In vitro and in vivo characterization of novel biodegradable polymers for application as drug-eluting stent coatings.

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.

Influence of surfactant tail branching and organization on the orientation of liquid crystals at aqueous-liquid crystal interfaces.

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.

Promotion of peptide antimicrobial activity by fatty acid conjugation.

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.

Acylation of SC4 dodecapeptide increases bactericidal potency against Gram-positive bacteria, including drug-resistant strains.

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.

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban.

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 (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-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 alpha-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 PLB(1-25)K 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 PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K 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.

Beta-sheet is the bioactive conformation of the anti-angiogenic anginex peptide.

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 beta-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 beta-sheet is in fact the bioactive conformation of anginex. CD and NMR spectral analysis of the analogues indicate formation of a beta-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 beta-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.

Aqueous gel formation of a synthetic peptide derived from the beta-sheet domain of platelet factor-4.

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 /= 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.