Hydrogen peroxide generation of copper/ascorbate formulations on cotton: effect on antibacterial and fibroblast activity for wound healing application

Authors: Edwards, Judson - Vince; Prevost, Nicolette; Santiago Cintron, Michael; von Hoven, Terri; Condon, Brian; QURESHI, HUZAIFAH - Virginia Commonwealth University; YAGER, DORNE - Virginia Commonwealth University

Submitted to: Molecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2018
Publication Date: 9/19/2018
Citation: Edwards, J.V., Prevost, N.T., Santiago Cintron, M., Von Hoven, T., Condon, B.D., Qureshi, H., Yager, D.R. 2018. Hydrogen peroxide generation of copper/ascorbate formulations on cotton: effect on antibacterial and fibroblast activity for wound healing application. Molecules. 23(9):1-16. https://doi.org/10.3390/molecules23092399.
DOI: https://doi.org/10.3390/molecules23092399

Interpretive Summary: For centuries bleached and scoured cotton has been employed in wound dressings. However the use of unbleached, greige cotton fibers in dressings, has been relatively unexplored. Wound dressings that generate low levels of H2O2 (5-50 micromolar) are thought to be advantageous to wound healing, i.e., low level generation of H2O2 (5-50 micromolar) is associated with enhanced cell signaling and proliferation in wounds. Whereas, hydrogen peroxide levels above 50 micromolar are considered bacteriostatic or antibacterial. Here the ability of nonwoven greige cotton to generate H2O2 at levels associated with enhanced cell proliferation and antibacterial activity is elucidated based on supplementation with copper and ascorbic acid. An assessment of both copper nanoparticles and ascorbic acid on cotton revealed that by simple addition of copper and ascorbic acid to unbleached cotton bacteriostatic activity is achievable. Moreover ascorbic acid alone combines with trace levels of copper in unbleached cotton to generate antibacterial levels of hydrogen peroxide. The formulations are discussed in light of current and past literature on the effect copper and hydrogen peroxide exert as antibacterial molecules and on fibroblasts. It was shown that copper nanoparticles synthesized on the cotton fibers using a simple technique do not leach from nonwoven greige cotton when subjected to aqueous conditions similar to a highly exudative wound. The work should advance a simple process for conferring antibacterial activity to cotton dressing made from unbleached cotton.

Technical Abstract: 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 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 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 milligrams copper per gram of cotton. The copper/ascorbate cotton analog formulations were characterized spectroscopically, copper titer quantified with ICP analysis and probed for peroxide production through assessment with amplex red. Hydrogen peroxide levels produced sustained hydrogen peroxide production. All analogs demonstrated antibacterial activity. Notably, treatment of unbleached cotton with low levels of ascorbate (~2mg/gram cotton) resulted in 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.