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.

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.

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.