)s reported in this paper was investigated by means of DSC and DMA analysis. PVC blends were also analyzed by determining their swellability and the amount of extractables in aqueous media. By comparison purposes, the chain-extension product of PCL1250, that is, PCL polycarbonate, was also synthesized and characterized. The results obtained demonstrated that the copolymers with shortest PEG segment length, i.e. PEG150, 400, and 600, give the best results in terms of miscibility with PVC and lead to blends with maximum resistance to extraction by water. Therefore, they represent, in principle, good substitutes for low-molecular-weight, leachable PVC plasticizers, such as di(ethylhexyl) phthalate.     

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Synthesis and characterization of the novel transfection reagent poly(amino ester glycol urethane)

Poly(ester urethane) (PEU) is a class of biodegradable polymer that has been applied as tissue-engineering scaffolds with minimum toxicity. Despite its unique biocompatibility, there have been no reports in modifying the PEU backbone to design a soluble, PEU-based DNA carrier. We have developed a method of incorporating tertiary amines and poly(ethylene glycol) (PEG) into PEU to synthesize a soluble poly(amino ester glycol urethane) (PaEGU) as a novel transfection reagent. Parallel to this, we have synthesized poly(amino ester) (PaE) and poly(amino ester urethane) (PaEU) as the control polymers. The test transfection reagent PaEGU and the control PaE were similar in their properties of being soluble and buffering pH in water and their capabilities of self-assembling with DNA and transfecting the target cells. Significantly, PaEGU exhibited faster hydrolysis kinetics than PaE, half-lives of 19 and 36 h for PaEGU and PaE, respectively, underlying PaEGU's unique property of low cytotoxicity. However, in comparison to PaEGU, the other control polymer, PaEU, was not readily dissolved in water, indicating the importance of PEG units in PaEGU in increasing polymer hydrophilicity. This study demonstrated a useful synthesis scheme for the PEU-based transfection reagent PaEGU. The combination of tertiary amine, ester, PEG, and urethane units in the polymer backbone constitutes a feasible approach for the future design of low-toxicity gene transfer vectors.     

Synthesis, characterization, and morphology studies of biodegradable amphiphilic poly[(R)-3-hydroxybutyrate]-alt-poly(ethylene glycol) multiblock copolymers

Novel biodegradable amphiphilic alternating block copolymers based on poly[(R)-3-hydroxybutyrate] (PHB) as biodegradable and hydrophobic block and poly(ethylene glycol) (PEG) as hydrophilic block (PHB-alt-PEG) were successfully synthesized through coupling reaction. Their chemical structures have been characterized by using gel permeation chromatography, (1)H nuclear magnetic resonance, and Fourier transform infrared spectroscopy. Differential scanning calorimetry (DSC) analysis revealed that both PHB and PEG blocks in PHB-alt-PEG block copolymers can crystallize to form separate crystalline phase except in those with a short PEG block (M(n) 600) only PHB crystalline phase has been observed. However, due to the mutual interference from each other, the melting transition of both PHB and PEG crystalline phases shifted to lower temperature with lower crystallinity in comparison with corresponding pure PHB and PEG. The crystallization behavior of PHB block and PEG block has also been studied by X-ray diffraction, and the results were in good agreement with those deduced from DSC study. The surface morphologies of PHB-alt-PEG block copolymer thin films spin-coated on mica have been visualized by atomic force microscopy with tapping mode, indicating formation of laterally regular lamellar surface patterns. Static water contact angle measurement revealed that surface hydrophilicity of these spin-coated thin films increases with increasing PEG block content.     

Plasticization of poly(L-lactide) with poly(propylene glycol)

A new plasticizer for poly(L-lactide) (PLA)-poly(propylene glycol) (PPG) is proposed. The advantage of using PPG is that it does not crystallize, has low glass transition temperature, and is miscible with PLA. PLA was plasticized with PPGs with nominal Mw of 425 and 1000 g/mol. Poly(ethylene glycol) (PEG), long known as a plasticizer for PLA, with nominal Mw of 600 g/mol, was also used to plasticize PLA for comparison. The thermal and tensile properties of PLA and PLA with 5-12.5 wt % of the plasticizers were studied. In blends of PLA with PPGs the glass transition temperature was lower than that of neat PLA. Both PPGs enhanced the crystallizability of PLA albeit less than PEG. All of the plasticizers increased also the ability of PLA to plastic deformation which was reflected in a decrease of yield stress and in an increase of elongation at break. The effect was enhanced by the higher PPG content and also by lower molecular weight of PPG. A phase separation occurred only in the blend containing 12.5 wt % of PPG with higher molecular weight. The evidences of crazing were found in deformed samples of PLA with low plasticizer content, whereas the samples with higher content of plasticizers crystallized due to deformation.     

Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living nitrogen-fixing bacterium accumulates poly(3-hydroxybutyric acid) [PHB, 69% of cell dry weight (CDW)] when grown on glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV with 19.2 mol% 3HV] when grown on glucose and valerate. Use of ethylene glycol (EG) and/or polyethylene glycols (PEGs) of low molecular weight as sole carbon source were detrimental to A. chroococcum growth and polymer yields. PEG-200, however, in the presence of glucose was incorporated into the polyhydroxyalkanoate (PHA) polymer. Addition of PEG-200 (150 mM) to culture medium during mid-log phase growth favored increased incorporation of EG units (12.48 mol%) into the PHB polymer. In two-step culture experiments, where valerate and PEG simultaneously were used in fresh medium, EG was incorporated most effectively in the absence of glucose, leading to the formation of a copolymer containing 18.05 mol% 3HV and 14.78 mol% EG. The physico-mechanical properties of PEG-containing copolymer (PHBV–PEG) were compared with those of the PHB homopolymer and the PHBV copolymer. The PHBV–PEG copolymer appeared to have less crystallinity and greater flexibility than the short-chain-length (SCL) PHA polymers.     

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB–DEG, PHB–TEG, PHB–PEG, and PHB–HV–PEG copolymers using short-chain PEGs (with n?≤?9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB–HV–PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.     

The Terpolymer Produced by Azotobacter Chroococcum 7B: Effect of Surface Properties on Cell Attachment

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by ¹H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.     

Biodegradable films of partly branched poly(l-lactide)-co-poly(epsilon-caprolactone) copolymer: modulation of phase morphology, plasticization properties and thermal depolymerization

We report on the modulation of phase morphology, plasticization properties, and thermal stability of films of partly branched poly(l-lactide)-co-poly(epsilon-caprolactone) copolymer (PLLA-co-PCL) with additions of low molecular weight compounds, namely, triethyl citrate ester, diethyl phthalate, diepoxy polyether (poly(propylene glycol) diglycidyl ether), and with epoxidized soybean oil (ESO). The PLLA-co-PCL/polyether films showed significant stability against thermal depolymerization, high film flexibility, and good plasticizing properties, probably due to cross-linking and chain branching formation between diepoxy groups with both the end carboxyl and hydroxyl groups of the PLLA copolymer (initially present or generated during the degradation process) to produce primary ester and ether bonds, respectively. Diethyl phthalate and triethyl citrate ester were found to be efficient plasticizers for PLLA copolymer in terms of glass transition and mechanical properties, but the more water-soluble plasticizer triethyl citrate induced a dramatic loss in the molecular weight of the copolymer. Although ESO cannot play the role of a plasticizer, it substantially stabilizes and retards thermal depolymerization of the PLLA copolymer matrix, possibly because of a reaction between epoxy groups with the end carboxyl and hydroxyl groups of the PLLA copolymer. The presence of ESO in PLLA-co-PCL/ESO/triethyl citrate blends enhanced the compatibility and miscibility of the plasticizer with the PLLA copolymer matrix, considerably improved the mechanical properties (elongation at break), and substantially stabilized the copolymer against thermal depolymerization. It seems likely that the epoxy groups interact not only with the end hydroxyl and carboxyl group of the copolymer but as well with the hydroxyl group of triethyl citrate plasticizer to produce a new ether bond (C-O-C) as the cross-linking unit. On the other hand, for PLLA-co-PCL/ESO/polyether blends, (80/10/10) epoxidized oil distorts the compactness of the blend by diminishing the proposed entanglements between carboxyl, hydroxyl, and diepoxy groups of polyether and reduces the high elongation properties otherwise observed in the PLLA-co-PCL/polyether films. The multicomponent approach toward modulating poly(l-lactide)-co-poly(epsilon-caprolactone) copolymer films using epoxy compounds and plasticizers and the insight into the nature of various PLLA matrixes presented here offer advantages to a broad engineering of PLLA copolymer films having desirable physical properties and multiphase behavior for efficient uses in future technical applications.     

Sulfonamide-based pH- and temperature-sensitive biodegradable block copolymer hydrogels

Novel pH- and temperature-sensitive biodegradable poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol) (PCLA-PEG) block copolymers were synthesized with oligomeric sulfamethazine (OSM) end groups (OSM-PCLA-PEG-PCLA-OSM). Aqueous solutions of these block copolymers have shown sol-gel transition behavior upon both temperature and pH changes under physiological conditions (37 degrees C, pH 7.4). The sol-gel transition of these block copolymer solutions was fine-tuned by controlling the PEG length, the hydrophobic to hydrophilic block ratio (PCLA/PEG), and the molecular weight of the sulfamethazine oligomer. Since changes in temperature do not induce gel formation in this pH- and temperature-sensitive block copolymer solution, this hydrogel can be employed as an injectable carrier using a long guide catheter into the body. In addition, the pH of the block copolymer solution showed no change following PCLA degradation over 1 month, and no indication of gel collapse was observed on addition of buffer solution. As such, these properties make the OSM-PCLA-PEG-PCLA-OSM hydrogel an ideal candidate for use as an injectable carrier for certain protein-based drugs known to denature in low-pH environments.     

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.     

Decisive role of hydrophobic side groups of polypeptides in thermosensitive gelation

Thermosensitive hydrogels based on PEG and poly(l-glutamate)s bearing different hydrophobic side groups were separately synthesized by the ring-opening polymerization (ROP) of l-glutamate N-carboxyanhydrides containing different alkyl protected groups, that is, methyl, ethyl, n-propyl, and n-butyl, using mPEG(45)-NH(2) as macroinitiator. The resulting copolymers underwent sol-gel transitions in response to temperature change. Interestingly, the polypeptides containing methyl and ethyl showed significantly lower critical gelation temperatures (CGTs) than those bearing n-propyl and butyl side groups. Based on the analysis of (13)C NMR spectra, DLS, circular dichroism spectra, and ATR-FTIR spectra, the sol-gel transition mechanism was attributed to the dehydration of poly(ethylene glycol) and the increase of β-sheet conformation content in the polypeptides. The in vivo gelation test indicated that the copolymer solution (6.0 wt %) immediately changed to a gel after subcutaneous injection into rats. The mass loss of the hydrogel in vitro was accelerated in the presence of proteinase K, and the MTT assay revealed that the block copolymers exhibited no detectable cytotoxicity. The present work revealed that subtle variation in the length of a hydrophobic side group displayed the decisive effect on the gelation behavior of the polypeptides. In addition, the thermosensitive hydrogels could be promising materials for biomedical applications due to their good biocompatibility, biodegradability, and the fast in situ gelation behavior.     

Bio-based polymeric composites comprising wood flour as filler

The increasing effort on development of bio-based polymeric materials in recent years is motivated by the basic concept of meeting the sustainability criteria for industrial development in the third millennium. Within this framework, our research group is currently involved in assessing the potentiality of some agro-industrial overproduction and byproducts in the formulation of eco-compatible bio-based polymeric materials displaying, among others, the propensity to biodegrade under controlled environment conditions. In the present work, beech wood flour (Bwf) composites were prepared from plasticized poly(3-hydroxybutyrate) (PHB). The type of plasticizer [tri(ethylene glycol) bis(2-ethylhexanoate) (TEGB) and poly(ethylene glycol) (PEG200)] and the amount [5 and 20 wt %] were selected as independent variables in a factorial design. Thermal and mechanical properties of 90 wt % PHB composites were investigated. Incorporation of PEG200 was found to compromise thermal stability of PHB as demonstrated by the higher decrease on the onset decomposition temperature (T(d)) and the drop in its average molecular weight (M(w)). The present study underlines the fact that TEGB/PHB/beech wood flour composites can be optimized to obtain new materials for disposable items.     

Polyhedral oligomeric silsesquioxane (POSS) suppresses enzymatic degradation of PCL-based polyurethanes

In this Article, we studied the enzymatic hydrolytic biodegradation behavior of a novel multiblock thermoplastic polyurethane (TPU) system, which incorporates polyhedral oligomeric silsesquioxane (POSS) into linear biodegradable thermoplastic polyurethanes containing poly(ε-caproactone) (PCL) and polyethylene glycol (PEG) blocks. The biodegradation behavior of POSS-PCL-PEG TPUs was characterized by proton nuclear magnetic resonance spectroscopy ((1)H NMR), differential scanning calorimetry (DSC), tensile tests, scanning electron microscopy (SEM), and wavelength dispersive X-ray spectrometry (WDS) after enduring 22-day accelerated enzymatic hydrolytic degradation tests. POSS incorporation significantly suppressed in vitro enzymatic hydrolytic degradation of PCL-PEG-based multiblock TPUs by a surface passivation mechanism. WDS observations revealed that the covalently bonded POSS moieties developed a near-continuous and robust POSS-layer after initial degradation, which prevented ester bonds of PCL from enzymatic attack, thereby inhibiting further degradation. These striking results provide a new strategy to fabricate the polyester-based biostable thermoplastic polyurethanes (TPUs) of potential use in long-term surgical implants.     

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.     

Microbial degradation of polyethers

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.     

Synthesis and characterization of triblock copolymers of methoxy poly(ethylene glycol) and poly(propylene fumarate)

Amphiphilic block copolymers were synthesized by transesterification of hydrophilic methoxy poly(ethylene glycol) (mPEG) and hydrophobic poly(propylene fumarate) (PPF) and characterized. Four block copolymers were synthesized with a 2:1 mPEG:PPF molar ratio and mPEGs of molecular weights 570, 800, 1960, and 5190 and PPF of molecular weight 1570 as determined by NMR. The copolymers synthesized with mPEG of molecular weights 570 and 800 had 1.9 and 1.8 mPEG blocks per copolymer, respectively, as measured by NMR, representing an ABA-type block copolymer. The number of mPEG blocks of the copolymer decreased with increasing mPEG block length to as low as 1.5 mPEG blocks for copolymer synthesized with mPEG of molecular weight 5190. At a concentration range of 5-25 wt % in phosphate-buffered saline, copolymers synthesized with mPEG molecular weights of 570 and 800 possessed lower critical solution temperatures (LCST) between 40 and 45 degrees C and between 55 and 60 degrees C, respectively. Aqueous solutions of copolymer synthesized with mPEG 570 and 800 also experienced thermoreversible gelation. The sol-gel transition temperature was dependent on the sodium chloride concentration as well as the mPEG block length. The copolymer synthesized from mPEG 570 had a transition temperature between 40 and 20 degrees C with salt concentrations between 1 and 10 wt %, while the sol-gel transition temperatures of the copolymer synthesized from mPEG molecular weight 800 were higher in the range 75-30 degrees C with salt concentrations between 1 and 15 wt %. These novel thermoreversible copolymers are the first biodegradable copolymers with unsaturated double bonds along their macromolecular chain that can undergo both physical and chemical gelation and hold great promise for drug delivery and tissue engineering applications.     

Studies on intracellular degradation of polyhydroxyalkanoic acid-polyethylene glycol copolymer accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [PHB] when grown in glucose containing nitrogen-free Stockdale medium. The same medium supplemented with valerate alone and valerate plus polyethylene glycol (PEG) leads to the accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV] and PEG containing PHBV-PEG polymers, respectively. The intracellular degradation of these polymers as studied in carbon-free Stockdale medium showed a rapid degradation of PHB followed by PHBV, while it was least in case of PHBV-PEG. The rate of such degradation was 44.16, 26.4 and 17.0 mg h(-1)l(-1) for PHB, PHBV and PHBV-PEG, respectively. During the course of such of PHBV and PHBV-PEG degradation the 3HB mol% of polymers decreased significantly with increase of 3HV mol fraction, the EG mol% in PHBV-PEG, however, remained constant. After 50h of degradation the decrease in intrinsic viscosity and molecular mass of PHBV-PEG were 37.5 and 43.6%, respectively. These values appeared low compared to PHB and PHBV. Moreover, the increasing EG content of polymer retarded their extent of degradation. Presence of PEG, particularly of low molecular weight PEG was inhibitory to intracellular PHA depolymerise (i-PHA depolymerase) activity and the relative substrate specificity of the i-PHA depolymerase of MAL-201 appeared to be PHB > PHBV > PHBV-PEG.