Identifying the selectivity of antimicrobial peptides to cell membranes by sum frequency generation spectroscopy.

Cationic amphiphilic peptides have been engineered to target both Gram-positive and Gram-negative bacteria while avoiding damage to other cell types. However, the exact mechanism of how these peptides target, bind, and disrupt bacterial cell membranes is not understood. One specific peptide that has been engineered to selectively capture bacteria is WLBU2 (sequence: RRWVRRVRRWVRRVVRVVRRWVRR).

Sequential and competitive adsorption of peptides at pendant PEO layers.

Earlier work provided direction for development of responsive drug delivery systems based on modulation of the structure, amphiphilicity, and surface density of bioactive peptides entrapped within pendant polyethylene oxide (PEO) brush layers. In this work, we describe the sequential and competitive adsorption behavior of such peptides at pendant PEO layers. Three cationic peptides were used for this purpose: the arginine-rich, amphiphilic peptide WLBU2, a peptide chemically identical to WLBU2 but of scrambled sequence (S-WLBU2), and the non-amphiphilic peptide poly-L-arginine (PLR).

Activity retention after nisin entrapment in a polyethylene oxide brush layer.

The cationic, amphiphilic peptide nisin is an effective inhibitor of gram-positive bacteria whose mode of action does not encourage pathogenic resistance, and its proper incorporation into food packaging could enhance food stability, safety, and quality in a number of circumstances. Sufficiently small peptides have been shown to integrate into otherwise nonfouling polyethylene oxide (PEO) brush layers in accordance with their amphiphilicity and ordered structure, including nisin, and we have recently shown that nisin entrapment within a PEO layer does not compromise the nonfouling character of that layer. In this work we test the hypothesis that surface-bound, pendant PEO chains will inhibit displacement of entrapped nisin by competing proteins and, in this way, prolong retention of nisin activity at the interface.

Binding interactions of bacterial lipopolysaccharide and the cationic amphiphilic peptides polymyxin B and WLBU2.

Passage of blood through a sorbent device for removal of bacteria and endotoxin by specific binding with immobilized, membrane-active, bactericidal peptides holds promise for treating severe blood infections. Peptide insertion in the target membrane and rapid/strong binding is desirable, while membrane disruption and release of degradation products to the circulating blood is not. Here we describe interactions between bacterial endotoxin (lipopolysaccharide, LPS) and the membrane-active, bactericidal peptides WLBU2 and polymyxin B (PmB).

Concentration effects on peptide elution from pendant PEO layers.

In earlier work, we have provided direction for development of responsive drug delivery systems based on modulation of structure and amphiphilicity of bioactive peptides entrapped within pendant polyethylene oxide (PEO) brush layers. Amphiphilicity promotes retention of the peptides within the hydrophobic inner region of the PEO brush layer. In this work, we describe the effects of peptide surface density on the conformational changes caused by peptide-peptide interactions, and show that this phenomenon substantially affects the rate and extent of peptide elution from PEO brush layers.

Blood protein repulsion after peptide entrapment in pendant polyethylene oxide layers.

A number of sufficiently small peptides have been shown to integrate into polyethylene oxide (PEO) brush layers in accordance with their amphiphilicity and ordered structure. Those results have suggested that responsive drug delivery systems based on peptide-loaded PEO layers can be controlled by modulation of solution conditions and peptide amphiphilicity. However, the presence of entrapped peptide may compromise the protein repulsive character of the PEO layer, and in this way reduce the viability of a medical device coating based on such an approach.

Preparation and evaluation of PEO-coated materials for a microchannel hemodialyzer.

The marked increase in surface-to-volume ratio associated with microscale devices for hemodialysis leads to problems with hemocompatibility and blood flow distribution that are more challenging to manage than those encountered at the conventional scale. In this work stable surface modifications with pendant polyethylene oxide (PEO) chains were produced on polydimethylsiloxane (PDMS), polycarbonate microchannel, and polyacrylonitrile membrane materials used in construction of microchannel hemodialyzer test articles. PEO layers were prepared by radiolytic grafting of PEO-polybutadiene-PEO (PEO-PB-PEO) triblock polymers to the material surfaces.

Adsorption, structural alteration and elution of peptides at pendant PEO layers.

An experimentally based, quantitative understanding of the entrapment and function of small peptides within PEO brush layers does not currently exist. Earlier work provided a rationale for expecting that an ordered, compact peptide will enter the PEO phase more readily than a peptide of similar size that adopts a less ordered, less compact form, and that amphiphilicity will promote peptide retention within the hydrophobic region of the PEO brush. Here we more deliberately describe criteria for peptide integration and structural change within the PEO brush, and discuss the reversibility of peptide entrapment with changing solvent conditions.

Quantifying nisin adsorption behavior at pendant PEO layers.

The antimicrobial peptide nisin shows potent activity against Gram-positive bacteria including the most prevalent implant-associated pathogens. Its mechanism of action minimizes the opportunity for the rise of resistant bacteria and it does not appear to be toxic to humans, suggesting good potential for its use in antibacterial coatings for selected medical devices. A more quantitative understanding of nisin loading and release from polyethylene oxide (PEO) brush layers will inform new strategies for drug storage and delivery, and in this work optical waveguide lightmode spectroscopy was used to record changes in adsorbed mass during cyclic adsorption-elution experiments with nisin, at uncoated and PEO-coated surfaces.

Structural attributes affecting peptide entrapment in PEO brush layers.

A more quantitative understanding of peptide loading and release from polyethylene oxide (PEO) brush layers will provide direction for development of new strategies for drug storage and delivery. In this work we recorded selected effects of peptide structure and amphiphilicity on adsorption into PEO brush layers based on covalently stabilized Pluronic(®)F 108. Optical waveguide lightmode spectroscopy and circular dichroism measurements were used to characterize the adsorption of poly-l-glutamic acid, poly-l-lysine, and the cationic amphiphilic peptide WLBU2, to the brush layers.

Molecular origins of surfactant-mediated stabilization of protein drugs.

Loss of activity through aggregation and surface-induced denaturation is a significant problem in the production, formulation and administration of therapeutic proteins. Surfactants are commonly used in upstream and downstream processing and drug formulation. However, the effectiveness of a surfactant strongly depends on its mechanism(s) of action and properties of the protein and interfaces.

Detection of nisin and fibrinogen adsorption on poly(ethylene oxide) coated polyurethane surfaces by time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Stable, pendant polyethylene oxide (PEO) layers were formed on medical-grade Pellethane® and Tygon® polyurethane surfaces, by adsorption and gamma-irradiation of PEO-polybutadiene-PEO triblock surfactants. Coated and uncoated polyurethanes were challenged individually or sequentially with nisin (a small polypeptide with antimicrobial activity) and/or fibrinogen, and then analyzed with time-of-flight secondary ion mass spectrometry (TOF-SIMS). Data reduction by robust principal components analysis (PCA) allowed detection of outliers, and distinguished adsorbed nisin and fibrinogen.

A novel enzymatic microreactor with Aspergillus oryzae β-galactosidase immobilized on silicon dioxide nanosprings.

The use of silicon dioxide (SiO(2) ) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm(2) × 60 μm thick) was functionalized with γ-aminopropyltriethoxysilane, then treated with N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (--SH) groups.

Synthesis and evaluation of heparin immobilized "side-on" to polystyrene microspheres coated with end-group activated polyethylene oxide.

Thiol (-SH) groups were introduced into unfractionated heparin by reaction of carboxyl groups in its uronic acid residues with 3,3'dithiobis(propanoic)hydrazide. Thiolated heparin derivatives were then linked to pyridyl disulfide-activated polyethylene oxide-polypropylene oxide-polyethylene oxide triblocks, which had previously been coated onto the surfaces of 1.15 microm polystyrene microspheres.

Nisin adsorption to polyethylene oxide layers and its resistance to elution in the presence of fibrinogen.

The adsorption and elution of the antimicrobial peptide nisin at silanized silica surfaces coated to present pendant polyethylene oxide chains was detected in situ by zeta potential measurements. Silica microspheres were treated with trichlorovinylsilane to introduce hydrophobic vinyl groups, followed by self assembly of the polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock surfactant Pluronic F108, or an F108 derivative with nitrilotriacetic acid end groups. Triblock-coated microspheres were gamma-irradiated to covalently stabilize the PPO-surface association.

Synthesis and anticoagulant activity of heparin immobilized "end-on" to polystyrene microspheres coated with end-group activated polyethylene oxide.

Thiol groups were introduced to unfractionated heparin (UFH) and end-aminated heparin (HepNH(2)) by reaction with 2-iminothiolane under conditions favoring selective modification of terminal over primary amines. End-thiolated heparin retained anticoagulant activity as shown by the activated partial thromboplastin time (aPTT) and anti-Factor Xa (anti-FXa) assays. Thiolated heparins were linked to pyridyl-disulfide activated poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers adsorbed to 1.

Activation of immobilized lipase in non-aqueous systems by hydrophobic poly-DL-tryptophan tethers.

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule ("tether") between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most "long" linkers (e.