Nicolette Prevost

Kinetic and structural analysis of fluorescent peptides on cotton cellulose nanocrystals as elastase sensors

Human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) are serine proteases with destructive proteolytic activity. Because of this activity, there is considerable interest in elastase sensors. Herein we report the synthesis, characterization, and kinetic profiles of tri- and tetrapeptide substrates of elastase as glycine-esterified fluorescent analogs of cotton cellulose nanocrystals (CCN). The degree of substitution of peptide incorporated in CCN was 3-4 peptides per 100 anhydroglucose units. Glycine and peptide-cellulose-nanocrystals revealed crystallinity indices of 79 and 76%, respectively, and a crystallite size of 58.5 Å. A crystallite model of the peptide-cellulose conjugate is shown. The tripeptide conjugate of CCN demonstrated five-fold greater efficiency in HNE than the tripeptide in solution judged by its kcat/Km of 33,515. The sensor limits of detection at 2mg of the tri- and tetrapeptide CCN conjugates over a 10 min reaction time course were 0.03 U/mL PPE and 0.05 U/mL HNE, respectively.

Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing). Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze). A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration.

Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton

Nanocellulosic aerogels (NA) provide a lightweight biocompatible material with structural properties, like interconnected high porosity and specific surface area, suitable for biosensor design. We report here the preparation, characterization and activity of peptide-nanocellulose aerogels (PepNA) made from unprocessed cotton and designed with protease detection activity. Low-density cellulosic aerogels were prepared from greige cotton by employing calcium thiocyanate octahydrate/lithium chloride as a direct cellulose dissolving medium. Subsequent casting, coagulation, solvent exchange and supercritical carbon dioxide drying afforded homogeneous cellulose II aerogels of fibrous morphology. The cotton-based aerogel had a porosity of 99% largely dominated by mesopores (2–50 nm) and an internal surface of 163 m2·g−1. A fluorescent tripeptide-substrate (succinyl-alanine-proline-alanine-4-amino-7-methyl-coumarin) was tethered to NA by (1) esterification of cellulose C6 surface hydroxyl groups with glycidyl-fluorenylmethyloxycarbonyl (FMOC), (2) deprotection and (3) coupling of the immobilized glycine with the tripeptide. Characterization of the NA and PepNA included techniques, such as elemental analysis, mass spectral analysis, attenuated total reflectance infrared imaging, nitrogen adsorption, scanning electron microscopy and bioactivity studies. The degree of substitution of the peptide analog attached to the anhydroglucose units of PepNA was 0.015. The findings from mass spectral analysis and attenuated total reflectance infrared imaging indicated that the peptide substrate was immobilized on to the surface of the NA. Nitrogen adsorption revealed a high specific surface area and a highly porous system, which supports the open porous structure observed from scanning electron microscopy images. Bioactivity studies of PepNA revealed a detection sensitivity of 0.13 units/milliliter for human neutrophil elastase, a diagnostic biomarker for inflammatory diseases. The physical properties of the aerogel are suitable for interfacing with an intelligent protease sequestrant wound dressing.

Electrokinetic properties of functional layers in absorbent incontinence nonwoven products

Incontinence control through the use of well designed nonwoven materials is a rapidly growing area of interest. Analysis of the streaming zeta potential, absorbance capacity and moisture content measurements of absorbent layers in incontinence materials is a useful approach to evaluation and design. Using this approach, electrokinetic properties can be used to demonstrate the role of fiber surface polarity, swelling, and water uptake in the mechanism of incontinence control. By applying electrochemical double layer analysis to functional layers of absorbent incontinence products, the polar charge differences between cover stock, the acquisition/distribution layer (ADL) and the absorbent core were characterized. The aqueous fiber polarity is characterized from pH titration plots that give zeta plateau (ζplateau) values for each absorbent layer. The ζplateau value assigns the relative hydrophilic/hydrophobic (amphiphilic) character of the cover stock and ADL. Delta zeta (Δζ) and moisture content are applied to determine the functional value of fluid acquisition due to swelling and moisture absorption. Structure/function mechanisms are proposed for urine uptake relative to volume, pH and fluid transport in the cover stock and ADL of heavy, moderate, light incontinence pads and adult incontinence underwear. Using an electrokinetic analysis as a model to describe the mechanism of urine transport in absorbent incontinence materials makes possible the distinction of absorbent material design differences based on fiber charge, swelling, and absorption capacity. The electrokinetic model approach to absorbent incontinence material analysis and design is discussed for its potential applications.

Electrokinetic and Hemostatic Profiles of Nonwoven Cellulosic/Synthetic Fiber Blends with Unbleached Cotton

Greige cotton contains waxes and pectin on the outer surface of the fiber that are removed when bleached, but these components present potential wound dressing functionality. Cotton nonwovens blended with hydrophobic and hydrophilic fibers including viscose, polyester, and polypropylene were assessed for clotting activity with thromboelastography (TEG) and thrombin production. Clotting was evaluated based on TEG measurements: R (time to initiation of clot formation), K (time from end of R to a 20 mm clot), α (rate of clot formation according to the angle tangent to the curve as K is reached), and MA (clot strength). TEG values correlate to material surface polarity as measured with electrokinetic parameters (ζplateau, Δζ and swell ratio). The material surface polarity (ζplateau) varied from −22 to −61 mV. K values and thrombin concentrations were found to be inversely proportional to ζplateau with an increase in material hydrophobicity. An increase in the swell ratios of the materials correlated with decreased K values suggesting that clotting rates following fibrin formation increase with increasing material surface area due to swelling. Clot strength (MA) also increased with material hydrophobicity. Structure/function implications from the observed clotting physiology induced by the materials are discussed.

Electrokinetic analysis of hydroentangled greige cotton–synthetic fiber blends for absorbent technologies

Through nonwoven hydroentanglement of greige cotton blends with polyester and nylon, varying degrees of fiber surface polarity, swelling, and absorbance can be achieved. Electrokinetic properties of nonwoven blends made with Ultra Clean™ cotton (100% greige or virgin cotton) and polyester or nylon in 40:60 and 60:40 ratios demonstrated distinct differences in charge, swell, and per-cent moisture uptake capability. An electrochemical double layer analysis of charge based on a pH titration (pH 1.5–11 in 1 mM KCl) was employed to measure the relative fiber and fabric surface polarity (ζplateau), which ranged from −60 to −26 millivolts. A linear relationship of fiber swelling (Δζ) and per cent moisture content is apparent when greige cotton and synthetic fibers are blended. Water contact angles revealed that the cotton/synthetic fiber blends were hydrophobic (contact angle >80°) while retaining significant absorbency. The greige cotton/synthetic nonwoven materials, however, possess absorbent properties characterized by varying degrees of moisture uptake, fiber polarity, and swelling attributes similar to absorbent fluid transport materials present in the layers of incontinence products. Electrokinetic properties of the blended greige cotton/synthetic nonwovens are correlated to absorbent incontinence materials.

Induction of Low-Level Hydrogen Peroxide Generation by Unbleached Cotton Nonwovens as Potential Wound Dressing Materials

Greige cotton is an intact plant fiber. The cuticle and primary cell wall near the outer surface of the cotton fiber contains pectin, peroxidases, superoxide dismutase (SOD), and trace metals, which are associated with hydrogen peroxide (H2O2) generation during cotton fiber development. Traditionally, the processing of cotton into gauze involves scouring and bleaching processes that remove the components in the cuticle and primary cell wall. The use of unbleached, greige cotton fibers in dressings, has been relatively unexplored. We have recently determined that greige cotton can generate low levels of H2O2 (5–50 micromolar). Because this may provide advantages for the use of greige cotton-based wound dressings, we have begun to examine this in more detail. Both brown and white cotton varieties were examined in this study. Brown cotton was found to have a relatively higher hydrogen peroxide generation and demonstrated different capacities for H2O2 generation, varying from 1 to 35 micromolar. The H2O2 generation capacities of white and brown nonwoven greige cottons were also examined at different process stages with varying chronology and source parameters, from field to nonwoven fiber. The primary cell wall of nonwoven brown cotton appeared very intact, as observed by transmission electron microscopy, and possessed higher pectin levels. The levels of pectin, SOD, and polyphenolics, correlated with H2O2 generation.

Hydrogen Peroxide Generation of Copper/Ascorbate Formulations on Cotton: Effect on Antibacterial and Fibroblast Activity for Wound Healing Application

Greige cotton (unbleached cotton) is an intact plant fiber that retains much of the outer cotton fiber layers. These layers contain pectin, peroxidases, and trace metals that are associated with hydrogen peroxide (H2O2) generation during cotton fiber development. When greige cotton is subjected to a nonwoven hydroentanglement process, components of the outer cotton fiber layers are retained. When hydrated, this fabric can generate H2O2 (5–50 micromolar). This range has been characterized as inducing accelerated wound healing associated with enhanced cell signaling and the proliferation of cells vital to wound restoration. On the other hand, H2O2 levels above 50 micromolar have been associated with bacteriostatic activity. Here, we report the preparation and hydrogen peroxide activity of copper/ascorbate formulations, both as adsorbed and in situ synthesized analogs on cotton. The cooper/ascorbate-cotton formulations were designed with the goal of modulating hydrogen peroxide levels within functional ranges beneficial to wound healing. The cotton/copper formulation analogs were prepared on nonwoven unbleached cotton and characterized with cotton impregnation titers of 3–14 mg copper per gram of cotton. The copper/ascorbate cotton analog formulations were characterized spectroscopically, and the copper titer was quantified with ICP analysis and probed for peroxide production through assessment with Amplex Red. All analogs demonstrated antibacterial activity. Notably, the treatment of unbleached cotton with low levels of ascorbate (~2 mg/g cotton) resulted in a 99 percent reduction in Klebsiella pneumoniae and Staphylococcus aureus. In situ synthesized copper/ascorbate nanoparticles retained activity and did not leach out upon prolonged suspension in an aqueous environment. An assessment of H2O2 effects on fibroblast proliferation are discussed in light of the copper/cotton analogs and wound healing.

Physico- and bio-activities of nanoscale regenerated cellulose nonwoven immobilized with lysozyme

Lysozyme-cellulose conjugates are of wide interest for food packaging, tissue scaffolding, wound healing, and antimicrobial applications. Here a recycled cotton-based source of regenerated cellulose in combination with carboxylated carbon nanotubes and graphene oxide was configured as nonwoven nanofibrous mats through electrospinning and utilized to immobilize lysozyme. Scanning electron microscopy, Fourier transform-infrared spectra, thermal-gravimetric analysis, tensile test, and antibacterial assessments were conducted to characterize and determine physical and bioactive properties of the nonwoven nanofibrous mats. The resulted cellulose-lysozyme conjugates were found to have robust bioactivity with no indication of cell cytotoxicity. The study confirmed that the carbon-nanoparticle-modified cellulose nonwoven mats revealed a high antimicrobial activity after immobilization of lysozyme.