Structure-property relationships of copolymers obtained by ring-opening polymerization of glycolide and epsilon-caprolactone. Part 1. Synthesis and characterization

A series of copolymers with various compositions were synthesized by bulk ring-opening polymerization of glycolide and epsilon-caprolactone, using stannous (II) octoate or zirconium (IV) acetylacetonate as initiator. Reaction time and temperature were varied so as to induce different chain microstructures. The resulting copolymers were characterized by (1)H NMR, SEC, DSC, and X-ray diffraction. The average lengths of glycolyl (L(G)) and caproyl sequences (L(C)) and the degree of randomness (R) were calculated and compared to the values of completely random chains. The concentration of CGC sequences was also obtained which resulted from transesterification reactions. Data showed that stannous (II) octoate leads to less transesterification than zirconium (IV) acetylacetonate, and lower temperatures lead to less transesterification than higher ones. The copolymers exhibited a more or less blocky chain structure because of the reactivity difference between glycolide and epsilon-caprolactone. The crystalline structure and thermal properties depend on both the composition and the chain microstructure. PGA- and PCL-type crystallites were obtained for copolymers with intermediate compositions.     

Degradation of N5-(2-hydroxyethyl)-L-glutamine and L-glutamic acid homopolymers and copolymers by papain

The rate of degradation of poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG), poly(L-glutamic acid) (PGA) and poly[HEG-co-GA] random copolymers by papain was measured in the pH range 4.0-7.5, employing the gel permeation chromatography method. The effect of the degree of ionization on the polymer conformation was measured by circular dichroism (c.d.). PHEG, which is uncharged, had a random coil conformation and an almost constant degradation rate within the whole pH interval. The ionization of PGA increased with increasing pH and was accompanied by conformational transition from helix to random coil. The hydrolysis of PGA by papain depended on pH with the optimum at about pH 5, indicating that both the high content of helix (at pH less than 5) and increasing charge density (at pH greater than 5), decreased the degradation rate. Contrary to PGA, pH profiles of the degradation rate of poly[HEG-co-GA] copolymers are monotonous and do not decrease at pH less than 5. In the copolymers the HEG residues act as a helix breaker and limit the formation of helical conformation. The role of structural features of a macromolecular substrate, i.e. the charge, helical conformation and the nature of amino acid residues, in the interaction between enzyme and polymer is discussed.     

Scavenging of Active Oxygen Species by Glycated Proteins

Scavenging of active oxygen species by glycated proteins was investigated. Glycated proteins were prepared from bovine serum albumin (BSA), insulin, and lysozyme incubated with glucose. Glycated BSA at concentration of 0.5% scavenged 34% of hydroxyl radicals by ESR experiments using DMPO as a spin-trapping reagent. The ability to scavenge hydroxyl radicals by glycated BSA was higher than that by BSA. Hydrogen peroxides also were largely scavenged with an increase in the concentration of glycated proteins. However, the ability to scavenge superoxides by glycated BSA was lower than that by BSA because glycated proteins produced superoxides. Experiments using model compounds such as Amadori compound and caproyl pyrraline suggested that the scavenging ability of glycated proteins against hydroxyl radicals depends on Maillard reaction products in the advanced stage, while the ability against hydrogen peroxides is dependent upon Maillard reaction products in the early stage and brown pigments.     

Synthesis, characterization, and degradation behavior of amphiphilic poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(epsilon-caprolactone)

A series of biodegradable amphiphilic graft polymers were successfully synthesized by grafting poly(epsilon-caprolactone) (PCL) sequences onto a water-soluble poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA) backbone. The graft copolymers were prepared through the ring-opening polymerization of epsilon-caprolactone (CL) initiated by the macroinitiator PHEA with pendant hydroxyl groups without adding any catalyst. By controlling the feed ratio of the macroinitiator to the monomer, the copolymers with different branch lengths and properties can be obtained. The successful grafting of PCL sequences onto the PHEA backbone was verified by FTIR, 1H NMR, and combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. The hydrolytic degradation and enzymatic degradation of these graft copolymers were investigated. The results show the hydrolytic degradation rate increases with increasing content of hydrophilic PHEA backbone. While the enzymatic degradation rate is affected by two competitive factors, the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the hydrophilicity which depends on the copolymer composition. In situ observation of the degradation under polarizing light microscope (PLM) demonstrates the different degradation rates of different regions in the polymer samples.     

Influence of network structure on the degradation of photo-cross-linked PLA-b-PEG-b-PLA hydrogels

Triblock copolymers of functionalized poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) (PLA-b-PEG-b-PLA) have been widely investigated as precursors for fabricating resorbable polymeric drug delivery vehicles and tissue engineering scaffolds. Previous studies show degradation and erosion behavior of PLA-b-PEG-b-PLA hydrogels to rely on macromer chemistry as well as structural characteristics of the cross-linked networks. In this research, the degradation kinetics of diacrylated PLA-b-PEG-b-PLA copolymers as soluble macromers and cross-linked gels are directly compared as a function of macromer concentration, buffer pH, and ionic strength. The pseudo first-order rate constants for degradation of soluble macromers increase with water concentration and show a minimum at intermediate pH values, but are insensitive to ionic strength. The degradation rate constants for covalently cross-linked gels display a greater sensitivity to local water concentration and a minimum at lower pH values than corresponding soluble macromers. In addition, ionic strength significantly affects the rate of gel degradation due to the direct correlation between the degree of network ionization and gel water content.     

Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics

The degradation of plant cell walls by ruminants is of major economic importance in the developed as well as developing world. Rumen fermentation is unique in that efficient plant cell wall degradation relies on the cooperation between microorganisms that produce fibrolytic enzymes and the host animal that provides an anaerobic fermentation chamber. Increasing the efficiency with which the rumen microbiota degrades fiber has been the subject of extensive research for at least the last 100 years. Fiber digestion in the rumen is not optimal, as is supported by the fact that fiber recovered from feces is fermentable. This view is confirmed by the knowledge that mechanical and chemical pretreatments improve fiber degradation, as well as more recent research, which has demonstrated increased fiber digestion by rumen microorganisms when plant lignin composition is modified by genetic manipulation. Rumen microbiologists have sought to improve fiber digestion by genetic and ecological manipulation of rumen fermentation. This has been difficult and a number of constraints have limited progress, including: (a) a lack of reliable transformation systems for major fibrolytic rumen bacteria, (b) a poor understanding of ecological factors that govern persistence of fibrolytic bacteria and fungi in the rumen, (c) a poor understanding of which glycolyl hydrolases need to be manipulated, and (d) a lack of knowledge of the functional genomic framework within which fiber degradation operates. In this review the major fibrolytic organisms are briefly discussed. A more extensive discussion of the enzymes involved in fiber degradation is included. We also discuss the use of plant genetic manipulation, application of free-living lignolytic fungi and the use of exogenous enzymes. Lastly, we will discuss how newer technologies such as genomic and metagenomic approaches can be used to improve our knowledge of the functional genomic framework of plant cell wall degradation in the rumen.     

Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity

The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biodegradation of copoly(L-aspartic acid/L-glutamic acid) in vitro.

The preparation of copolypeptides consisting of L-aspartic acid and L-glutamic acid was performed to determine the effects of copolymer composition and sequential distributions on the rate of degradation by papain in a PECF (pseudoextracellular fluid) at pH 4.75 and 7.40, at 37.0 degrees C, to simulate in vivo polymer degradation. Random copolymers consisting of beta-benzyl L-aspartate and gamma-benzyl L-glutamate were synthesized by the N-carboxyanhydride method. Water-soluble copolymers were obtained by successive reactions of side chains by anhydrous HBr treatment. All the samples were found to be degraded by random chain scission with papain. Further, the degradation data for the samples followed the Michaelis-Menten rate law, being the first order in papain concentration. The nature of side chains are important to the rate of degradation by papain and it was controlled by the comonomer composition as well as the sequential distribution of comonomers in the copolymer chains.     

Exogenously triggered, enzymatic degradation of photopolymerized hydrogels with polycaprolactone subunits: experimental observation and modeling of mass loss behavior

Degradation plays an important role in the evolution of the extracellular matrix secreted by chondrocytes encapsulated in PEG-based hydrogels. For this study, macromonomers were synthesized by methacrylating both ends of polycaprolactone-b-poly(ethylene glycol)-b-polycaprolactone (PEG-CAP) tri-block copolymers. These divinyl molecules were photopolymerized to form hydrogels with PEG-CAP crosslinks that were subsequently degraded upon exogenous addition of a lipase enzyme. The rate of degradation and subsequent mass loss depends on both the length of the polycaprolactone units and the concentration of enzyme. Control gels that did not receive lipase did not significantly degrade on the time scale of these experiments. A model was developed to predict mass loss using enzyme kinetics and a previously described statistical treatment of bulk network degradation. The model was used to predict mass loss profiles at the specific conditions used, and also to demonstrate the importance of potential changes in reaction rate and enzyme stability on temporal mass loss.     

Stereocomplexes of enantiomeric lactic acid and sebacic acid ester-anhydride triblock copolymers

A systematic study on the synthesis, characterization, degradation, and drug release of d-, l-, and dl-poly(lactic acid) (PLA)-terminated poly(sebacic acid) (PSA) and their stereocomplexes is reported. PLA-terminated sebacic acid polymers were synthesized by melt condensation of the acetate anhydride derivatives of PLA oligomers and sebacic anhydride oligomers to yield ABA triblock copolymers of molecular weights between 3000 and 9000 that melt at temperatures between 35 and 80 degrees C. Pairs of the corresponding enantiomeric ABA copolymers composed of l-PLA-PSA-l-PLA and d-PLA-PSA-d-PLA were solvent mixed to form stereocomplexes. The formed stereocomplexes exhibited higher crystalline melting temperature than the enantiomeric polymers, which indicate stereocomplex formulation. The PLA terminals had a significant effect on the polymer degradation and drug release rate. PSA with up to 20% w/w of PLA terminals degraded and released the incorporated drug for more than 3 weeks as compared with 10 days for PSA homopolymer.     

Matrix metabolism rate differs in functionally distinct tendons.

Tendon matrix integrity is vital to ensure adequate mechanical properties for efficient function. Although historically tendon was considered to be relatively inert, recent studies have shown that tendon matrix turnover is active. During normal physiological activities some tendons are subjected to stress and strains much closer to their failure properties than others. Tendons with low safety margins are those which function as energy stores such as the equine superficial digital flexor tendon (SDFT) and human Achilles tendon (AT). We postulate therefore that energy storing tendons suffer a higher degree of micro-damage and thus have a higher rate of matrix turnover than positional tendons. The hypothesis was tested using tissue from the equine SDFT and common digital extensor tendon (CDET). Matrix turnover was assessed indirectly by a combination of measurements for matrix age, markers of degradation, potential for degradation and protein expression. Results show that despite higher cellularity, the SDFT has lower relative levels of mRNA for collagen types I and III. Non-collagenous proteins, although expressed at different levels per cell, do not appear to differ between tendon types. Relative levels of mRNA for MMP1, MMP13 and both pro-MMP3 and MMP13 protein activity were significantly higher in the CDET. Correspondingly levels of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were higher in the CDET and tissue fluorescence lower suggesting more rapid turnover of the collagenous component. Reduced or inhibited collagen turnover in the SDFT may account for the high level of degeneration and subsequent injury compared to the CDET.     

Synthesis, properties, and biological activity of poly[di(sodium carboxylatoethylphenoxy)phosphazene

A new water-soluble polyphosphazene polyelectrolyte containing carboxylate functionalities, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) was synthesized via reaction of macromolecular substitution. The polymer was characterized using (1)H, (31)P NMR, and gel permeation chromatography with multiangle laser light scattering detection. PCEP was shown to undergo hydrolytic degradation in aqueous solutions, as indicated by the decrease in the molecular weight and the release of side groups. A series of incompletely substituted copolymers of PCEP containing varying amounts of residual chlorine atoms was also prepared. The rate of degradation for such copolymers increased with the rise in the content of chlorine atoms. In vivo studies demonstrated high potency of PCEP as a vaccine immunoadjuvant. The new polyphosphazene was also shown to be capable of forming microspheres in aqueous solutions via reactions of ionic complexation with physiologically occurring amines, such as spermine.     

Computer modelling study on the compatibility of PEO–PMMA block copolymers

The compatibility of six kinds of designed poly(ethylene oxide)-block-poly(methyl methacrylate) (PEO-b-PMMA) copolymers was studied at 270, 298 and 400 K by mesoscopic modelling. The values of order parameter depended on both the structures of block copolymers and the simulation temperatures, while the values of order parameter of the long chains were higher than those of the short ones; temperature showed a more obvious effect on long chains than on short ones. These plain copolymers doped with PEO or PMMA homopolymer showed different order parameter values. When the triblock copolymer was composed of the same component at both ends and was doped with a homopolymer with the same component as that in the middle or the end of triblock copolymer, the values of order parameter depended on the simulation temperature, such as A5B6A5 doped with B6 at 400 K and A5B6A5 doped with A5 at 270 K, it showed the highest order parameter values. The study of copolymers doped with nanoparticles showed that the mesoscopic phase was influenced by not only the properties of the nanoparticles, such as the size and density, but also the compositions of copolymers. Among them, increasing the size of doped nanoparticles was the most significant effect on changing the phase morphologies.     

Photocurable liquid biodegradable copolymers: in vitro hydrolytic degradation behaviors of photocured films of coumarin-endcapped poly(epsilon-caprolactone-co-trimethylene carbonate)

Coumarin-endcapped tetrabranched liquid copolymers composed of epsilon-caprolactone and trimethylene carbonate (TMC), prepared using pentaerythritol or four-branched poly(ethylene glycol) (PEG) as an initiator, were ultraviolet irradiated to produce photocured solid biodegradable copolymers. The hydrolytic degradation behaviors of photocured films were determined from the weight loss of the films. The initial hydrolysis rate (determined for up to 24 h using a quartz crystal microbalance) was enhanced with aqueous solutions of higher pH. The hydrolysis rate in the early period of immersion was increased with an increase in TMC content, whereas that in the later period (week order) decreased with a increase in TMC content. This inverse relation of composition dependence on the hydrolysis rate between the early and late periods was discussed. Topological measurements using scanning electron microscopy and atomic force microscopy as well as depth profiles of the fluorescein-stained hydrolyzed layer showed that for the pentaerythritol-initiated copolymer, irrespective of copolymer composition, hydrolysis occurred at surface regions and surface erosion proceeded with immersion time. For PEG-based copolymers, both surface erosion and bulk degradation occurred simultaneously. The hydrolyzed surfaces became highly wettable with water and exhibited noncell adhesivity.     

Polymeric membranes for guided bone regeneration

In this review, different barrier membranes for guided bone regeneration (GBR) are described as a useful surgical technique to enhance bone regeneration in damaged alveolar sites before performing implants and fitting other dental appliances. The GBR procedure encourages bone regeneration through cellular exclusion and avoids the invasion of epithelial and connective tissues that grow at the defective site instead of bone tissue. The barrier membrane should satisfy various properties, such as biocompatibility, non-immunogenicity, non-toxicity, and a degradation rate that is long enough to permit mechanical support during bone formation. Other characteristics such as tissue integration, nutrient transfer, space maintenance and manageability are also of interest. In this review, various non-resorbable and resorbable commercially available membranes are described, based on expanded polytetrafluoroethylene, poly(lactic acid), poly(glycolic acid) and their copolymers. The polyester-based membranes are biodegradable, permit a single-stage procedure, and have higher manageability than non-resorbable membranes; however, they have shown poor biocompatibility. In contrast, membranes based on natural materials, such as collagen, are biocompatible but are characterized by poor mechanical properties and stability due to their early degradation. Moreover, new approaches are described, such as the use of multi-layered, graft-copolymer-based and composite membranes containing osteoconductive ceramic fillers as alternatives to conventional membranes.     

Effects of nanoparticles on the compatibility of PEO-PMMA block copolymers

The compatibility of six kinds of designed poly(ethylene oxide)-block-poly(methyl methacrylate) (PEO-b-PMMA) copolymers was studied at 270, 298 and 400 K via mesoscopic modeling. The values of the order parameters depended on both the structures of the block copolymers and the simulation temperature, while the values of the order parameters of the long chains were higher than those of the short ones; temperature had a more obvious effect on long chains than on the short ones. Plain copolymers doped with poly(ethylene oxide) (PEO) or poly(methyl methacrylate) (PMMA) homopolymer showed different order parameter values. When a triblock copolymer had the same component at both ends and was doped with one of its component polymers as a homopolymer (such as A5B6A5 doped with B6 or A5 homopolymer), the value of its order parameter depended on the simulation temperature. The highest order parameter values were observed for A5B6A5 doped with B6 at 400 K and for A5B6A5 doped with A5 at 270 K. A study of copolymers doped with nanoparticles showed that the mesoscopic phase was influenced by not only the properties of the nanoparticles, such as the size and density, but also the compositions of the copolymers. Increasing the size of the nanoparticles used as a dopant had the most significant effect on the phase morphologies of the copolymers.     

Separation of amino acids and peptides by temperature induced phase partitioning. Theoretical model for partitioning and experimental data

There is a strong interest in use of ‘smart polymers’ in separation systems. These are polymers which can react on external influence, such as temperature or pH change. With such polymers it is possible from the outside to affect the properties of a separation system. Amphiphilic copolymers show drastic changes in solubility properties, such as self-association and phase separation, at e.g. temperature increase. The random copolymers of ethylene oxide and propylene oxide units (EOPO-polymers) can form aqueous two-phase systems above the copolymer cloud point temperature. Two phases are formed, one consisting of 40–60% polymer in water and the other of almost 100% water. Amino acids and peptides can be partitioned in the thermoseparating systems. The partitioning strongly depends on the solute hydrophobicity, where aromatic amino acids and peptides are partitioned to the polymer phase and hydrophilic to the water phase. Salt effects can be used to enhance the partitioning of charged molecules. The thermodynamic driving forces which govern the partitioning of molecules in a thermoseparated aqueous phase system is described with use of the Flory-Huggins theory for polymer solutions. Expressions are derived which show the entropic and enthalpic effects on solute partitioning. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

马尾松人工林林窗边缘效应对樟和红椿凋落叶难降解物质分解的影响

采用凋落物分解袋, 以四川低山丘陵区马尾松人工林人工砍伐形成的7个不同面积的林窗边缘(100、225、400、625、900、1225、1600 m²)为研究对象, 以林下为对照, 研究了2种乡土树种——樟和红椿凋落叶难降解物质(木质素、纤维素、总酚、缩合单宁)在不同大小林窗边缘的降解动态特征.结果表明:马尾松人工林林下与不同大小林窗边缘相比较,红椿凋落叶中除纤维素外,其余难降解物质的降解率以及樟凋落叶木质素降解率均显著高于林下.在全年分解过程中, 2种凋落叶4种难降解物质的降解率总体均呈现持续上升的趋势.其中,缩合单宁降解最快,其次是总酚和纤维素,而木质素降解最慢.随林窗面积的增大, 红椿凋落叶除纤维素外,其余难降解物质在中型林窗边缘(400、625 m²)具有相对较高的降解率,而樟凋落叶的木质素在625 m²林窗边缘时也表现出较高的降解率.在凋落叶分解过程中,难降解物质降解率与凋落叶袋内温度和凋落物质量均呈显著相关.中型林窗(400～625 m²)对凋落物分解过程中难降解物质的降解具有更显著的边缘效应, 而这种边缘效应与物种有一定关联.     

Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol)

Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).