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At least 109 records · Page 6Linked to original sources

Enzyme-induced biodegradation of polycarbonate-polyurethanes: dependence on hard-segment chemistry.

Polycarbonate urethanes (PCNUs) have been used as a replacement for traditional biomedical polyether-urethanes due to their reported resistance to oxidative biodegradation. However, relatively little is known about their hydrolytic stability in the presence of inflammatory derived enzymes. This has in part motivated the current study relating to the effect of hard segment chemistry and the microdomain structures generated by such chemistry, on the cholesterol esterase (CE) catalyzed hydrolysis of PCNUs. The bulk structures of the studied materials were characterized using gel permeation chromatography (GPC), differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), Fourier transform infrared spectroscopy (FTIR) for their bulk structures, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) for their subsurface structures. 14C-labeled PCNUs were incubated with CE (400 units/mL), for a period of 10 weeks (pH 7.0 at 37 degrees C), and radiolabel release was used to monitor the degradation. The results showed that all of the polymers synthesized in this study were susceptible to CE-catalyzed hydrolytic degradation, and that the extent of degradation was highly dependent on the nature of hard segment interactions within the polymer and at the surface. More specifically, the degree of phase separation and soft segment crystallinity were found to be less important in comparison to the hydrogen bonding among the carbonate and urethane linkages. The rank of the different chemical groups' susceptibility to hydrolysis was as follows: nonhydrogen bonded carbonate > nonhydrogen bonded urethane > hydrogen bonded carbonate > hydrogen bonded urethane. The findings suggest that the degree of hydrogen bonding, when processed into a polyurethane material could be an important parameter to consider in the design of new biostable polyurethane products.

Biocompatible Materials↗

Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices.

Single photon burst techniques were used to detect double-stranded DNA molecules in poly(methylmethacrylate) (PM MA) and polycarbonate (PC) microfluidic devices. A confocal epi-illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono-intercalating dye, TOPRO-5, which possessed an absorption maximum at 765 nm allowing excitation with a solid-state diode laser and fluorescence monitoring in the near-infrared (IR). Near-IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing lambda-DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 microm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PM MA-based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths > -120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 microm widths compared to those of 50 microm, indicating improved sampling efficiency for the narrower channels without significantly deteriorating detection efficiency. Attempts were made to do single molecule sizing of lambda-DNA, M13 (7.2 kbp) and pUC19 (2.7 kbp) using photon burst detection. While the average number of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To demonstrate the sensitivity of single molecule analysis in the near-IR using polymer microfluidic devices, the near-IR chromophore, NN382, wasanalyzed using ourconfocal imager. A detection efficiency of 94% for single NN382 molecules was observed in the PC devices.

Bacteriophage lambda↗

Tyrosine-derived polycarbonates: backbone-modified "pseudo"-poly (amino acids) designed for biomedical applications.

Starting from L-tyrosine (Tyr) and its metabolites desaminotyrosine (Dat) and tyramine (Tym), four structurally related model dipeptides were prepared: Dat-Tym (neither N- or C-terminus present), Z-Tyr-Tym (N-terminus protected by benzyloxycarbonyl), Dat-Tyr-Hex (C-terminus protected by a hexyl ester group), and Z-Tyr-Tyr-Hex (both N- and C-termini present, protected by benzyloxycarbonyl and hexyl ester, respectively). The model dipeptides were used as monomers in the synthesis of polycarbonates. The polymerization reaction in the presence of either phosgene or triphosgene proceeded via the phenolic hydroxyl groups. Polymers with molecular weights of 105,000-400,000 da (by gel permeation chromatography, relative to polystyrene standards) were obtained. The physicomechanical properties (solubility, mechanical strength, glass transition and decomposition temperature, processibility) of the polymers were determined, and an attempt was made to correlate the polymer properties with the nature of the N- and C-terminus protecting groups. The presence of the urethane bond at the N-terminus protecting group was found to reduce solubility, ductility, and processibility, probably due to interchain hydrogen bonding. The presence of a C-terminus alkyl ester group increased solubility and processibility. Thus, the most promising candidate polymer for biomedical applications was obtained from Dat-Tyr-Hex, the monomer carrying a C-terminus protecting group only. Since very similar results had recently been obtained for a series of structurally related polyiminocarbonates, the structure property correlations seem to be generally valid.

Carbonates↗

Fabrication of a gold microelectrode for amperometric detection on a polycarbonate electrophoresis chip by photodirected electroless plating.

A novel method of photoresist-free micropatterning coupled with electroless gold plating is described for the fabrication of an integrated gold electrode for electrochemical detection (ED) on a polycarbonate (PC) electrophoresis microchip. The microelectrode layout was photochemically patterned onto the surface of a PC plate by selective exposure of the surface coated without photoresist to 254 nm UV light through a chromium/quartz photomask. Thus, the PC plate was selectively sensitized by formation of reactive chemical moieties in the exposed areas. After a series of wet chemistry reactions, the UV-exposed area was activated with a layer of gold nanoparticles that served as a seed to catalyze the electroless plating. The gold microelectrode was then selectively plated onto the activated area by using an electroless gold plating bath. Nonselective gold deposition on the unwanted areas was eliminated by sonication of the activated PC plate in a KSCN solution before electroless plating, and the adhesion of the plated electrodes to the PC surface was strengthened with thermal annealing. Compared with the previously reported electroless plating technique for fabrication of microelectrodes on a microchip, the present method avoided the use of a membrane stencil with an electrode pattern to restrict the area to be wet-chemically sensitized. The CE with integrated ED (CE-ED) microchip was assembled by thermal bonding an electrode-plated PC cover plate to a microchannel-embossed PC substrate. The novel method allows one to fabricate low-cost, electrode-integrated, complete PC CE-ED chips with no need of a clean room. The fabricated CE-ED microchip was demonstrated for separation and detection of model analytes, including dopamine (DA) and catechol (CA). Detection limits of 0.65 and 1.03 microM were achieved for DA and CA, respectively, and theoretical plate number of 1.4 x 10(4) was obtained for DA. The plated gold electrode can be used for about 4 h, bearing usually more than 100 runs before complete failure.

Catechols↗

Biodegradation of polycarbonate-based polyurethanes by the human monocytes-derived macrophage and U937 cell systems.

The prominent cell type found on implanted medical devices during the chronic inflammatory response is the monocyte-derived macrophage (MDM). Using an activated in vitro cell system, it was possible to show that MDMs possess esterolytic activities that may contribute to the degradation of polyurethanes. In the present study, the U937 cell line was paralleled to the MDM cell system in order to validate the use of a cell line that could expedite studies on biomaterial biocompatibility and biostability. Using 12-o-tetradecanoylphorbol 13-acetate (PMA), the optimum differentiation time for the U937 cells was 72 h based on biodegradation, degradative potential, and (35)S-methionine uptake. After activation of the cells by resuspending from tissue culture polystyrene plates and reseeding onto a (14)C-labeled polycarbonate-based polyurethane(PCNU), both U937 cells and the MDMs elicited comparable radiolabel release (measure of polymer breakdown) and esterase activity (measure of degradative potential) at 48 h. There was no difference in the effect on radiolabel release and esterase activity elicited by both cell types with inhibitors of protein synthesis, esterase activity, and phospholipase A(2). This established that both cell types likely used similar hydrolytic activities and signaling pathways to cause degradation of the PCNU. Immunoblotting demonstrated that both cell systems secreted monocyte-specific esterase and cholesterol esterase enzymes previously shown to degrade PCNUs. The U937 cell system is more convenient and reproducible than MDMs for pursuing possible biological pathways elucidating the mechanism of polyurethane biodegradation. Once established with U937s, the pathways can then be validated with the more physiologically relevant human MDM cell system.

Aristolochic Acids↗

Poly(ethylene glycol) enhances cell motility on protein-based poly(ethylene glycol)-polycarbonate substrates: a mechanism for cell-guided ligand remodeling.

The regulation of cell motility on ligand-adsorbed poly(ethylene glycol) (PEG)-based polymeric biomaterials is governed by variables that are not well characterized. In this report, we examined keratinocyte migratory responsiveness to PEG-variant tyrosine-derived polycarbonates adsorbed with equivalent levels of the cell adhesion ligand, fibronectin. The equivalently adsorbed ligand adopted differential distributions, confirmed via atomic force microscopy, and the total number of exposed cell-binding domains (CBD), quantified through immunosorbent fluorometry, varied as a function of PEG concentration. Specifically, the CBD exposure was maximized at 4 mol % PEG and diminished at 8 mol % PEG, suggesting, based on our previous work (Tziampazis et al., Biomaterials 2000;21:511-520), that activation of cell adhesion and motility could be potentially promoted through increased CBD exposure at intermediate levels of PEG. This was confirmed through cell migration studies wherein cell speed values increased from 11 to 22 microm/h as the PEG concentration was increased from 0 to 4 mol %. Unexpectedly, however, high cell motility rates were sustained at 8 mol % PEG despite diminished levels of initial CBD exposure beyond 4 mol % PEG, suggesting that factors other than the initial CBD exposure may additionally have a role in activating cell migration at higher levels of PEG. Through studies of direct ligand mobility, cell-ligand-polymer interactions via atomic force microscopy, and CBD variation and integrin receptor roles in ligand remodeling, we offer evidence that cell motility is enhanced by a new mechanism for the regimen of higher PEG concentration: upon cell attachment and spreading, the ligand exhibits greater "slippage" at the polymer interface, and undergoes cell-engendered remodeling, which further activates cell motility, likely through enhanced exposure of hitherto encrypted sites for cell binding and signaling.

Antibodies↗

Role of protein kinase C in the monocyte-derived macrophage-mediated biodegradation of polycarbonate-based polyurethanes.

Polycarbonate-polyurethanes (PCNUs) elicit a foreign body reaction during the initial tissue contact, partly mediated by the respiratory burst in monocytes, during which protein kinase C (PKC) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase. Using an in vitro cell system, monocytes were differentiated into monocyte-derived macrophages (MDMs) and then reseeded onto three PCNUs (HDI431, HDI321, or MDI321): hexane (HDI) or 4,4-methylene bis-phenyl (MDI) diisocyanates synthesized with poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 14C-labeled butanediol (BD) in the ratios 4:3:1 or 3:2:1 (diisocyanate/PCN/BD). MDM-mediated degradation was assessed by radiolabel release in the presence of a PKC activator (phorbol myristate acetate), inhibitor (H7), and a catalase/peroxidase inhibitor (NaN3). Activating PKC decreased biodegradation and esterase activity in MDMs on HDI431 and HDI321 but not MDI321, whereas H7 and NaN3 inhibited the MDM degradation of MDI321 only. Pretreatment of the PCNUs with H2O2 inhibited esterase-mediated radiolabel release from HDI431 and HDI321 but stimulated radiolabel release from MDI321. The difference in the effect of H2O2 on the HDI versus MDI PCNUs contributes to explaining the effect of PKC activation on material degradation. Understanding the mechanism by which this pathway is linked to PCNU chemistry may assist in designing materials with tailored biodegradation rates.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Cellular response to phase-separated blends of tyrosine-derived polycarbonates.

Two-dimensional thin films consisting of homopolymer and discrete compositional blends of tyrosine-derived polycarbonates were prepared and characterized in an effort to elucidate the nature of different cell responses that were measured in vitro. The structurally similar blends were found to phase separate after annealing with domain sizes dependent on the overall composition. The thin polymer films were characterized with the use of atomic force microscopy (AFM), water contact angles, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) and significant changes in roughness were measured following the annealing process. Genetic expression profiles of interleukin-1beta and fibronectin in MC3T3-E1 osteoblasts and RAW 264.7 murine macrophages were measured at several time points, demonstrating the time and composition-dependent nature of the cell responses. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) depicted upregulation of the fibronectin gene copy numbers in each of the blends relative to the homopolymers. Moreover, the interleukin-1beta expression profile was found to be compositionally dependent. The data suggest strongly that optimal composition and processing conditions can significantly affect the acute inflammatory and extracellular matrix production responses.

Animals↗

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: a study using fluorescence measurements through polycarbonate supports.

We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.

Animals↗

Problems in the use of polycarbonate diffusion chambers for syngeneic pancreatic islet transplantation in rats.

Islets of Langerhans have been enclosed in polycarbonate diffusion chambers and transplanted intraperitoneally to syngeneic streptozotocin diabetic rats. Direct implantation of 1100--1400 islets in these chambers failed to reverse diabetes during a period of 12 weeks, and viable islet tissue was not recoverable at the end of this period. Islets placed in chambers which had been implanted 3--12 weeks previously similarly failed to lower blood glucose of diabetic recipients, as a result of lack of survival of the islets. Insulin infusion into chambers previously implanted in vivo, I125 insulin diffusion studies in chambers recovered 6--8 weeks after implantation, and scanning electron microscopy of the recovered membranes all indicated that the pores were not totally occluded. The failure of islet transplantation via chambers in this simple syngeneic model has discouraging implications for their use as a means of avoiding allograft rejection.

Animals↗

Polyester and polycarbonate synthesis by in vitro enzyme catalysis.

Enzyme technology has significantly expanded in scope and impact over the past 10 years to include organic transformations in non-traditional environments. This is in part due to an increased understanding and capability of using enzyme catalysis in a wide variety of organic solvents, at interfaces, and at high temperatures and pressures. This review focuses on a relatively new but rapidly expanding research activity where in vitro enzyme catalysis is used for the synthesis of non-natural polyesters and polycarbonates. The inclination to use of enzymes for polymer synthesis has been fueled by a desire to carry out these reactions in the absence of heavy metals, at lower temperatures, and with increased selectivity. Aspects of this work that include enzyme-catalyzed step-growth condensation reactions, chain-growth ring-opening polymerizations, and corresponding transesterification of macromolecular substrates are discussed.

Acylation↗

Tyrosine derived polycarbonate membrane is useful for guided bone regeneration in rabbit mandibular defects.

Standardized bilateral through-and-through defects (12x6 mm) were created extraorally in the mandibular angle of 18 New Zealand White rabbits. Animals were divided in to three groups (n=6) according to the intended healing time. On the left side, defects were covered with a poly(desaminotyrosyl-tyrosine-ethyl ester carbonate) (PDTE carbonate) membrane wrapped around the inferior border of the mandible and fixed with bioabsorbable sutures. On the right side, the defects were filled with a mesh made of bioactive glass 13-93 and 3 wt% chitosan. The defects were covered with the same membranes. Periosteal flap was sutured over the membrane. Radiographically, bone ingrowth was seen in all specimens at 12 weeks postoperatively. At 24 weeks, completely ossified area remained approximately at the same level as at 12 weeks, but the non-ossified area decreased to almost zero. However, the bioactive glass mesh did not improve the results. Nevertheless, enveloping the defect with PDTE carbonate membrane seemed to play a crucial role in new bone formation. Based on these results, we conclude that tyrosine polycarbonate is a promising new material for guided bone regeneration.

Animals↗

Fractionation and analysis of mitochondria with polycarbonate membrane filters.

Polycarbonate membrane filters were used to fractionate mitochondrial populations depending on their aggregation or association with other subcellular structures. Isolated rat liver mitochondria penetrated through filters which have pore sizes larger than 1 micron. In contrast, mitochondria which were induced to aggregate in vitro by incubation at low pH were retained by the filters and thus could be separated from the single or small aggregates of mitochondria. Use of this membrane filtration method to analyze release of mitochondria from isolated hepatocytes showed that treatment with digitonin at concentrations only sufficient to lyse the plasma membrane did not release mitochondria. Homogenization or sonication following digitonin treatment released 25-50% of the mitochondria, but only a small fraction was intact. A high yield of intact mitochondria was released from digitonin-treated cells by a brief treatment with a low concentration of the proteolytic enzyme nagarse. Thus, this membrane filtration method provides a simple and rapid approach to analyze the extent of mitochondrial aggregation and association with other subcellular structures.

Animals↗

Preparation of unilamellar liposomes of intermediate size (0.1-0.2 mumol) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes.

Liposomes can be prepared by a combination of reverse phase evaporation and sequential extrusion through polycarbonate membranes. The vesicles have diameters in the range 0.05-0.5 micron and are mostly unilamellar as indicated by electon microscopy, capture volume, and availability of reactive groups to periodate oxidation. Sequential extrusion leads to a decrease in the encapsulation efficiency by 2-4-fold, depending upon the lipid composition. The inclusion of cholesterol at a 1 : 1 molar ratio of cholesterol-to-phospholipid increases both the mean size and the size heterogeneity of the liposomes as measured by negative-stain electron microscopy. The mean size of vesicles with an equal molar ratio of cholesterol-to-phospholipid after extrusion through a 0.1 micron membrane is 0.140 micron. Vesicles composed of phosphatidylglycerol/phosphatidylcholine (1 : 4) have a mean size of 0.08 micron after extrusion through a 0.1 micron membrane. The intermediate-size (0.1-0.2 micron) vesicles formed by this process have an aqueous space-to-lipid ratio of 3 : 5 and capture between 12 and 25% of the aqueous phase. The procedure is relatively simple, rapid, and yields almost quantitative recovery of vesicles that encapsulate a large percentage of the total aqueous space.

Carbonates↗

Rapid diffusion of the lipid phosphorus of phosphatidylglycerol liposomes through polycarbonate membranes is caused by the oxidation of the unsaturated fatty acids.

The lipid phosphorus of phosphatidylglycerol liposomes was found to diffuse extensively, after a lag time of 1 to 2 days, through a 0.1 micron pore size polycarbonate membrane in a two compartment system. Diffusion occurred when either multilamellar or large unilamellar vesicles were studied, even if they were sedimented to eliminate any smaller particles. The lipid of liposomes prepared under sterile conditions also diffused extensively. Diffusion appeared to be related to the age of the vesicles, and could be eliminated by incorporating antioxidants into the liposomes, or by using liposomes prepared from saturated phospholipids (C14 or larger). This indicated that diffusion was caused by phospholipid oxidation, which was confirmed by HPLC analysis. Phospholipid phosphorus that diffused through a membrane appeared more polar, as indicated by its capacity to distribute into the upper phase of a two phase extraction. Phospholipid phosphorus diffusion was preceded by the complete loss of liposomes contents, indicated by the complete diffusion of encapsulated carboxyfluorescein through the membrane. Oxidation of the lipid could be prevented by inclusion of either butylated hydroxytoluene or alpha-tocopherol in the membrane. The best retention of liposomal contents was achieved when both antioxidants and cholesterol were included in the liposome preparation. The antioxidant incorporated in the liposomes remained effective in protecting the phospholipids upon storage at 4 degrees C for 2 months. The inclusion of EDTA in the suspension medium retarded the rapid oxidation, suggesting that the presence of trace amounts of heavy metal ions in the buffer catalyzed the oxidation. Phospholipid oxidation was most effectively inhibited by the presence of serum or chemically defined medium, suggesting that oxidation of liposomal lipids in a biological environment may be minimized if appropriate steps are taken.

Antioxidants↗

Rapid quantitation of neutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly.

A neutrophil chemotaxis assay was developd which permits rapid, quantitative assessment of migration across a membrane filter. The critical factor in the assay was the use of a 10 microns thick polycarbonate membrane without the usual polyvinylpyrrolidone coating. Migrated neutrophils remain adherent to the uncoated membrane, whereas 20-50% fall off polyvinylpyrrolidone-coated membranes. A major advantage of the method is that neutrophil chemotaxis can be readily quantified, since the migrated cells adhere to the membrane surface and are in one optical plane for counting. A 25 mm x 80 mm membrane sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 microliters of attractant per assay well. Neutrophil chemotaxis was complete within 10-20 min at 37 degrees C, with 20-30% of the cells migrating to N-formyl-methionyl-leucyl-phenylalanine and 40-50% migrating to complement derived C5a.

Carbonates↗

Immunofluorescent screening of monoclonal antibodies to surface antigens of animal and plant cells bound to polycarbonate membranes.

We have developed an immunofluorescent screening method in which animal or plant cells are immobilized without fixation on polycarbonate (Nuclepore) membranes. The technique does not involve centrifugation, and virtually no cells are lost during processing. Direct microscopic analysis of antibody binding provides an extremely sensitive and reproducible screening system for hybridoma supernatants.

Animals↗

Use of sparse-pore polycarbonate (Nuclepore) membrane for the measurement of chemotaxis of polymorphonuclear leukocytes. Comparison of 'micro-Boyden' chambers and estimation of 'drop-off' of cells.

Various modified Boyden chambers having upper and lower compartments of small volume ('micro-Boyden' chambers) were tested for their suitability for use with the recently described 'sparse-pore' polycarbonate (Nuclepore) membrane for the measurement of chemotaxis of polymorphonuclear leukocytes in vitro towards N-formyl-methionyl-leucyl-phenylalanine. Previously reported 'open-well' and 'blind-well' chambers, as well as a new chamber having a lower compartment made from perspex and an upper compartment made from silicone rubber were tested. The most satisfactory results were provided by the new chamber, and this is attributed to the easy, accurate filling of the lower compartment, the lack of distortion of membrane, and the reliability of the seals around the edges of the membrane which can be achieved with this chamber. Full humidification during assembly was necessary for obtaining maximal response of the cells through the 'sparse-pore' membrane in all types of micro-Boyden chamber. 'Drop-off' of polymorphonuclear leukocytes from the lower surface of the 'sparse-pore' membrane during incubation was also studied in these experiments and found to be slight.

Chemotaxis, Leukocyte↗