Biosynthesis and properties of medium-chain-length polyhydroxyalkanoates with enriched content of the dominant monomer.

When grown in a nonanoic acid-limited chemostat at a dilution rate of 0.25 h(-1), Pseudomonas putida KT2440 produced poly(3-hydroxynonanoate-co-3-hydroxyheptanoate) containing 68 mol % 3-hydroxynonanoate (C9) and 32 mol % 3-hydroxyheptanoate (C7). Under the same conditions, but in the presence of acrylic acid, a fatty acid ß-oxidation inhibitor, the C9 monomer content increased to 88 mol %. Cofeeding glucose (3.9 g L(-1)) and nonanoic acid (2.9 ± 0.1 g L(-1)) in continuous culture with 0.2 g L(-1) of acrylic acid in the feed, further increased the C9 content to 95 mol %. A yield of PHA from nonanoic acid of 0.93 mol mol(-1) was attained. PHA with a 3-hydroxyoctanoate (C8) content of 98 mol % was produced with the same cofeeding methodology from octanoic acid. As the dominant monomer content increased, the melting point of the poly(3-hydroxynonanoate) copolymers increased from 46 to 63 °C and that of the poly(3-hydroxyoctanoate) copolymers from 54 to 62 °C. All copolymer compositions resulted in elongation to break values of about 1300%, but tensile strength at break and Young's modulus both increased with increasing amounts of the dominant monomer.

Microbial medium chainlength poly[(R)-3-hydroxyalkanoate] shows liquid crystal behaviour.

Medium chainlength (mcl) polyhydroxyalkanoates (PHAs) are a class of polymers receiving attention because of their potential as renewable, biodegradable and high tech properties. Unlike most short chain PHAs, mcl-PHAs are low crystallinity and elastomeric in character. In this paper we wish to point out that in their broad properties mcl-PHAs might be classified as thermotropic liquid crystals with dynamic conformational disorder and long range orientational order. As the characterization of mcl-PHAs progresses, their similarities to liquid crystalline elastomers are noteworthy. Wunderlich coined the acronym CONDIS from the words "conformational disorder" to categorize this type of liquid crystal. Thermal analysis reveals a T(g) of -40 to -45°C with several T(m) peaks. The chemistry of the elastomer from (13)C NMR confirms the poly(3-hydroxynonanoate), PHN, composition of the starting material along with two other samples containing double bonds: PHNU-18 and PHNU-31 where the numeral stands for the percent of double bonds.

Application of polyhydroxyalkanoate granules for sizing of paper.

Polyhydroxyalkanoates (PHAs) are characterized by the chemistry of the biodegradable inclusions inside the microbial membrane. They are produced by a wide variety of bacteria, where they function as energy and carbon storage materials. This intracellular Bioplastic forms a stable latex suitable for surface treatments of paper such as sizing and coating. In this work, we compare native granules and artificial granules made from market poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate), P(3HB-co-3HV), for their ability as sizing agent. Paper sizing was assayed by measuring the resistance of sized paper to penetration by aqueous fluids. Our results indicate that the sizing effect of PHAs is dependent on several factors, such as, paper drying temperature, drying time, pressure, and polymer composition, that is, homopolymer, random copolymer, and texture of granules. The sizing efficiency of the copolymer is generally poor compared to the PHB homopolymer. In addition to water permeability, the tensile strength of sized paper was measured and physical properties of granule suspensions were recorded using SEM microscopy, X-ray diffraction, and dynamic light scattering.

Polyester-based micelles and nanoparticles for the parenteral delivery of taxanes.

Taxanes are potent antimitotic agents that have demonstrated efficacy in a wide range of malignancies. Due to their poor water-solubility, these cytostatic drugs were first formulated with low molecular weight surfactants, e.g. Cremophor EL (CrEL) and Tween 80 (polysorbate 80), which are known to exhibit serious adverse effects in humans. In recent years, there has been growing interest in the design of more biocompatible formulations for both paclitaxel and docetaxel. Polymer-based drug carriers represent an attractive venue given the diversity in the array of existing polymers. Most notably, biopolyesters are vastly employed in the field of biomedical research given their biocompatibility and biodegradability. Polyester-based micelles and nanoparticles have been applied to the parenteral delivery of taxanes with varying degrees of success. Block copolymer micelles possess a unique core-shell structure generated through the self-assembly of amphiphilic copolymers in aqueous media. Although these systems have shown greatly enhanced tolerability compared to formulations based on low molecular weight surfactants, in some cases their failure to retain their cargo following parenteral administration has hindered their capacity to target taxanes to solid tumours. While polyester-based nanoparticles possess comparatively greater stability and drug targeting capacity, they frequently display a significant burst effect whereby a major portion of the cargo is immediately discarded from the carrier upon injection. This review focuses on the current application of polyester-based micelles and nanoparticles to the tumour targeting of taxanes. The preparation, loading efficiencies, release kinetics, cytotoxicity and in vivo behaviour of these systems is discussed in detail.

Membrane formation and drug loading effects in high amylose starch tablets studied by NMR imaging.

Cross-linked high amylose starch is used as an excipient in the preparation of pharmaceutical tablets for the sustained release of drugs. NMR imaging with contrast enhanced by proton density and by self-diffusion coefficient was used to follow the water uptake and swelling, two critical parameters controlling the drug release of the cross-linked starch tablets containing 10 wt % of ciprofloxacin and of acetaminophen, respectively. The drug-loaded tablets were studied in a H2O/D2O mixture at 37 degrees C in comparison to the tablets without any drug loading. The diffusion of water in the tablets all showed a Fickian behavior, but the kinetics of water uptake was faster in the case of the drug-loaded tablets. The formation of a membrane at the water/tablet interface was observed.

Study of hydration of cross-linked high amylose starch by solid state 13C NMR spectroscopy.

Starch is subjected to chemical treatments such as cross-linking or hydroxypropylation to meet the material requirements for food uses or controlled release in the pharmaceutical industries. In this work, two types of cross-linking formulations have been employed for the preparation of high amylose starch for use as an excipient for sustained drug release. The structural differences and chain dynamics of the modified starches in the dry and hydrated states have been compared by the use of variable contact time cross polarization-magic angle spinning solid state (13)C NMR spectroscopy.

Graft copolymers of methyl methacrylate and poly([R]-3-hydroxybutyrate) macromonomers as candidates for inclusion in acrylic bone cement formulations: Compression testing.

Graft copolymers of methyl methacrylate and biodegradable, biocompatible bacterial poly([R]-3-hydroxybutyrate) (PHB) blocks were synthesized and evaluated as possible constituents in acrylic bone cements for use in orthopaedic applications. The copolymers were produced by conventional free radical copolymerization and incorporated in one commercially available acrylic bone cement brand, Antibiotic Simplex (AKZ). Cements with formulations containing 6.7 and 13.5 wt % of PMMA-graft-PHB were prepared. The morphology of the graft copolymer particles was suggested to influence the ability of the modified cement to be processed. Formulations containing more than about 20 wt % of the graft copolymer resulted in cement doughs that, both after first preparation and several hours later, were either sandy or soft spongy in texture and, thus, would be unacceptable for use in orthopaedic applications. The morphologies of the powders and the volumetric porosity (p) and ultimate compressive strength (UCS) of the cured cements were determined. Micro computed tomography showed that the cements presented average porosities of 13.5-16.9%. It was found that, while the powder particle shape and size for the experimental cements were markedly different from those of AKZ, there was no significant difference in either p or UCS for these cements. The latter was determined to be about 85 MPa for the modified cements and 84 MPa for Antibiotic Simplex. Furthermore, the UCS of all the cements exceeded the minimum level for acrylic bone cements, as stipulated by ASTM F-451.

Imaging of high-amylose starch tablets. 3. Initial diffusion and temperature effects.

The penetration of water into cross-linked high amylose starch tablets was studied at different temperatures by nuclear magnetic resonance (NMR) imaging, which follows the changes occurring at the surface and inside the starch tablets during swelling. It was found that the swelling was anisotropic, whereas water diffusion was almost isotropic. The water proton image profiles at the initial stage of water penetration were used to calculate the initial diffusion coefficient. The swelling and water concentration gradients in this controlled release system show significant temperature dependence. Diffusion behavior changed from Fickian to Case II diffusion with increasing temperature. The observed phenomena are attributed to the gelatinization of starch and the pseudo-cross-linking effect of double helix formation.

Self-assembly of precursors in single-crystal growth of biopolymers.

Self-assembly of precursors in dilute solution single-crystal growth of poly[(R)-3-hydroxyvalerate] (PHV) and the fungal polysaccharide mycodextran were studied by transmission electron microscopy, especially at the early stages of crystallization. Precursors for PHV, such as small primary nuclei and tiny square tiles, consolidate to a large square crystal composed of orthogonally arranged tiles. By contrast, the precursors of mycodextran were lath-shaped, which suggests that the crystal growth is mainly in the longitudinal direction. Needlelike precursors were observed either free or as protrusions at the lath ends. In general, single-crystal shape and morphology are dependent on the type of precursor and on the manner of crystallization.

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology.

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.

Synthesis and properties of graft copolymers based on poly(3-hydroxybutyrate) macromonomers.

Graft copolymers of poly(methyl methacrylate) with poly(3-hydroxybutyrate), PHB, segments as long side chains were prepared by the macromonomer method. PHB macromonomers were prepared from the esterification of oligomers with 2-hydroxyethyl methacrylate at their carboxylic acid end. Esterification products displayed low polydispersity indices (ca. 1.2) and a functionality of over 83%, with a Mn of 2,020. Using free radical polymerization methods, the macromonomers were copolymerized with methyl methacrylate to yield graft (comb type) copolymers at different comonomer feed ratios. The graft copolymers contained from 0.5 to 14 mol-% of PHB blocks, with a glass transition temperature decreasing from 100 to 3 degrees C.

Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli.

Physical properties of chiral poly(thioesters), PTEs, prepared by engineered Escherichia coli, were examined by GPC, 13C CP/MAS solid-state NMR, X-ray diffraction, and thermal analysis. Microbial homopolymers of PTEs, poly(3-mercaptopropionate), PMP, and poly(3-mercaptovalerate), PMV, showed different solubility characteristics compared to poly(hydroxyalkanoates), PHAs. Generally, PTEs required higher temperatures for dissolution. Poly(3-mercaptobutyrate), PMB, and PMV dissolve in chloroform, and the molecular weight values were revealed by GPC as 176,000 and 165,000, respectively. The density values for PMP and PMB were 1.42 and 1.27 g/cm3, respectively. These values are similar to those for oxygen analogues. The NMR spectra for PTEs showed that carbonyl carbons are greatly shifted downfield by the sulfur atoms in the chain backbone compared to the PHA family. X-ray powder diffraction data indicated that PTEs are crystalline materials, but they do not crystallize as well as in the PHA family. The melting point, Tm, for PMP was 170 degrees C, which is about 100 degrees C higher than the equivalent oxygen analogue, poly(3-hydroxypropionate), PHP, and almost the same as that of bacterial poly(3-hydroxybutyrate), PHB. According to thermal analysis, only the PMP sample had enhanced heat stability, e.g., the decomposition temperature for PMP was 277 degrees C at 5% weight loss, whereas the values for PHP and PHB were 233 and 260 degrees C at the same weight loss, respectively.

Cocrystallization model for synthetic biodegradable poly(butylene adipate-co-butylene terephthalate).

Synthetic biodegradable poly(butylene adipate-co-butylene terephthalate), P(BA-co-BT), with 56 mol % butylene adipate, BA, was characterized by solid-state NMR spectroscopy, thermal analysis, X-ray diffraction, computer modeling, and polarization microscopy. The NMR study showed the presence of BA and butylene terephthalate, BT. T(1C) NMR measurements proved that some BA and BT units were in crystalline regions. Thermal analysis showed one glass-transition temperature and a single diffuse melting endotherm corresponding to a large melting-point depression of about 100 degrees C compared with poly(butylene terephthalate), PBT. These results suggest that there is only one crystalline phase. An X-ray fiber diagram of a stretched film could be indexed with the same unit cell as that for PBT. Computer modeling showed that the adipate unit fits into the crystal structure of PBT by adopting a TTGTG dihedral angle sequence in the crystalline conformation proposed for the cocrystallization model. The predicted fiber diagram from the proposed model qualitatively agrees with the experimental one. Polarization microscopy revealed that the spherulite growth rate of P(BA-co-BT) was similar to that for poly(butylene adipate), PBA.

NMR imaging of high-amylose starch tablets. 2. Effect of tablet size.

Carbohydrate polymers are widely used for pharmaceutical applications such as the controlled release of drugs. The swelling and water mobility in high-amylose starch tablets are important parameters to be determined for these applications. They have been studied at different time intervals by nuclear magnetic resonance imaging (NMRI) after the immersion of the samples in water. These tablets have a hydrophilic matrix, which swells anisotropically and forms a hydrogel in water. NMRI shows clearly the anisotropy of the water penetration and the swelling along the radial and axial dimensions of the tablets. Empirical relationships are established to describe the kinetics of water penetration and swelling of the tablets. Results show that water uptake and tablet swelling strongly depend on the size of the tablets. Gravimetric measurements of water uptake were also performed in comparison with the NMRI results.

One-step synthesis of amphiphilic diblock copolymers from bacterial poly([R]-3-hydroxybutyric acid).

Catalyzed transesterification in the melt is used to produce diblock copolymers of poly([R]-3-hydroxybutyric acid), PHB, and monomethoxy poly(ethylene glycol), mPEG, in a one-step process. Bacterial PHB of high molecular weight is depolymerized by consecutive and partly simultaneous reactions: pyrolysis and transesterification. The formation of diblocks is accomplished by the nucleophilic attack from the hydroxyl end-group of the mPEG catalyzed by bis(2-ethylhexanoate) tin. The resulting diblock copolymers are amphiphilic and self-assemble into sterically stabilized colloidal suspensions of PHB crystalline lamellae.

Characterization of polyacrylamide-stabilized Pfl phage liquid crystals for protein NMR spectroscopy.

A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the 2H NMR water signal and by the measurement of 1H-15N residual dipolar couplings (RDC) in the archeal translation elongation factor 1beta. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.

NMR imaging of high-amylose starch tablets. 1. Swelling and water uptake.

Pharmaceutical tablets made of modified high-amylose starch have a hydrophilic polymer matrix into which water can penetrate with time to form a hydrogel. Nuclear magnetic resonance imaging was used to study the water penetration and the swelling of the matrix of these tablets. The tablets immersed in water were imaged at different time intervals on a 300 MHz NMR spectrometer. Radial images show clearly the swelling of the tablets and the water concentration profile. The rate constants for water diffusion and the tablet swelling were extracted from the experimental data. The water diffusion process was found to follow case II kinetics at 25 degrees C. NMR imaging also provided spin density profiles of the water penetrating inside the tablets.

Thermal degradation of poly(3-hydroxyalkanoates): preparation of well-defined oligomers.

The thermal degradation of the biodegradable bacterial polyesters poly(3-hydroxybutyrate), PHB, poly(3-hydroxyvalerate), PHV, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), 0-21 mol % of hydroxyvalerate, was studied. At moderately low temperatures (170-200 degrees C), the main product is a well-defined oligomer, especially a 500-10,000 g/mol macromolecule, which contains one unsaturated end group, predominantly a trans-alkenyl end group, as well as a carboxylic end group. The process was studied regarding the effect of the copolymer composition and reaction time at 190 degrees C. During the first few hours of reaction, the thermal degradation of PHB and PHV followed a kinetic model of random scission, but eventually auto-acceleration of the pyrolysis was detected, probably due to the influence of the crotonate end groups of the oligomers formed. Ten-time scale-up experiments on a Brabender instrument were successfully undertaken.

Characterization of microbial polythioesters: physical properties of novel copolymers synthesized by Ralstonia eutropha.

Various samples of polythioesters with different contents of 3-mercaptopropionic acid (3MP) or 3-mercaptobutyric acid (3MB) as one comonomer and with 3-hydroxybutyric acid (3HB) as the second constituent were produced by cultivating cells of Ralstonia eutropha strain H16 in mineral salts medium containing 3MP or 3MB plus gluconate as carbon sources. Fermentations were done also at the 30-L scale. The various samples were cast as films from chloroform and the following were recorded: melting point, solid-state NMR, X-ray diffraction. The copolyester poly(3HB-co-3MP) displayed mutiple melting peaks corresponding to separate phases rich in 3MP and 3HB. The copolyester poly(3HB-co-3MB) displayed very low crystallinity and melting points higher than that of poly(3HB) when the 3HB content was 40% or less.