Abstract
In chronic wounds, elevated human neutrophil elastase (HNE) is a destructive protease that has been proposed as a biomarker. Numerous wound dressing designs have been introduced in an effort to lower HNE levels. The clinical detection of HNE as a point of care biomarker or an in situ colorimetric adjuvant to chronic wound dressings presents potential advantages in the management of chronic wounds. A colorimetric approach to the detection of HNE using peptide conjugated cotton cellulose nanocrystals (CCN) is reported here. For this purpose a HNE tripeptide substrate, n-Succinyl-Alanine–Alanine-Valine-para-nitroanilide (Suc-Ala–Ala-Val-pNA), was covalently attached to glycine esterified CCN and compared with a similar tetrapeptide analog for colorimetric HNE sensor activity. Visible HNE activity was significantly higher on CCN tripeptide conjugates when compared with similar analogs synthesized on paper. Upon enzymatic release of para-nitroaniline (pNA) from the Glycine-CCN conjugate of succinyl-Ala–Ala-Val-pNA, amplification of the colorimetric response from pNA with reactive dyes enhanced visible absorption of the chromogen. Two color amplifying dyes that react with pNA were compared for their ability to enhance the visual sensor response to HNE activity. The colorimetric detection of HNE with CCN tripeptide conjugates was sensitive at HNE levels previously reported in chronic wound fluid (0.05 U/mL HNE). The HNE sensor and the chromogen amplifying dyes were interfaced with 50 and 10 kD dialysis cellulose membranes (DCM) to model filtration of HNE and chromogen (pNA) from a model wound dressing surface before and after sensor reactivity. The detection sensitivity to HNE activity was assessed with the CCN-tripeptide conjugate interfaced at the DCM surface distal and proximal to a dressing surface. The HNE sensor interfaced proximal to the dressing surface was most efficient with 10 kD membrane filtration of pNA and subsequent reaction with amplifying dyes. When interfaced with the 10 kD cellulose membrane, elastase sensor activity remained sensitive to 0.05 U/mL HNE. The nanocellulose surface properties, performance and design issues of the biosensor approach are discussed.
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References
Adhiraan N, Shanmugasundaram N, Babu M (2007) Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: development and characterization. J Microencapsul 24:659–671
Araujo AC, Song Y, Lundeberg J, Stahl PL, Brumer H (2012) Activated paper surfaces for the rapid hybridization of DNA through capillary transport. Anal Chem 84:3311–3317
Ballerstadt R, Schultz J (1996) Sensor methods for use with microdialysis and ultrafiltration. Adv Drug Deliv Rev 21:225–238
Barazzouk S, Daneault C (2012) Tryptophan-based peptides grafted onto oxidized nanocellulose. Cellulose 19:481–493
Barros SC, Martins JA, Marcos JC, Cavaco-Paulo A (2012) Characterization of potential elastase inhibitor-peptides regulated by a molecular switch for wound dressing applications. Enzym Microb Technol 50:107–114
Blackwell HE (2006) Hitting the SPOT: small-molecule macroarrays. Adv Comb Synth 10:203–212
Cheng CM, Martinez AW, Gong J, Mace CR, Phillips ST, Carrilho E, Mirica KA, Whitesides GM (2010) Paper-based Elisa. Angew Chem Int Ed 49:4771–4774
Clark RAF (1996) Wound repair: overview and general considerations. In: Richard AFC (ed) The molecular and cellular biology of wound repair. Plenum, New York, pp 3–35
Cullen B, Smith R, McCulloch E, Silcock D, Morrison L (2002) Mechanism of action of PROMOGRAN, a protease modulating matrix, for the treatment of diabetic foot ulcers. Wound Reg Rep 10:16–25
Dargaville TR, Farrugia BL, Broadbent JA, Pace S, Upton Z (2013) Sensors and imaging for wound healing: a review. Biosens Bioelectron 41:30–42
Das G, Talukdar P, Matile S (2002) Fluorometric detection of enzyme activity with synthetic supramolecular pores. Science 298:1600–1602
Diegelmann RF (2003) Excessive neutrophils characterize chronic pressure ulcers. Wound Repair Regen 11:490–495
Dufresne A (2008) Polysaccharide nanocrystal reinforced nanocomposites. Can J Chem 86:484–494
Edwards JV, Batiste SL, Gibbins EM, Goheen SC (1999a) Synthesis and activity of NH2- and COOH- terminal elastase recognition sequences on cotton. J Pept Res 54:536–543
Edwards JV, Bopp AF, Batiste S, Ullah AJ, Cohen IK, Diegelmann RF, Montante SJ (1999b) Inhibition of elastase by synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release. Wound Rep Reg 7:106–118
Edwards JV, Yager DR, Cohen IK, Diegelmann RF, Montante S, Bertoniere N, Bopp AF (2001) Modified cotton gauze dressings that selectively absorb neutrophil elastase activity in solution. Wound Rep Reg 9:50–58
Edwards JV, Bopp AF, Batiste SL, Goynes WR (2003) Human neutrophil elastase inhibition with a novel cotton-alginate wound dressing formulation. J Biomed Mat Res (A) 66:433–440
Edwards JV, Caston-Pierre S, Bopp AF, Goynes W (2005) Detection of human neutrophil elastase with peptide-bound cross-linked ethoxylate acrylate resin analogs. J Pept Res 66:160–168
Edwards JV, Howley P, Davis R, Mashchak A, Goheen SC (2007) Protease inhibition by oleic acid transfer from chronic wound dressing to albumin. Int J Pharm 340:42–51
Edwards JV, Caston-Pierre S, Howley P, Condon B, Arnold J (2008) A bio-sensor for human neutrophil elastase employs peptide-para-nitroanilide cellulose conjugates. Sens Lett 6:518–523
Edwards JV, Prevost NT, Condon B, French A, Wu Q (2012) Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose I and II cotton nanocrystalline preparations. Cellulose 19:495–506
Eming SA, Smola-Hess S, Kurschat P, Hirche D, Krieg T, Smola H (2006) A novel property of povidon-iodine: inhibition of excessive protease levels in chronic non-healing wounds. J Invest Dermatol 126:2731–2733
Eyley S, Thielemans W (2011) Imidazolium grafted cellulose nanocrystals for ion exchange applications. Chem Comm 47:4177–4179
Fleming K, Gray DG, Matthews S (2001) Cellulose crystallites. Chem Eur J 7:1831–1835
Friedman RB, Kwaan HC, Szczecinski M (1977) Improved sensitivity of chromogenic substrate assays for urokinase and plasmin. Thromb Res 12:37–46
Grinnell F, Zhu M (1994) Identification of neutrophil elastase as the proteinase in burn wound fluid responsible for the degradation of fibronectin. J Invest Dermatol 103:155–161
Grinnell F, Zhu M (1996) Fibronectin degradation in chronic wounds depends on the relative levels of elastase, 1-proteinase inhibitor and 2-macroglobulin. J Invest Dermatol 106:335–341
Habibi Y, Chanzy H, Vignon MR (2006) Tempo-mediated surface oxidation of cellulose whiskers. Cellulose 13:679–687
Habibi Y, Lucia LA, Rojas OJ (2010) Cellulose nanocrystals: chemistry, self-assembly, and applications. Chem Rev 110:3479–3500
Hasmann A, Gewessler U, Hulla E, Schneider KP, Binder B, Francesko A, Tzanov T, Schintler M, Palen JV, Guebitz GM, Wehrschuetz-Sigl E (2011) Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid. Exp Dermatol 20:508–513
He J-L, Wu Z-S, Zhang S-B, Shen G-L, Yu R-Q (2010) Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection. Talanta 80:1264–1268
Henares M, Takashi N, Yoshida S, Terabe F, Mizutani R, Sikizawa R, Hisamoto H (2007) Integration of multianalyte sensing function on a capillary-assembled microchip: simultaneous determination of ion concentration and enzymatic activities by “drop-and-sip” technique. Anal Chem 79:908–915
Herrick SE, Sloan P, McGurk MM, Freak L, McCollum CN, Ferguson MW (1992) Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers. Am J Pathol 141:1085–1095
Herrick S, Ashcroft G, Ireland G, Horan M, McCollum C, Ferguson M (1997) Up-regulation of elastase in acute wounds of healthy aged humans and chronic venous leg ulcers are associate with matrix degradation. Lab Investig 77:281–288
Hofer HP, Kukovetz E, Petek W, Schweighofer F, Wildburger R, Schaur RJ (1996) Released PMN elastase: an indicator of postsurgical uneventful wound healing and early inflammatory complications. A contribution to the search for objective criteria in wound healing monitoring. Injury 26:103–106
International Consensus (2011) The role of proteases in wound diagnostics. An expert working review. Wounds International, London
James TJ, Hughes MA, Cherry GW, Taylor PT (2003) Simple biochemical markers to assess chronic wounds. Wound Rep Reg 11:172–176
Klemm D, Kramer F, Moritz S, Lindstrom T, Ankerfors M, Gray D, Dorris A (2011) Nanocelluloses: a new family of nature-based materials. Angew Chem Int Ed 50:5438–5466
Kwaan HC, Friedmann RB, Szczecinski M (1978) Amplification of color yield of chromogenic substrates using p-dimethylaminocinnamaldehyde. Thromb Res 13:5–13
Lam EL, Male KB, Male Chong JH, Leung ACW, Loung JHT (2012) Applications of functionalized and nanoparticle-modified nanocrystalline cellulose. Trends Biotechnol 30:283–290
Leetsma-Vogt G, Rhemrev-Boom MM, Tissen RG, Venema K, Korf J (2004) The potential of biosensor technology in clinical monitoring and experimental research. Biomed Mater Eng 14:455–464
Meyer-Ingold W, Eichner W, Ettner N, Schink M (2000) Wound coverings for removal of interfering factors from wound fluid. United States Patent 6,156,334
Mihranyan A, Nyholm L, Bennett AEG, Stromme M (2008) A novel high specific surface area conducting paper material composed of polypyrrole and Cladophora cellulose. J Phys Chem B 112:12249–12255
Pelton R (2009) Bioactive paper provides a low-cost platform for diagnostics. Trends Anal Chem 28:925–942
Ramanathan M, Luckarift HR, Sarsenova A, Wild JR, Ramanculov EK, Olsen EV, Simonian AL (2009) Lysozyme-mediated formation of protein-silica nano-composites for biosensing applications. Colloids Surf B Biointerfaces 73:58–64
Rayment EA, Dargaville TR, Shooter GK, George GA, Upton Z (2008) Attenuation of protease activity in chronic wound fluid with bisphosphonate-functionalized hydrogels. Biomaterials 29:1785–1795
Samir MASA, Alloin F, Sanchez JY, Kissi NE, Dufresne A (2004) Preparation of cellulose whisker reinforced nanocomposites from an organic medium suspension. Macromolecules 37:1386–1393
Samir MASA, Alloin F, Dufresne A (2005) Review of recent research into cellulose whiskers, their properties and their application in nanocomposite field. Biomacromolecules 6:612–626
Schultz GS, Ladwig G, Wysocki A (2005) Extracellular matrix: review of its roles in acute and chronic wounds. http://www.worldwidewounds.com/2005/August/Schultz/ Extrace-Matric-Acute-Chronic-Wounds.html, World Wide Wounds, p 13
Shen B, Shimmon S, Smith MM, Ghosh PJ (2003) Biosensor analysis of the molecular interactions of pentosan polysulfate and of sulfated glycosaminoglycans with immobilized elastase, hyaluronidase and lysozyme using surface plasmon resonance technology. Pharm Biomed Anal 31:83–93
Siqueira G, Bras J, Dufresne A (2010) New process of chemical grafting of cellulose nanoparticles with a long chain isocyanate. Langmuir 26:402–411
Siro I, Plackett D (2010) Microfibrillated cellulose and new nanocomposite materials: a review. Cellulose 17:459–494
Sjo P (2012) Neutrophil elastase inhibitors: recent advances in the development of mechanism-based and nonelectrophilic inhibitors. Future Med Chem 4:651–660
Sorde N, Das G, Matile S (2003) Enzyme screening with synthetic multifunctional pores: focus on biopolymers. Proc Natl Acad Sci USA 100:11964–11969
Stair JL, Watkinson M, Krause S (2009) Sensor materials for the detection of proteases. Biosens Bioelectron 24:2113–2118
Steuerwald AJ, Villeneuve JD, Sun L, Stenken JA (2006) In vitro characterization of an in situ microdialysis sampling assay for elastase activity detection. J Pharm Biomed Anal 40:1041–1047
Subiros-Funosas R, Prohens R Barbas R, El-Faham A, Albericio F (2009) Oxyma: an efficient additive for peptide synthesis to replace the benzotriazole-based HOBt and HOAt with a lower risk of explosion. Chem Eur J 15:9394–9403
Touzinsky GF, Gordon SH (1979) Degree of substitution of cellulose derivatives containing n different substituent groups. Carbohydr Res 69:327–329
Trejo-‘O’reilly JA, Cavaille JY, Gandini A (1997) The surface chemical modification of cellulosic fibres in view of their use in composite materials. Cellulose 4:305–320
Trengrove NJ, Stacey MC, Macauley S, Bennett N, Gibson F, Burslem F, Murphy G, Schultz G (1999) Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers. Wound Rep Reg 7:442–452
Vachon DJ, Yager DR (2006) Novel sulfonated hydrogel composite with the ability to inhibit proteases and bacterial growth. J Biomed Mater Res 76A:35–43
Wiegand C, Schonfelder U, Abel M, Ruth P, Kaatz M, Hipler U-C (2010) Protease and proinflammatory cytokine concentrations are elevated in chronic compared to acute wound and can be modulated by collagen type I in vitro. Arch Dermatol Res 302:419–428
Wright JB, Lam K, Buret AG, Olson ME, Burrell RE (2002) Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing. Wound Reg Rep 10:141–151
Yager DR, Nwomeh BC (2003) The proteolytic environment of chronic wounds. Wound Rep Reg 11:490–495
Yager DR, Chen SM, Ward SI, Olutoye OO, Diegelmann RF, Cohen IK (1997) Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Rep Reg 5:23–32
Yue Y, Zhou C, French AD, Xia G, Han G, Wang Q, Wu Q (2012) Comparative properties of cellulose nano-crystals from native and mercerized cotton fibers. Cellulose 19:1173–1187
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Edwards, J.V., Prevost, N., Sethumadhavan, K. et al. Peptide conjugated cellulose nanocrystals with sensitive human neutrophil elastase sensor activity. Cellulose 20, 1223–1235 (2013). https://doi.org/10.1007/s10570-013-9901-y
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DOI: https://doi.org/10.1007/s10570-013-9901-y