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.

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.

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.

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.

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 (ζ_plateau, Δζ and swell ratio). The material surface polarity (ζ_plateau) varied from −22 to −61 mV. K values and thrombin concentrations were found to be inversely proportional to ζ_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.