Search

We have used a series of in vitro and in vivo tests to assess the suitability of two new degradable polymers for application as coatings for drug-eluting stents. The first is a family of urethane-linked multi-block copolymers (MBCP) that comprise blocks of lactide, glycolide, epsilon-caprolactone and/or poly(ethylene glycol) chain-extended with 1,4-butanediisocyanate (SynBiosys polymers). The second is a family of maltodextrin (MD) modified with fatty acid sidechains to yield a hydrophobic polymer (Eureka() SOLO polymers). We coated stainless-steel stents with two representative urethane-linked MBCPs and one hydrophobic MD polymer alone or in combination with the anti-restenotic drug sirolimus. Urethane-linked MBCPs formed uniform coatings on the stent substrates, withstood crimping and expansion on balloon catheters, completely released sirolimus from the coating within 30 days, and degraded within 30-60 days in PBS. The hydrophobic MD polymer formed uniform coatings, exhibited somewhat slower release of sirolimus (approx. 85% within 30 days), degraded within 60 days in PBS when sirolimus was incorporated in the coating, but showed very slow degradation in the absence of drug. We implanted stents coated with urethane-linked MBCPs or hydrophobic MD polymers in a porcine coronary artery model and used histological analysis at 28- and 90-day end-points to assess the biological response to the materials. Measures of stenosis and inflammation for urethane-linked MBCP and hydrophobic MD polymer coatings were not statistically different from bare metal controls at 28 and 90 days, suggesting that the polymers show good vascular biocompatibility. Endothelialization was nearly complete at 28 days and complete at 90 days for all formulations. Urethane-linked MBCP polymer-only and drug-eluting coatings and hydrophobic MD drug-eluting coatings were nearly completely degraded within 90 days in vivo whereas roughly half of hydrophobic MD polymer-only coatings remained after 90 days. Taken together, our in vitro and in vivo results suggest that SynBiosys urethane-linked MBCP and Eureka SOLO hydrophobic MD polymer families possess the physical and chemical properties and vascular biocompatibility necessary for further investigation for use in the next generation of drug-eluting stents.

The tools of surface science have enabled a thorough understanding of the ordering of liquid crystalline materials in contact with the surfaces of solids. This report moves beyond these past studies of solid–liquid crystal interfaces by describing the emergence of a set of experimental observations that revolve around the ordering of liquid crystals at interfaces to immiscible aqueous phases. The self-assembly of surfactants, lipids, proteins and synthetic polymers at these interfaces has recently been shown to lead to a rich but poorly understood spectrum of orientational ordering behaviors of liquid crystalline materials. It is also evident that the ordering of the liquid crystals influences the organization of the molecules that assemble at these interfaces. In particular, recent experiments have revealed the influence of the molecular structure and interfacial organization of adsorbed surfactants on the ordering of the liquid crystals. The time-dependent behaviors of liquid crystals, which can reorganize on time scales of tens of milliseconds, have been used to follow dynamic phenomena at these interfaces, including the adsorption, desorption and spatial reorganization of surfactants. At these same aqueous–liquid crystal interfaces, phospholipids have been shown to assemble into monolayers that mimic dynamic properties of biological membranes. Specific protein binding events and enzyme-catalyzed reactions at phospholipid-decorated interfaces of liquid crystals lead to changes in the organization of the phospholipids that are reported as ordering transitions in the liquid crystals. Larger amphiphilic molecules, including polymers and proteins, have also been shown to assemble at aqueous–liquid crystal interfaces and to couple to the ordering of the liquid crystal. For example, functional amphiphilic polymers impart a means to reversibly control the ordering of liquid crystals in response to changes in solution conditions (e.g., pH). These observations, when combined, suggest that interfaces formed between liquid crystals and aqueous phases represent a fundamentally interesting and technologically promising class of interfaces for chemical and biological sensing, active control of interfacial assemblies and realization of stimuli-responsive materials. These interfaces define a rich set of scientific challenges that warrant the attention of the surface science community.

We report use of thin films of the nematic liquid crystal E7 supported on chemically functionalized surfaces to indicate the presence of vapors of the organophosphorous nerve agents sarin (GB), soman (GD), tabun (GA) and VX. The surfaces were prepared by the deposition of metal perchlorate salts onto carboxylic acid-terminated self-assembled monolayers. When using surfaces prepared from aluminum perchlorate salts, the nematic film of E7 underwent a transition from a perpendicular orientation to a tilted or planar orientation upon exposure to vapors of GB, GD, GA or VX generated from a small drop of agent placed on a piece of filter paper. The orientational transition of the liquid crystal was readily apparent as an optical signal that was visible to the naked eye. The agent VX, which has the lowest vapor pressure of these organophosphorous nerve agents (∼140 ppb), was reported by the liquid crystal within 60 s. The agents GB, GD, and GA, which have higher vapor pressures, triggered responses in the liquid crystal that were evident within 15 s, 15 s, and 60 s, respectively. By preparing surfaces from perchlorate salts of aluminum(III), zinc(II) and iron(III) it was possible to distinguish between GB, VX and either GD or GA, thus demonstrating the feasibility of designing surfaces patterned with metal salts to detect and positively identify chemical warfare agents.

Differential scanning calorimetry (DSC) was used to characterize interactions of synthetic LCs, 4‐pentyl‐4′‐cyanobiphenyl (5CB) and TL205 (a mixture of cyclohexane‐fluorinated biphenyls and fluorinated terphenyls) with simple mimics of cell membranes. The investigation was motivated by reports that living cells can be placed into contact with TL205 without apparent toxicity, whereas contact of cells with 5CB leads to cell death. The tendency was examined for 5CB and TL205 to spontaneously partition into and influence the organization for model cell membranes composed of phospholipids. Upon contact of an aqueous dispersion of DPPC liposomes with neat LC for 4 h, 5CB partitioned into the liposomes at a weight ratio of 5:1 DPPC:5CB, whereas TL205 partitioned at a ratio of 310:1 DPPC:TL205. DSC endotherms indicated that the 5CB spontaneously partitioned into the liposomes was far more perturbing than TL205. DSC endotherms of DPPC bilayers containing the same concentration of either 5CB or TL205 also revealed ...

We have investigated the culture of human embryonic stem cells (hESCs) on interfaces of the thermotropic liquid crystal, TL205, that are decorated with thin films of the extracellular matrix, Matrigel. hESCs seeded at the liquid-crystal/Matrigel interface survive for weeks, and cell colonies grow over this time. The cells show levels of differentiation comparable to that observed for cells on Matrigel-coated glass controls. Polarized and fluorescence microscopy reveal that the orientational order of the liquid crystal is coupled to the presence and organization of Matrigel. This enables straightforward imaging of the reorganization of Matrigel by the hESCs through changes in the appearance of the liquid crystal when observed using polarized light microscopy. The coupling between Matrigel and TL205 thus provides a simple tool for monitoring the reorganization of the Matrigel film over time. Our results suggest new approaches to the culture of cells and measurements of cell–extracellular-matrix interactions.

Recent studies have reported on the self-assembly of surfactants and phospholipids at interfaces between aqueous phases and thermotropic liquid crystals. These studies have been enabled by an experimental system that permits the preparation of stable and easily-imaged interfaces between liquid crystals and aqueous phases. The influence of the molecular structure of surfactants and their interfacial organization on the ordering of the liquid crystals has been revealed. Because the liquid crystals reorder on time-scales of seconds or less, the time-dependent behavior of the liquid crystals can be used to follow dynamic phenomena at these interfaces, including the adsorption and desorption of surfactants. At these same aqueous-liquid crystal interfaces, phospholipids have been shown to organize into assemblies that possess the mobility of biological membranes and are coupled to the order within the liquid crystal. Specific protein binding events and enzyme-catalyzed reactions at phospholipid-decorated interfaces of liquid crystals lead to changes in the organization of the phospholipids that are “reported” as ordering transitions in the liquid crystal.

We have examined the influence of two aspects of surfactant structure--tail branching and tail organization--on the orientational ordering (so-called anchoring) of water-immiscible, thermotropic liquid crystals in contact with aqueous surfactant solutions. First, we evaluated the influence of branches in surfactant tails on the anchoring of nematic liquid crystals at water-liquid crystal interfaces. We compared interfaces that were laden with one of three linear surfactants (sodium dodecyl sulfate, sodium dodecanesulfonate, and isomerically pure linear sodium dodecylbenzenesulfonate) to interfaces laden with branched sodium dodecylbenzenesulfonate. We carried out these experiments at 60 degrees C, above the Krafft temperatures of all the surfactants studied, and used the liquid crystal TL205 (a mixture of cyclohexane-fluorinated biphenyls and fluorinated terphenyls), which forms a nematic phase at 60 degrees C. Linear surfactants caused TL205 to assume a perpendicular orientation (homeotropic anchoring) above a threshold concentration of surfactant and parallel orientation (planar anchoring) at lower concentrations. In contrast, branched sodium dodecylbenzenesulfonate caused planar anchoring of TL205 at all concentrations up to the critical micelle concentration of the surfactant. Second, we used sodium dodecanesulfonate and a commercial linear sodium dodecylbenzenesulfonate to probe the influence of surfactant tail organization on the orientations of liquid crystals at water-liquid crystal interfaces. Commercial linear sodium dodecylbenzenesulfonate, which comprises a mixture of ortho and para isomers, has been previously characterized to form less ordered monolayers than sodium dodecanesulfonate at oil-water interfaces at room temperature. We found sodium dodecanesulfonate to cause homeotropic anchoring of both TL205 and 4'-pentyl-4-cyanobiphenyl (5CB, nematic at room temperature), whereas commercial linear sodium dodecylbenzenesulfonate caused predominantly planar and tilted orientations of both TL205 and 5CB. These results, when combined, lead us to conclude that (1) interactions between the aliphatic tails of surfactants and liquid crystals largely dictate the orientations of liquid crystals at aqueous-liquid crystal interfaces, (2) the interactions that orient the liquid crystals at these interfaces are sensitive to the branching and degree of disorder in the surfactant tails, and (3) differences in the chemical composition of TL205 and 5CB, most notably fluorination of TL205, lead to subtle differences in the orientations of these two nematic liquid crystals.

Three peptides, YGAA[KKAAKAA](2) (AKK), KLFKRHLKWKII (SC4), and YG[AKAKAAKA](2) (KAK), were conjugated with lauric acid and tested for the effect on their structure, antibacterial activity, and eukaryotic cell toxicity. The conjugated AKK and SC4 peptides showed increased antimicrobial activity relative to unconjugated peptides, but the conjugated KAK peptide did not. The circular dichroism spectrum of AKK showed a significantly larger increase in its alpha-helical content in the conjugated form than peptide KAK in a solution containing phosphatidylethanolamine/phosphotidylglycerol vesicles, which mimics bacterial membranes. The KAK and AKK peptides and their corresponding fatty acid conjugates showed little change in their structure in the presence of phosphatidylcholine vesicles, which mimic the cell membrane of eukaryotic cells. The hemolytic activity of the KAK and AKK peptides and conjugates was low. However, the SC4 fatty acid conjugate showed a large increase in hemolytic activity and a corresponding increase in helical content in the presence of phosphatidylcholine vesicles. These results support the model of antimicrobial peptide hemolytic and antimicrobial activity being linked to changes in secondary structure as the peptides interact with lipid membranes. Fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes.

Field emission (FE) cathodes made from carbon nanotube (CNT) fibers have demonstrated high emission currents, low turn-on voltages, long lifetimes and offer considerable potential for use as electron sources for vacuum electronic devices. CNT fibers were fabricated by wet-spinning of pre-made CNTs1 and consist of CNT fibrils held together by van der Waals forces. The fibers were 10–100 µm in diameter and their morphology was controlled by fabrication method, processing conditions, as well as purity, size, and type of the CNT starting material. Thermal and electrical conductivity was measured with the 3-omega method, and fiber density was determined with transmission electron microscopy. Wide angle x-ray diffraction was used to measure fiber alignment. Fibers with the highest density, alignment, thermal and electrical conductivity had the best field emission performance. Recent results have demonstrated a single 5 mm long fiber with a 20 µm diameter emitting 5.2 mA before failure, and 6.1 mA when treated with Boron Nitride2. Residual gas analysis (RGA) was used to identify the species desorbed during field emission and model was developed for the transition from adsorbate-enhanced FE at low bias to FE from pure CNTs at high bias3. Infrared images of CNT fibers during FE have been captured with an InGaAs array camera, showing temperatures of ∼600°C while emitting currents of ∼4mA. Mechanisms of fiber heating during FE, to include Joule and Nottingham effects, were investigated. Calculated thermal performance based on measured thermal conductivity is presented, and comparisons between observed and calculated thermal performance are discussed.

We used residual gas analysis (RGA) to identify the species desorbed during field emission (FE) from a carbon nanotube (CNT) fiber. The RGA data show a sharp threshold for H2 desorption at an external field strength that coincides with a breakpoint in the FE data. A comprehensive model for the gradual transition of FE from adsorbate-enhanced CNTs at low bias to FE from CNTs with reduced H2 adsorbate coverage at high bias is developed which accounts for the gradual desorption of the H2 adsorbates, alignment of the CNTs at the fiber tip, and importance of self-heating effects with applied bias., Comment: 18 pages 3 figures

New approaches to the culture of cells and measurements of cell–extracellular-matrix interactions are suggested in the Full Paper by Abbott and co-workers on p. 618. The culture of human embryonic stem cells (hESCs) on interfaces of a thermotropic liquid crystal that are decorated with thin films of the extracellular matrix, Matrigel, are investigated. The cover image shows the polarized light microscopy image of hESCs cultured for three days on a Matrigel-coated film of liquid crystal hosted within a TEM grid. The hESCs reorganized the Matrigel, leading to patterned orientations of the liquid crystal. We have investigated the culture of human embryonic stem cells (hESCs) on interfaces of the thermotropic liquid crystal, TL205, that are decorated with thin films of the extracellular matrix, Matrigel. hESCs seeded at the liquid-crystal/Matrigel interface survive for weeks, and cell colonies grow over this time. The cells show levels of differentiation comparable to that observed for cells on Matrigel-coated glass controls. Polarized and fluorescence microscopy reveal that the orientational order of the liquid crystal is coupled to the presence and organization of Matrigel. This enables straightforward imaging of the reorganization of Matrigel by the hESCs through changes in the appearance of the liquid crystal when observed using polarized light microscopy. The coupling between Matrigel and TL205 thus provides a simple tool for monitoring the reorganization of the Matrigel film over time. Our results suggest new approaches to the culture of cells and measurements of cell–extracellular-matrix interactions.

Recent experiments have shown that liquid crystals can be used to image mammalian cell membranes and to amplify structural reorganization in phospholipid-laden liquid crystal-aqueous interfaces. In this work, molecular dynamics simulations were employed to explore the interactions between commonly used liquid crystal-forming molecules and phospholipid bilayers. In particular, umbrella sampling was used to obtain the potential of mean force of 4-cyano-4′-pentylbiphenyl (5CB) and 4′-(3,4-difluor-phenyl)-4-pentyl-bicylohexyl (5CF) molecules partitioning into a dipalmitoylphosphatidylcholine bilayer. In addition, results of simulations are presented for systems consisting of a fully hydrated bilayer with 5CB or 5CF molecules at the lowest (4.5mol %) and highest (20mol %) concentrations used in recent laboratory experiments. It is found that mesogens preferentially partition from the aqueous phase into the membrane; the potential of mean force exhibits highly favorable free energy differences for partitioning (−18kBT for 5CB and −26kBT for 5CF). The location and orientation of mesogens associated with the most stable free energies in umbrella sampling simulations of dilute systems were found to be consistent with those observed in liquid-crystal-rich bilayers. It is found that the presence of mesogens in the bilayer enhances the order of lipid acyl tails, and changes the spatial and orientational arrangement of lipid headgroup atoms. These effects are more pronounced at higher liquid-crystal concentrations. In comparing the behavior of 5CB and 5CF, a stronger spatial correlation (i.e., possibly leading to aggregation) is observed between 5CB molecules within a bilayer than between 5CF molecules. Also, the range of molecular orientations and positions along the bilayer normal is larger for 5CB molecules. At the same time, 5CF molecules were found to bind more strongly to lipid headgroups, thereby slowing the lateral motion of lipid molecules.

We have conjugated dodecyl and octadecyl fatty acids to the N-terminus of SC4, a potently bactericidal, helix-forming peptide 12-mer (KLFKRHLKWKII), and examined the bactericidal activities of the resultant SC4 ‘peptide-amphiphile’ molecules. SC4 peptide-amphiphiles showed up to a 30-fold increase in bactericidal activity against Gram-positive strains (Staphylococcus aureus, Streptococcus pyogenes and Bacillus anthracis), including S. aureus strains resistant to conventional antibiotics, but little or no increase in bactericidal activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fatty acid conjugation improved endotoxin (lipopolysaccharide) neutralization by 3- to 6-fold. Although acylation somewhat increased lysis of human erythrocytes, it did not increase lysis of endothelial cells, and the haemolytic effects occurred at concentrations 10- to 100-fold higher than those required for bacterial cell lysis. For insight into the mechanism of action of SC4 peptide-amphiphiles, CD, NMR and fluorescence spectroscopy studies were performed in micelle and liposome models of eukaryotic and bacterial cell membranes. CD indicated that SC4 peptide-amphiphiles had the strongest helical tendencies in liposomes mimicking bacterial membranes, and strong membrane integration of the SC4 peptide-amphiphiles was observed using tryptophan fluorescence spectroscopy under these conditions; results that correlated with the increased bactericidal activities of SC4 peptide-amphiphiles. NMR structural analysis in micelles demonstrated that the two-thirds of the peptide closest to the fatty acid tail exhibited a helical conformation, with the positively-charged side of the amphipathic helix interacting more with the model membrane surface. These results indicate that conjugation of a fatty acid chain to the SC4 peptide enhances membrane interactions, stabilizes helical structure in the membrane-bound state and increases bactericidal potency.

We have used synthetic lipidated peptides (“peptide-amphiphiles”) to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB1–52) and its cytoplasmic (PLB1–25K: phospholamban residues 1–25 plus a C-terminal lysine), and transmembrane (PLB26–52) domains, and a 38-residue model α-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of α-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB1–25K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB1–25 amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB1–25K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 283–292, 2003

Anginex is a designed peptide 33mer that functions as a cytokine-like agent to inhibit angiogenesis. Although this short linear peptide has been shown by NMR and CD to form a nascent β-sheet conformation in solution, the actual bioactive structure formed upon binding to its receptor on the surface of endothelial cells could be quite different. By using a series of double-cysteine disulphide-bridged analogues, we provide evidence in the present study that the β-sheet is in fact the bioactive conformation of anginex. CD and NMR spectral analysis of the analogues indicate formation of a β-sheet conformation. Three functional assays, endothelial cell proliferation, apoptosis and in vitro angiogenesis, were performed on all analogues. As long as the placement of disulphide bonds preserved the β-strand alignment, as in the proposed bioactive conformation, bioactivities were preserved. Knowledge of the bioactive conformation of anginex will aid in the design of smaller molecule mimetics of this potent anti-angiogenic peptide.

We observed gelation of a 23-residue peptide derived from the beta-sheet domain of platelet factor-4 (PF4(24)(-)(46)). The gels were primarily heterogeneous mixtures of 50-200 microm spherical aggregates in a less-dense gel matrix. Infrared and circular dichroism spectroscopies showed gelation involving the conversion of PF4(24)(-)(46) from random coil to beta-sheet. We used aggregation-induced NMR resonance broadening to show that temperature, pH, and ionic strength influenced PF4(24)(-)(46) gelation rates. Under identical solution conditions, gel formation took days at T/= 20 degrees C but only 30 min at T/= 50 degrees C. Gelation was most rapid at pH values near the pK(a) of the central His35 residue. Increases in solution ionic strength reduced the critical gelation concentration of PF4(24)(-)(46). Our results suggest that PF4(24)(-)(46) gels by a process combining aspects of both heat-set and beta-fibril gelation mechanisms.

Bacterial resistance to conventional antibiotics is an ever increasing problem in the treatment of infectious disease. The basic problem is that conventional antibiotics, which generally operate by inhibiting some bacterial enzyme(s), can be rendered ineffective by overuse, natural selection and induced bacterial mutations. A novel class of antibiotics that functions by disrupting the superstructure of bacterial membranes rather than by inhibiting any specific enzyme has presented itself as the future for antibiotics. Most of these bactericidal agents are peptides or peptide-based molecules that have been either isolated from natural sources or synthetically designed. Here we review recent research into the development of peptides as bacterial membrane-disintegrating agents.