Degradation properties of co-continuous calcium-phosphate-polyester composites

Co-continuous composites consisting of a porous calcium phosphate matrix (hydroxyapatite, HA, or β-tricalcium phosphate, TCP) filled with poly(D,L-lactide) (PDLLA) were produced with two different methods: in situ polymerization of D,L-lactide monomer inside the matrix, or infiltration of the matrix with molten polymer. The influence of the calcium phosphate matrix as well as the manufacturing method on the degradation were investigated with accelerated in vitro studies at 42 °C in pH 7.4 phosphate-buffered saline (PBS), with some controls at 37 °C. The results show that samples produced with the infiltration method had higher initial molecular weights leading to a later onset of mass loss. Heterogenous polymer degradation was still present in the composites, as indicated by molecular weight distributions and glass transition temperature measurements. The calcium phosphate matrix delayed degradation, with evidence from X-ray microtomography suggesting that the polymer degrades more slowly in proximity to the matrix.     

Morphology and Release Profiles of Biodegradable Microparticles Containing Rhamnolipid Biosurfactant

In an effort to expand the technology of bioremediation of hydrophobic organic compounds, microencapsulation technology was investigated as a method of biosurfactant delivery to contaminated sites. Microparticles are composed of active or inactive materials encapsulated in a polymer coating designed for controlled release of the encapsulated substance. Surface morphology and release profiles of microparticles containing rhamnolipid biosurfactant were investigated for development of a controlled release bioremediation scheme. The evaluation was conducted under laboratory conditions with 45 mg/ml concentration of biosurfactant and a representative environmental medium; using artificial salt water (35 ppt) and deionized water medium as a control. The microparticles were prepared by the water–in–oil–in–water double emulsion solvent evaporation method. The surface morphology was examined after initial preparation, at 0, 15 and 31 days incubation, using light microscopy. Light microscopic images revealed smooth, spherical microparticles that degraded over time in the media. Results indicated that microparticle degradation occurred mostly in the salt water environment, suggesting that the presence of salts (Na⁺ and Cl^? ions) in the water enhanced microparticle degradation. The deionized water environment achieved polymeric degradation that was similar to what was generally reported in the literature. Biosurfactant release was evaluated for polymer molecular weights (M_w) 40, 80, and 200 kDa, in salt water and deionized water media, each of which showed a high initial burst release of biosurfactant, followed by pulse releases that occurred over the 31 day period. The highest level of biosurfactant release of all the molecular weights tested occurred in the M_w 80 kDa. The release from M_w 40 kDa and M_w 200 kDa was not significantly different (P > 0.05). The results showed that this technology may be useful for enhancing bioremediation of residual hydrophobic organic contaminants (HOC) in estuarine and marine environments.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils.

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or a copolymer of 90% 3-hydroxybutyric acid and 10% 3-hydroxyvaleric acid was studied in soils incubated at a constant temperature of 15, 28, or 40 degrees C for up to 200 days. In addition, hydrolytic degradation in sterile buffer at temperatures ranging from 4 to 55 degrees C was monitored for 98 days. Degradation was measured through loss of weight (surface erosion), molecular weight, and mechanical strength. While no weight loss was recorded in sterile buffer, samples incubated in soils were degraded at an erosion rate of 0.03 to 0.64% weight loss per day, depending on the polymer, the soil, and the incubation temperature. The erosion rate was enhanced by incubation at higher temperatures, and in most cases the copolymer lost weight at a higher rate than the homopolymer. The molecular weights of samples incubated at 40 degrees C in soils and those incubated at 40 degrees C in sterile buffer decreased at similar rates, while the molecular weights of samples incubated at lower temperatures remained almost unaffected, indicating that molecular weight decrease is due to simple hydrolysis and not to the action of biodegrading microorganisms. The degradation resulted in loss of mechanical properties. From the samples used in the biodegradation studies, 295 dominant microbial strains capable of degrading P (3HB) and the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer in vitro were isolated and identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.     

Hydrolytic Degradation of Poly(3-Hydroxybutyrate) and Its Copolymer with 3-Hydroxyvalerate of Different Molecular Weights in vitro

The hydrolytic degradation of polymer films of poly(3-hydroxybutyrate) of different molecular weights and its copolymers with 3-hydroxyvalerate (9 mol % 3-hydroxyvalerate in the poly(3-hydroxybutyrate) chain) of different molecular weights was studied in model conditions in vitro. The changes in the physicochemical properties of the polymers were investigated using different analytical techniques: viscometry, differential scanning calorimetry, gravimetrical method, and water contact angle measurement for polymers. The data showed that in a period of 6 months the weight of polymer films decreased insignificantly. The molecular weight of the samples was reduced significantly; the largest decline (up to 80% of the initial molecular weight of the polymer) was observed in the high-molecular-weight poly(3-hydroxybutyrate). The surface of all investigated polymers became more hydrophilic. In this work, we focus on a mathematical model that can be used for the analysis of the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by noncatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. Thus, the studied polymers can be used to develop biodegradable medical devices such that they can perform their functions for a long period of time.     

In vivo degradation and tissue response to poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide)

Low molecular weight poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide) (PEKCDLLA) is being considered as a viscous liquid, injectable depot for localized drug delivery. This polymer degrades in vitro via surface erosion, which is potentially advantageous for the proposed application. However, the in vivo degradation rate and mechanism, and tissue response, to polymers based on 5-ethylene ketal ε-caprolactone have not yet been reported. The purpose of this study was to measure the in vivo weight loss and change in polymer properties and assess the tissue response to PEKCDLLA after subcutaneous injection in rats. The tissue response was assessed histologically using Masson's trichrome staining and immunohistochemically by staining for CD68 positive cells. The polymer lost weight with time in a nearly linear fashion but did not exhibit significant changes in number average molecular weight, polydispersity index, and glass transition temperature or monomer ratio, consistent with a surface erosion process. The tissue response to the polymer was moderate and comparable to that reported in the literature for other degradable polymers used in clinical applications. These findings indicate that PEKCDLLA is a promising candidate for injectable drug delivery.     

Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium

A bacterial strain able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. From the cell-free supernatant of cultures grown on crude lambda-carrageenan, a kappa-carrageenase was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S 200 HR and ion-exchange chromatography on DEAE--Sepharose-CL6B. The purified kappa-carrageenase was detected as a single protein upon SDS/PAGE. Its molecular mass was estimated at 40 kDa. Activity was observed against kappa-carrageenan over the pH range 5.0-8.5 and was optimal at pH 7.2 in Tris buffer or 7.0 in Mops buffer. The enzyme activity remained stable at 30 degrees C, but only for up to 1 h at 40 degrees C. Analysis of the degradation products of the kappa-carrageenase by gel filtration and 13C-NMR spectroscopy indicated that the enzyme degrades kappa-carrageenan down to the level of the kappa-neocarratetraose sulfate. The properties of this new enzyme are compared with those of previously characterized carrageenases.     

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.     

Synthesis of linear, beta-cyclodextrin-based polymers and their camptothecin conjugates

6(A),6(D)-Bis-(2-amino-2-carboxylethylthio)-6(A),6(D)-dideoxy-beta-cyclodextrin 1, a diamino acid derivative of beta-cyclodextrin, is synthesized and condensed with difunctionalized PEG comonomers to give linear, high molecular weight (M(w) over 50 kDa) beta-cyclodextrin-based polymers (2-4) with pendant functionality (carboxylate). 2-4 are all highly soluble in aqueous solutions (over 200 mg/mL). 20-O-trifluoroglycinylcamptothecin, 5a, and 20-O-trifluoroglycinylglycinylglycinylcamptothecin, 5b, are synthesized and conjugated to 2 to give polymer-camptothecin (CPT) prodrugs. The solubility of CPT is increased by more than three orders of magnitude when it is conjugated to 2. The rates of CPT release from the conjugates HGGG6 (high molecular weight polymer (M(w) 97 kDa), glyglygly linker and 6 wt % CPT loading) and HG6 (high MW polymer (M(w) 97 kDa), gly linker and 6 wt % CPT loading) in either mouse or human plasma are dramatically accelerated over the rates of pure hydrolysis at pH = 7.4, indicating the presence of enzymatic cleavage as a rate-determining step at this pH in the release of the CPT. The pH of aqueous solution has a large effect on hydrolysis rate of CPT from HGGG6 and HG6; the lower the pH, the slower the rate in the range at 4.1 相似文献   

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery

A biodegradable gene transfer vector has been synthesized by linking several low molecular weight (MW) polyethylenimine (PEI, 1200 Da) blocks using an oligo(L-lactic acid-co-succinic acid) (OLSA, 1000 Da). The resulting copolymer P(EI-co-LSA) (8 kDa) is soluble in water and degrades via base-catalyzed hydrolytic cleavage of amide bonds. With regard to its application as a gene transfer agent, the polymer showed an interesting pH dependency of degradation. At pH 5, when DNases are highly active, the degradation proceeds at a slower rate than at a physiological pH of 7.4. PEI and P(EI-co-LSA) spontaneously formed complexes with plasmid DNA. Whereas the complexes formed with PEI were not stable and aggregated, forming particles of up to 1 microm hydrodynamic diameter, P(EI-co-LSA) formed complexes, which were about 150 nm in size and of narrow size distribution. The latter complexes were stable, due to their high surface charge (zeta-potential + 18 mV). Similar to low MW PEI, the copolymer exhibited a low toxicity profile. At the same time, the copolymer showed a significant enhancement of transfection activity in comparison to the low MW PEI. This makes P(EI-co-LSA) a promising candidate for long-term gene therapy where biocompatibility and biodegradability become increasingly important.     

Purification and characterization of a neutral protease from rat-liver cytosolic fraction

Rat liver cytosol contains a neutral protease which degrades acetylated hemoglobin and some urea-denatured proteins maximally at pH 7.5. The enzyme was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA and o-phenanthroline, the metal-depleted enzyme can be reactivated by Co2+, Zn2+, Mn2+, or Mg2+, and it is not inhibited by reagents specific for carboxyl, seryl, or thiol proteases. The enzyme has an apparent molecular weight of 200,000 as determined on Sephacryl S-200 column chromatography, and electrophoresis in sodium dodecyl sulfate showed 3 protein bands corresponding to the molecular weights of 110,000, 74,000, and 40,000.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Allowing for polymer polydispersity--an essential condition for determination of aqueous pore diameters in cell walls and membranes using polymers

A method of allowing for polydispersion of polyethylene glycol (PEG) preparations was developed for the use of these preparations for the osmometrical evaluation of pore diameters with aqueous pores of Chara corallina cell walls as an example. The mass share of polyethylene glycol preparation fractions gamma p penetrating through the pores was determined using cellular "shadows", fragments of internodal cell walls tied up at the ends and filled with a 25% solution of nonpenetrating PEG 6000. When immersed into water, such "shadow" acquired a turgor (hydrostatic) pressure close to the cellular pressure and persistent over long time. The determination of gamma p for polyethylene glycols with different average molecular weights Mw was performed from the degree of pressure restoration after water was replaced by a 5-10% polymer solution. The kinetics of pressure changes was recorded using a mechanotronic dynamometer, which measures, in the quasi-isometric mode, the force necessary for partial compression of the "shadow" in its small fragment. By utilizing the dependence of the overall share of fractions with molecular weights Mi < Mk on Mk (data of [1]), we found that gamma p, for these polyethylene glycols corresponds to the threshold value of Mk = 800-1100 D (hydrodynamic radius of molecules rh = 0.85-1.05 nm). Thus, the effective diameter of the pores in the cell wall of Chara does not exceed 2.1 nm. It was shown that the smoothness of the sigmoid shape of the dependence of ionic channel conductivity on the Mw value of the polymer in the media is largely due to the polydispersion of polymer preparations, particularly, to the reduction in the share of fractions penetrating the channels as Mw is increased. The method normally used to estimate pore diameters in ionic channels which ignores the dispersion of polymer preparations, results in overestimated values.     

Degradation kinetics and mechanism of RH1, a new anti-tumor agent: A technical note

Summary and Conclusions  The degradation of RH1 in aqueous solution is found to be both acid and base catalyzed. The maximum stability is obtained in neutral pH but still degrades by 10% (t₉₀) after just 1 week. The stability profile at pH 5 was done, and 4 major degradation products were observed in acid solutions. LC-MS was performed and the molecular weights determined, from which a degradation mechanism was proposed. Degradation products I, II, and III form 2 isomers each depending on which aziridine group is hydrolyzed. No significant effect of light or the presence of antioxidants was observed, indicating that photodegradation and oxidation are not likely degradation reactions. Published: March 2, 2007     

Qualitative analysis of skeletal myosin as substrate of Ca2+-activated neutral protease: comparison of filamentous and soluble, native, and L2- deficient myosin

Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.     

Sensitivity of gelatin and albumin to irradiation at 230 NM.

Gelatin or poly-L-tyrosyl gelatin shows extensive degradation when exposed to ultraviolet radiation at a wavelength of 230 nm. Bovine serum albumin, a globular protein, exposed either to such ultraviolet radiation or to gamma-irradiation in the solid state resists the photolysis of peptide bonds. Molecular weights are determined by the ultracentrifugal "low speed" sedimentation equilibrium method. The effect of different speeds on the apparent average molecular weight of heterogenous material is clearly illustrated.     

Discovery of 2,3′-diindolylmethanes as a novel class of PCSK9 modulators

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of low density lipoprotein receptor (LDLR). Anti-PCSK9 agents have been approved for the treatment of hypercholesterolemia. We recently discovered a series of small-molecule PCSK9 modulators that contains a relatively small pharmacophore of 2,3′-diindolylmethane with molecular weights around only 250. These molecules can significantly lower the amount of PCSK9 protein in a cell-based phenotypic assay. Our SAR studies yielded compound 16 with a IC₅₀-value of 200 nM. No obvious cytotoxicity was observed at concentrations below 50 µM.     

Turnover of endogenous SsrA-tagged proteins mediated by ATP-dependent proteases in Escherichia coli

Formation and degradation of SsrA-tagged proteins enable ribosome recycling and elimination of defective products of incomplete translation. We produced an antibody against the SsrA peptide and used it to measure the amounts of SsrA-tagged proteins in Escherichia coli cells without interfering with tagging or altering the context of the tag added at the ends of nascent polypeptides. SsrA-tagged proteins were present in very small amounts unless a component of the ClpXP protease was missing. From the levels of tagged proteins in cells in which degradation is essentially blocked, we calculate that > or =1 in 200 translation products receives an SsrA tag. ClpXP is responsible for > or =90% of the degradation of SsrA-tagged proteins. The degradation rate in wild type cells is > or =1.4 min(-1) and decreases to approximately 0.10 min(-1) in a clpX mutant. The rate of degradation by ClpXP is decreased approximately 3-fold in mutants lacking the adaptor SspB, whereas degradation by ClpAP is increased 3-5-fold. However, ClpAP degrades SsrA-tagged proteins slowly even in the absence of SspB, possibly because of interference from ClpA-specific substrates. Lon protease degrades SsrA-tagged proteins at a rate of approximately 0.05 min(-1) in the presence or absence of SspB. We conclude that ClpXP, together with SspB, is uniquely adapted for degradation of SsrA-tagged proteins and is responsible for the major part of their degradation in vivo.     

Poly(ether-ester) conjugates with enhanced degradation

When a linear or a four arm star-shaped polyglycidol is used as macroinitiator, densely grafted poly(glycidol-graft-epsilon-caprolactone) and poly(glycidol-graft-L-lactide) and loosely grafted poly[(glycidol-graft-epsilon-caprolactone)-co-glycidol] copolymers have been synthesized by chemical or, in the latter case, by enzymatic catalyzed ring-opening polymerization of epsilon-caprolactone and L-lactide. The well-defined copolymers possess similar molecular weights, but differ in their architecture, microstructure and chemical composition. The hydrolytic degradation behavior was studied in a phosphate buffer solution at pH 7.4 and 37 degrees C for up to 90 days. After different time periods, the mass loss was determined and the degraded copolymers were analyzed by means of NMR, size exclusion chromatography, and scanning electron microscopy. Compared to linear poly(epsilon-caprolactone), poly[(glycidol-graft-epsilon-caprolactone)-co-glycidol] shows a change of the degradation mechanism and a tremendous enhancement of polymer degradation. As this effect is attributed to the high concentration of hydroxy groups at the polyglycidol backbone, this work points out a new possibility to tailor the degradation profiles of polyesters by the introduction of functionality into the polymeric material.