Assessment of Cellulose Nanofiber-Based Elastase Biosensors to Inflammatory Disease as a Function of Spacer Length and Fluorescence Response.

Inflammatory disease biomarker detection has become a high priority in point-of-care diagnostic research in relation to chronic wounds, with a variety of sensor-based designs becoming available. Herein, two primary aspects of biosensor design are examined: (1) assessment of a cellulose nanofiber (CNF) matrix derived from cotton ginning byproducts as a sensor transducer surface; and (2) assessment of the relation of spacer length and morphology between the CNF cellulose backbone and peptide fluorophore as a function of sensor activity for porcine pancreatic and human neutrophil elastases. X-ray crystallography, specific surface area, and pore size analyses confirmed the suitability of CNF as a matrix for wound care diagnostics. Based upon the normalized degree of substitution, a pegylated-linker connecting CNF transducer substrate to peptide fluorophore showed the greatest fluorescence response, compared to short- and long-chain alkylated linkers.

Preparation and Activity of Hemostatic and Antibacterial Dressings with Greige Cotton/Zeolite Formularies Having Silver and Ascorbic Acid Finishes.

The need for prehospital hemostatic dressings that exert an antibacterial effect is of interest for prolonged field care. Here, we consider a series of antibacterial and zeolite formulary treatment approaches applied to a cotton-based dressing. The design of the fabric formulations was based on the hemostatic dressing TACGauze with zeolite Y incorporated as a procoagulant with calcium and pectin to facilitate fiber adherence utilizing silver nanoparticles, and cellulose-crosslinked ascorbic acid to confer antibacterial activity. Infra-red spectra were employed to characterize the chemical modifications on the dressings. Contact angle measurements were employed to document the surface hydrophobicity of the cotton fabric which plays a role in the contact activation of the coagulation cascade. Ammonium Y zeolite-treated dressings initiated fibrin equal to the accepted standard hemorrhage control dressing and showed similar improvement with antibacterial finishes. The antibacterial activity of cotton-based technology utilizing both citrate-linked ascorbate-cellulose conjugate analogs and silver nanoparticle-embedded cotton fibers was observed against Staphylococcus aureus and Klebsiella pneumoniae at a level of 99.99 percent in the AATCC 100 assay. The hydrogen peroxide levels of the ascorbic acid-based fabrics, measured over a time period from zero up to forty-eight hours, were in line with the antibacterial activities.

A Comparison of Hemostatic Activities of Zeolite-Based Formulary Finishes on Cotton Dressings.

The need for affordable effective prehospital hemostatic dressings to control hemorrhage has led to an increased interest in new dressing design approaches. Here we consider the separate components of fabric, fiber, and procoagulant nonexothermic zeolite-based formulations on design approaches to accelerated hemostasis. The design of the fabric formulations was based on incorporation of zeolite Y as the principal procoagulant, with calcium and pectin to adhere and enhance the activity. Unbleached nonwoven cotton when combined with bleached cotton displays enhanced properties related to hemostasis. Here, we compare sodium zeolite with ammonium zeolite formulated on fabrics utilizing pectin with pad versus spray-dry-cure and varied fiber compositions. Notably, ammonium as a counterion resulted in shorter times to fibrin and clot formation comparable to the procoagulant standard. The time to fibrin formation as measured by thromboelastography was found to be within a range consistent with modulating severe hemorrhage control. The results indicate a correlation between fabric add-on and accelerated clotting as measured by both time to fibrin and clot formation. A comparison between the time to fibrin formation in calcium/pectin formulations and pectin alone revealed an enhanced clotting effect with calcium decreasing by one minute the time to fibrin formation. Infra-red spectra were employed to characterize and quantify the zeolite formulations on the dressings.

Ascorbic Acid as an Adjuvant to Unbleached Cotton Promotes Antimicrobial Activity in Spunlace Nonwovens.

The development of affordable, effective, and environmentally friendly barrier fabrics is a current goal in antimicrobial textile development. The discovery of new routes to achieve non-toxic naturally occurring molecules with antimicrobial activity is of interest in the development of materials that promote wound healing, improve hygiene, and offer protection against nosocomial infection. Highly cleaned and sterile unbleached cotton has constituents that produce hydrogen peroxide at levels commensurate with those that favor cell signaling in wound healing. Here, we show the antimicrobial and antiviral properties of spunlaced griege cotton-containing nonwovens treated with ascorbic acid formulations. The mechanism of action occurs through the promotion of enhanced hydrogen peroxide activity. The levels of hydrogen peroxide activity afford antimicrobial activity against Gram-negative and Gram-positive bacteria and antiviral activity against MS2 bacteriophages. Spun-bond nonwoven unbleached cotton was treated with ascorbic acid using traditional pad-dry-cure methods. An assessment of antibacterial and antiviral activity against Staphylococcus aureus, Klebsiella pneumoniae, and MS2 bacteriophages with the AATCC 100 test method showed a 99.99% inhibitory activity. An approach to the covalent attachment of ascorbic to cellulose through citric acid crosslinking chemistry is also discussed. Thus, a simple, low-cost approach to antimicrobial and antiviral cotton-based nonwovens applicable to dressings, nosocomial barrier fabrics, and face masks can be adopted by combining ascorbic acid with spunlace greige cotton nonwoven fabrics.

Detection of Human Neutrophil Elastase by Fluorescent Peptide Sensors Conjugated to TEMPO-Oxidized Nanofibrillated Cellulose.

Peptide-cellulose conjugates designed for use as optical protease sensors have gained interest for point-of-care (POC) detection. Elevated serine protease levels are often found in patients with chronic illnesses, necessitating optimal biosensor design for POC assessment. Nanocellulose provides a platform for protease sensors as a transducer surface, and the employment of nanocellulose in this capacity combines its biocompatibility and high specific surface area properties to confer sensitive detection of dilute biomarkers. However, a basic understanding of the spatiotemporal relationships of the transducer surface and sensor disposition is needed to improve protease sensor design and development. Here, we examine a tripeptide, fluorogenic elastase biosensor attached to TEMPO-oxidized nanofibrillated cellulose via a polyethylene glycol linker. The synthetic conjugate was found to be active in the presence of human neutrophil elastase at levels comparable to other cellulose-based biosensors. Computational models examined the relationship of the sensor molecule to the transducer surface. The results illustrate differences in two crystallite transducer surfaces ((110) vs. (1-10)) and reveal preferred orientations of the sensor. Finally, a determination of the relative (110) vs. (1-10) orientations of crystals extracted from cotton demonstrates a preference for the (1-10) conformer. This model study potentiates the HNE sensor results for enhanced sensor activity design.

Comparison of cellooligosaccharide conformations in complexes with proteins with energy maps for cellobiose.

Shapes (conformations) of cellulose molecules are described by their glycosidic linkage torsion angles ϕ and ψ. Although the torsions are known for cellulose in crystals, amorphous shapes are also interesting for understanding reactivity and physical properties. ϕ and ψ determination for unorganized matter is difficult; one approach is to study their range in many related molecules. For example, linkage torsions of cellulose should be similar to those in cellobiose. Herein, torsions were measured for cellooligosaccharides and lactose moieties complexed with proteins in the Protein Data Bank (PDB). These torsions were compared with ϕ/ψ maps based on quantum mechanics energies for solvated cellobiose and analogs lacking hydroxyl groups. Most PDB conformations corresponded to low map energies. Amorphous cellulose should be generally extended with individual linkages that would give 2- to 3-fold helices. The map for an analog lacking hydrogen bonding ability was more predictive for PDB linkages than the cellobiose map.

Antimicrobial and Hemostatic Activities of Cotton-Based Dressings Designed to Address Prolonged Field Care Applications.

INTRODUCTION: Developing affordable and effective hemostatic and antimicrobial wound dressings for prolonged field care (PFC) of open wounds is of interest to prevent infection, to prevent sepsis, and to conserve tissue viability. The need for an effective hemostatic dressing that is also antimicrobial is required of a hemostatic dressing that can be left in place for extended periods (days). This is particularly important in light of the existence of pathogens that have coagulopathy properties. Thus, dressings that provide effective hemostasis and reduction in the frequency of dressing changes, whereas exerting robust antimicrobial activity are of interest for PFC. Highly cleaned and sterile unbleached cotton has constituents not found in bleached cotton that are beneficial to the hemostatic and inflammatory stages of wound healing. Here, we demonstrate two approaches to cotton-based antimicrobial dressings that utilize the unique components of the cotton fiber with simple modification to confer a high degree of hemostatic and antimicrobial efficacy. METHODS: Spun bond nonwoven unbleached cotton was treated using traditional pad dry cure methods to add ascorbic acid, zeolite (NaY) with pectin, calcium chloride, and sodium carbonate/calcium chloride. Similarly, nanosilver-embedded cotton fiber was blended with pristine cotton fibers at various weight ratios to produce hydroentangled nonwoven fabrics. The resulting treated fabrics were assessed for hemostasis using thromboelastographic clotting assays and antimicrobial activity utilizing American Association of Textile Chemists and Colorists 100. RESULTS: Zeolite-containing dressings possessed significant hemostatic activity, whereas ascorbic acid- and silver-containing dressings reduced Gram-positive and Gram-negative organism numbers by several logs. CONCLUSION: Based on this study, a multilayered hemostatic dressing with antimicrobial properties is envisioned. This dressing would be safe, would be economical, and have a stable shelf-life that would be conducive for using PFC.

Clinical Translational Potential in Skin Wound Regeneration for Adipose-Derived, Blood-Derived, and Cellulose Materials: Cells, Exosomes, and Hydrogels.

Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.

Development of a Nonwoven Hemostatic Dressing Based on Unbleached Cotton: A De Novo Design Approach.

Minimally processed greige (unbleached) cotton fibers demonstrate enhanced clotting relative to highly processed United States Pharmacopeia (USP) type 7 bleached cotton gauze. This effect is thought to be due to the material surface polarity. We hypothesized that a textile could be constructed, conserving the hemostasis-accelerating properties of greige cotton, while maintaining structural integrity and improving absorbance. Spun bond nonwovens of varying surface polarity were designed and prepared based on ratios of greige cotton/bleached cotton/polypropylene fibers. A thromboelastographic analysis was performed on fibrous samples in citrated blood to evaluate the rate of fibrin and clot formation. Lee White clotting times were obtained to assess the material's clotting activity in platelet fresh blood. An electrokinetic analysis of samples was performed to analyze for material surface polarity. Hemostatic properties varied with composition ratios, fiber density, and fabric fenestration. The determinations of the surface polarity of cotton fabrics with electrokinetic analysis uncovered a range of surface polarities implicated in fabric-initiated clotting; a three-point design approach was employed with the combined use of thromboelastography, thrombin velocity index, Lee White clotting, and absorption capacity determinations applied to fabric structure versus function analysis. The resulting analysis demonstrates that greige cotton may be utilized, along with hydrophilic and hydrophobic fibers, to improve the initiation of fibrin formation and a decrease in clotting time in hemostatic dressings suitable to be commercially developed. Hydroentanglement is an efficient and effective process for imparting structural integrity to cotton-based textiles, while conserving hemostatic function.

Nanocellulose as a colorimetric biosensor for effective and facile detection of human neutrophil elastase.

Nanocellulose has functionalities suitable for efficient sensor transducer surface design including crystallinity, biocompatible and high specific surface area. Here we explore two forms of nanocellulose as transducer surfaces to enable colorimetric detection of human neutrophil elastase (HNE), and a wide range of inflammatory diseases. A deep eutectic solvent (DES) was utilized to mediate formation of cotton cellulose nanocrystals (DCNCs) employed to prepare a peptide-cellulose conjugate as a protease sensor of HNE. The tetrapeptide-cellulose analog on DCNC is contrasted with an analogous derivative of TEMPO-oxidized wood cellulose nanofibrils (WCNFs). DCNCs showed greater degree of substitution of HNE tetrapeptide and sensitivity to the elastase than WCNFs, despite the smaller surface area and pore sizes. XRD models revealed the higher crystallinity and larger crystallite sizes of DCNCs, indicating the well-arranged cellulose chains for immobilization of the tetrapeptide on (110) lattice reflections of cellulose crystals. The sensitivity of DCNCs-based colorimetric sensor was less than 0.005 U/mL, which would provide a convenient, sensitive sensor applicable for improved colorimetric point of care protease biomarker detection.

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.

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.

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.

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.

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  Î¶plateau 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.

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.