A Dual-Enzyme Hydrogen Peroxide Generation Machinery in

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A dual-enzyme hydrogen peroxide generation machinery in hydrogels supports antimicrobial wound treatment Daniela Huber, Gregor Tegl, Anna Mensah, Bianca Beer, Martina Baumann, Nicole Borth, Christoph Sygmund, Roland Ludwig, and Georg M. Guebitz ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 22, 2017

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ACS Applied Materials & Interfaces

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A Dual-Enzyme Hydrogen Peroxide Generation Machinery in Hydrogels supports Antimicrobial Wound Treatment

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Daniela Huber1, Gregor Tegl1*, Anna Mensah1, Bianca Beer1, Martina Baumann3, Nicole Borth3,4,

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Christoph Sygmund2, Roland Ludwig2 and Georg M Guebitz1,3

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1

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Sciences, Vienna, Konrad Lorenz Straße 20, 3430 Tulln an der Donau, Austria

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2

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Sciences, Muthgasse 18, 1190 Vienna, Austria

Institute of Environmental Biotechnology, BOKU−University of Natural Resources and Life Department of Food Science and Technology, BOKU−University of Natural Resources and Life

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3

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Austria

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4

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Muthgasse 18, 1190 Vienna, Austria

ACIB−Austrian Centre of Industrial Biotechnology, Konrad Lorenz Straße 20, 3430 Tulln, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU),

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*corresponding author: [email protected]

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Abstract

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The aging population and accompanying diseases like diabetes resulted in an increased

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occurrence of chronic wounds. Topical wound treatment with antimicrobial agents to inhibit

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bacterial invasion and promote wound healing is often associated with difficulties. Here, we

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investigated the potential of succinyl chitosan (SC) – carboxymethyl cellulose (CMC) hydrogels

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which constantly release clinically relevant levels of hydrogen peroxide (H2O2). CMC hydrogel

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matrix was in-situ converted by limited hydrolysis by a cellulase into substrates accepted by

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cellobiose dehydrogenase (CDH) for continuous production of H2O2 (30 µM over 24 h). This

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dual-enzyme catalyzed in situ H2O2 generation system proved its antimicrobial activity in a zone

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of inhibition (ZOI) assay best simulating the application as wound dressing and was found to be

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biocompatible towards mouse fibroblasts (95% viability). The hydrogels were thoroughly

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characterized regarding their rheological properties indicating fast gel formation (< 3 min) and

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moderate crosslinking (1,5% strain, G´ 10 Pa). Cooling (fridge conditions) was found to be the

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simple on/off switch of the enzymatic machinery which is of great importance regarding storage

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and applicability of the bioactive hydrogel. This robust and bioactive antimicrobial hydrogel

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system overcomes dosing issues of common topical wound treatments and constitutes a

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promising wound healing approach for the future.

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Keywords

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Cellobiose dehydrogenase; chronic wounds; infection; enzyme immobilization; rheology;

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biocompatible

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Introduction

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Chronic wounds are increasingly described as an epidemic with a rising amount of reported

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incidents not at least caused by an aging population and the remaining issue to stem diabetes

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and obesity. The increased occurrence of chronic wounds and the difficulty in treating this

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disease render them a great financial burden for our healthcare economy not considering the

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costs for long-term care of affected individuals 1. Chronic wounds can be defined as acute

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wounds that fail to heal, which is often a result of deleterious effects from other diseases

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up/downregulating mechanisms within the wound healing procedure 2. Another issue promoting

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their emergence is the lack of appropriate infection detection methods accurately assessing the

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wound status, although suitable infection biomarker are known 3. Current wound management

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procedures mostly rely on swabbing cleaning, dressing and debridement of the wound. Systemic

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treatment with antibiotics still administers these wound care methods, although the increasing

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issue of antibiotic resistances is known and reported in a substantial number of unsuccessful

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treatment trials 4. Topical wound treatment instead has the advantage of applying antimicrobials


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at the site of infection which requires less amounts of the antimicrobial, reduces the risk of

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systemic toxicity and the development of resistances

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commonly incorporated into ointments or wound dressings thereby slowly releasing the agent

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into the wound environment. Hydrogels gain attention as dressing materials due to their ease of

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preparation and multifold available strategies to give them desired properties like loading drugs,

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antimicrobial activities and many more 6. Commonly antiseptics are used for that purpose;

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however, only few candidates are available to date and proved effective in clinical trials 7.

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Hydrogen peroxide (H2O2) is the most prominent wound disinfectant used since decades in

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hospitals, which is effective on gram-negative and gram-positive bacteria 8. It is a strong

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oxidative agent commonly used as solution of 3% (v/v) for wound care and disinfection of

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surfaces and medical materials like implants. At this concentration H2O2 was found to completely

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inhibit bacterial growth

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long-term use of H2O2 at high concentrations

9

5

. Consequently, antimicrobials are

but only moderate cytotoxic effects to intact skin were observed after 10

. Antibacterial activity of H2O2 is observed at

11

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concentrations greater 10 µM

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generation systems that provide H2O2 above the threshold, yet below cytotoxic levels. These

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enzyme based systems utilized the ability of cellobiose dehydrogenase (CDH) to produce H2O2

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in the presence of cellobiose and molecular oxygen. When compared to glucose oxidase GOX ,

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CDH offers the advantage of accepting larger cello-oligomers as substrates

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of glucose to fuel GOX would concomitantly support growth of microorganisms. CDH was

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therefore immobilized on chitosan and enzyme functionalized chitosan particles were able to

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completely inhibit bacterial growth 13. The H2O2 levels generated by this system are known to act

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as chemoattractant on neutrophils and stimulate the proliferation of human fibroblasts beside the

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antimicrobial effect, which is of great interest promoting wound healing. A remaining issue of the

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described CDH based systems is the supply of the enzyme substrate which needs to be

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externally provided. Within this study, this flaw was eliminated by utilizing a hydrogel as CDH

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substrate by the aid of a mediating cellulase which partly degraded the matrix to suitable CDH

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substrates. Succinyl chitosan (SC) / carboxymethylcellulose (CMC) hydrogels were thus

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investigated for wound dressing applications. The combination of CDH and cellulases,

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encapsulated in the hydrogels, generated H2O2 over 24 h. The hydrogels produced H2O2 in

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clinically relevant concentrations is possible converting cellulose derived component of the

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hydrogel by the aid of enzymes and inhibited bacterial growth.

, which recently led to the development of in situ H2O2

32


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while the addition


1

Materials and Methods

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All chemicals were used in analytical grade. Media components and cellobiose were purchased

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from Carl Roth (Karlsruhe, Germany), all other chemicals were purchased from sigma-Aldrich

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(Steinheim, Germany). Chitosan was purchased from Sigma and had a number-average

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molecular weight of 200 kDa (as determined by SEC) and a degree of N-acetylation of 13% (1H

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NMR)

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from Sigma: 700 kDa and a degree of substitution (DS) of 0.9 (CMC 1), 250 kDa and DS 0.9

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(CMC 2), 250 kDa and DS 0.7 (CMC 3), 90 kDa and DS 0.7 (CMC 4); Staphylococcus aureus

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strain ATCC 25923 and Escherichia coli strain XL1 were used for the study of antimicrobial

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activities and were acquired from the culture collection of the Institute of Environmental

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Biotechnology from Graz University of Technology, Austria. The recombinant Myriococcum

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thermophilum cellobiose dehydrogenase (rMtCDH) was produced in Pichia pastoris as

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previously described

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purchased from Sigma-Aldrich containing 10% enzyme. Elastase (HNE) from human leukocyte

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was purchase from Sigma-Aldrich.

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All experiments were performed in triplicate, unless otherwise stated.

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. Four different carboxylmethyl celluloses were used within this study and all purchased

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. The lyophilized cellulose mix from Trichoderma longibrachiatum was

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Hydrolysis profile of carboxymethyl cellulose by cellulases

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Carboxymethyl celluloses (CMCs) with different molecular weights and degrees of substitution

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(DS) were investigated in terms of susceptibility towards hydrolysis by a cellulase preparation

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from Trichoderma longibrachiatum (TrlCel). To do so, 2 mL of a CMC solution (1% w/v, in H2O)

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was incubated with TrlCel (25 mg powder per g CMC) for 24 h at 37°C and 300 rpm. Afterwards

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the enzyme was inactivated by heating the solution to 95°C for 10 min. The experiment was

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performed with four CMCs with varying molecular weight and degree of substitution.

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The hydrolysis success was determined using the 3,5-dinitrosalicylic acid (DNS) method.16 A

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standard curve was plotted determining the absorbance of different concentrations of glucose at

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540 nm wavelength. The relative sugar content is given as glucose equivalents (mg/mL). CMC

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hydrolysis was further confirmed and quantified by size exclusion chromatography (SEC). The

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number average molecular mass (Mn) was determined using an Agilent 1100 Series

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Chromatography system equipped with an Agilent 1200 G1362A refractive index detector. A

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TSKgel G5000PWXL column was used for analysis (Tosoh Bioscience, Montgomeryville, PA,

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USA). Calibration was performed using a pullulan standard set (Fluka, Buchs, Switzerland). As

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mobile phase an acetate buffer was used consisting of 0.15 M acetic acid, 0.1 M sodium


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acetate, 0.4 mM sodium azide in ddH2O, previously filtered through an ExpressTMPlus filter with

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0.22 µm pore size (47 mm diameter, Millipore).

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Hydrogen peroxide production

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The CMC hydrolysis products should act as co-substrate for cellobiose dehydrogenase (CDH) to

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produce H2O2. A succinyl chitosan solution (0.75 mL, 5% w/v) was mixed with the CMC solution

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(0.25 mL, 1.5%). The mixture was incubated together with TrlCel (25 mg powder per g CMC)

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and CDH (1 mg/mL) at 37°C and 300 rpm. Samples were taken at regular time points to

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determine accumulated H2O2.

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The CDH activity was measured as a function of the H2O2 production using the Amplex Red

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assay, like previously published.13

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Synthesis of N-succinyl chitosan

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N-succinyl chitosan (SC) was published using a common approach.17 Chitosan (6.4 g) was

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dissolved in acidic acid (5% v/v, 500 mL) before ethanol was slowly added (500 mL) while

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stirring the solution. Afterwards succinic anhydride (70 g) dissolved in acetone (500 mL) was

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added and the mixture was stirred for 2 h. After completion of the reaction the pH was adjusted

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to 9 using sodium hydroxide. The resulting precipitate was isolated and salts were removed by

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dialysis (MWCO 10000) (Spectrum Labs, USA). The degree of substitution was analyzed by 1H

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NMR (in D2O)

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MHz for 1H and 100.63 MHz for 13C) equipped with a 5 mm observe broadband probe head

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with z-gradients.

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Preparation of succinyl chitosan / carboxymethyl cellulose hydrogels

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SC was dissolved in double distilled H2O (ddH2O) to obtain a 4% (w/v) solution and CMC was

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dissolved in ddH2O to obtain a 1.5% (w/v) solution. The two solutions were mixed in a ratio 3:1

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(SC:CMC), mixed well by vortexing and existing air bubbles were removed by centrifugation. To

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1 mL of polysaccharide solution 40 mg EDC and afterwards 40 mg NHS were added, both

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previously dissolved in 20 µL ddH2O. The solution was vortexed, casted in a petri dish and

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allowed to gel.

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Enzyme incorporation into the hydrogel

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When incorporating the enzymes into the hydrogel, the amount of crosslinking agent had to be

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adjusted. One µL of the respective enzyme solution in ddH2O was added to the already mixed

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polysaccharide solution to obtain a total amount of 25 µg CDH and 75 µg TrlCel per gel. After

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adding the enzymes, 45 mg EDC and 45 mg NHS per 1 mL of gel solution were added and the

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protocol was followed like previously described.

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using a Bruker Avance II 400 spectrometer (resonance fre- quencies 400.13



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Rheology

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The rheological measurements were conducted on a Malvern Kinexus rheometer (Malvern

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Instruments Ltd., UK) consisting of a heated plate-plate (diameter of 20 mm, gap 0.5 mm). The

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gelation time was determined with a frequency of 1 Hz and a strain of 0.1%. Further tested

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properties were adopted by Wang et al

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ranging from 0.01 to 150 Hz with a fixed strain of 0.1%. Furthermore, the change of the hydrogel

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properties over a strain ranging from 0.01 to 1000% was tested with a fixed frequency of 1 Hz.

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Determination of the antimicrobial activity

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The antimicrobial activity of the prepared hydrogels was determined by the zone of inhibition

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assay to test the effectiveness of the hydrogel to elute inhibitory levels of H2O2; the effect was

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assessed against E. coli and S. aureus. To do so, 80 µL of an overnight culture was spread in a

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MH agar plate (2*104 CFU/mL for E. coli, 7*103 CFU/mL for S. aureus) and a weighed amount of

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a hydrogel was placed on the agar. The plates were incubated at 37°C for 24 h and zones of

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inhibition were measured and images were taken.

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The antimicrobial effect of the hydrogels was quantified in liquid antimicrobial assays using a

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previously published protocol

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by plating culture medium at regular time points and counting colonies at suitable dilutions after

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24 h incubation. All experiments were performed in triplicate.

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Biofilm assay

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The inhibitory effect of the hydrogels on static biofilm formation was investigated in microtiter

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plate biofilm assays. Briefly, overnight cultures of E. coli and S. aureus were 1:10 diluted with

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MH broth and 200 µL were pipetted in sterile microtiter plates which have not been tissue culture

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treated. Hydrogel pieces of approx. 1 mm3 were added to the wells and incubated for 24 h at

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37°C. Biofilm formation was subsequently quantified using the crystal violet staining method

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and results were compared to positive controls lacking of hydrogels.

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Anti-inflammatory properties

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The anti-inflammatory potential of the hydrogels was tested by inhibition of the enzyme human

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neutrophil elastase (HNE). HNE activity was quantified by modifying the method of Löser et al 21.

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Therefore, 25, 50, and 100 mg of the hydrogels were weighed in and 470 µL of 1xPBS buffer

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with a pH of 7 was added. Then, 25 µL of elastase substrate (N-methoxysuccinyl-Ala-Ala-Pro-

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Val p-nitroanilide) was added with a final concentration of 1 mM. By adding 5 µL of HNE

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(enzyme acitivty: 5 mU mL-1) the reaction was started and the reaction mixture was further

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incubated for 1 h at 37 °C. In addition, controls were performed, where hydrogels without the

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dual-enzyme system and the HNE alone were incubated. The reaction was stopped by addition

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. A frequency sweep was performed with a frequency

13

. The colony forming units per mL (CFU / mL) were determined


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of 500 µL of trypsin inhibitor with a concentration of 0.2 mg mL-1. After vortexing, 200 µL of the

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reaction mixtures were transferred into a 96-well plate and the absorbance was measured at

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405 nm with a spectrophotometer (Tecan Infinite 200Pro, Switzerland). Elastase inhibition in %

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was calculated by means of the following equation,

5 ℎ % =

− × 100

6 7

with Absblank as the absorbance at 405 nm of the blank and Abssample of the hydrogels.

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Cytotoxicity

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The biocompatibility of the chitosan hydrogels was tested by adopting the procedure of Huber et 22

10

al

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conditions. The sterile samples were than incubated for 4 days in 14 ml DMEM media (Gibco,

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Thermo Fisher Scientific, US) with 10% fetal calve serum (FCS) at 37 °C and 5% CO2 to allow

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leaching into the media. Afterwards, the media was filtered through a 0.22 µm filter and the

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leach-out media was used for biocompatibility testing. The 3T3 mouse fibroblast cell line (ATCC,

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US) was incubated with the leach-out media in a 6-well plate (Greiner Holding AG; Austria). After

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4 and 7 days of incubation the cells were detached and the cell number and viability was

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measured with a ViCell instrument (Beckmann Coulter, US). A control was performed by using

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NIH 3T3 mouse fibroblast cell line with untreated media.

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Swelling properties

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The swelling behavior of the prepared hydrogels was determined by transferring pre-weighed

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amounts of the hydrogels in petri dishes and determination of the total weight. Afterwards 3 mL

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of sodium phosphate buffer (0,25 M, pH 7.4) or artificial wound fluid (AWF) were added to the

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gels. The composition of AWF was chosen like previously published 23. The gels were allowed to

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swell at room temperature and the weight of hydrogels was regularly determined. Therefore the

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swelling medium was removed and remaining liquid was carefully adsorbed using a filter paper.

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After the measurement new swelling medium was again added. The degree of swelling was

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calculated using the following formula

. Therefore, 0.75 mL of the hydrogel was prepared and UV-sterilized for 1 h under sterile

S=

Wt − W0 W0

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where W0 is the weight of the dry samples applied and Wt is the weight of the swollen samples

29

after a defined time.



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Storage stability

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The hydrogels with and without incorporated enzymes were investigated upon the stability of the

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geld and the change in enzyme activity over time. To do so, the hydrogels were stored at 4°C for

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30 d and for every time point taken a gel was prepared. The remaining enzyme activity was

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tested in terms of the H2O2 production, whereby the gels were incubated in sodium phosphate

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buffer (6 mL, 0.25 M, pH 7.4) for 24 h at 37°C and 200 rpm. The H2O2 concentration was

7

determined using the Amplex Red assay as previously described.

8


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1

Results and Discussion

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The continuous production of H2O2 by the aid of enzymes is a promising strategy to provide a

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well-known antimicrobial agent in targeted amounts as needed for the respective application.

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Enzymes for this purpose are preferably immobilized on a solid matrix like membranes and

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hydrogels, which impedes degradation by proteolytic enzymes, amongst others

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production of H2O2 is then triggered by the supply of the respective enzymes substrate, like

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cellobiose for CDH

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molecules, the stable incorporation of them in the enzyme environment (eg. the hydrogel) is

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challenging due to moderate leaching out of the matrices. However, for wound dressing

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applications leaching of compounds in the wound environment is a critical aspect and should be

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avoided.

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Within this study CDH is immobilized in a SC / CMC hydrogel. To overcome the issue of

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substrate supply, the CMC matrix of the hydrogel was partially hydrolysed by a cellulase (TrlCel)

14

to a substrate suitable for CDH to produce H2O2.

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Generation of H2O2 by CDH and TrlCel

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Different cellulases from Trichoderma were previously found to efficiently hydrolyze CMCs into

17

oligosaccharides

18

derivatives like cello oligosaccharides of varying chain length and CMC oligomers

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keep the H2O2 production upright over long time, the supply of the substrate for CDH has to be

20

assured and was investigated incubating different CMCs with CDH and TrlCel. No significant

21

difference in H2O2 production was observed applying the different CMCs (figure 1), although

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CMC 3 was more efficiently hydrolyzed by TrlCel when compared to the other CMCs, as

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analyzed by the DNS assay (data not shown). The slight difference in the DS (0.7 and 0.9) of the

24

CMCs and the consequently resulting oligomers did not influence the oxidation efficiency by

25

CDH. H2O2 levels were found to be clearly above the desired threshold of 10 µM at which

26

antimicrobial activity was described but did not exceed concentrations for which cytotoxic effects

27

were observed after long-term exposure 11.

24

. The

25

. Since most of the substrates of H2O2 producing enzymes are small

26

. CDH is known to not only accept cellobiose as substrate but many more 12

. In order to

28 29

Thus the multi-enzyme system produced the desired amounts of H2O2 promoting wound healing

30

and could be incorporated into a hydrogel system. Since the choice of the CMC derivative did

31

not influence hydrogen peroxide production, the CMC showing the best properties for hydrogel

32

formation was used in all further experiments for the development of suitable hydrogels.



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Figure 1: Production of H2O2 by CDH from CMC in the presence of TrlCel. TrlCel hydrolyzed CMC into oligomers, which were converted by CDH.

4 5

SC / CMC hydrogels

6

Hydrogels for medical applications must exclude toxic effects. The most prominent approach

7

obtaining biocompatible and biodegradable hydrogels is the use of natural materials such as

8

cellulose and chitosan

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conditions and frequently used in hydrogels and wound dressings

27

. CMC is an elegant cellulose derivative soluble at physiological 28

. Chitosan is presumably

10

used in biomedical formulations because of its moderate water solubility, in contrast to chitin. SC

11

is one of them and gained attention in the past years due to its good solubility in water and the

12

high number of functional groups facilitating its fabrication 29.

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Preparation and characterization

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The remaining (not modified) primary amines of SC were used to crosslink SC with each other

15

and with the carboxyl groups of CMC via carbodiimide chemistry using EDC / NHS. Prior to

16

crosslinking, SC was synthesized from chitosan in a common approach and resulted in a DS of

17

0.47 as determined by 1H NMR (figure S1). A hydrogel was formed shortly after the addition of

18

EDC / NHS to the polymer mixture whereby a consistent and robust gel was only obtained using

19

CMC 3. It is assumed that the increased viscosity of the solution, when using the other CMCs,

20

resulted in an insufficient distribution of the crosslinking agent. Due to the given results, CMC 3

21

was chosen for all further experiments within this study. The fast hydrogel formation was

22

obtained in non-buffered water solution, which is of special interest considering future

23

applications since the prepared hydrogels do not require extensive washing steps to remove

24

undesired reagents and salts. Common EDC coupling procedures are carried out at acidic pH

25

values. The crosslinking degree of the obtained hydrogel was determined via the ninhydrin ACS Paragon Plus Environment

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assay to be 68% and was further confirmed by FTIR (figure 2c). A complex spectrum was

2

obtained due to the co-polymeric nature of the hydrogel but an intensity increase of the

3

characteristic bands was observed after cross linking. The increase of the carbonyl stretching

4

band (Amide I) at 1653 cm-1 and the Amide III band at 1260 cm-1 proved chemical cross linking.

5

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Figure 2: SEM images of SC /CMC 3 hydrogel cross sections, (a) before EDC cross linking (b) after cross

8

linking of the solid gel. (c) ATR-FTIR spectrum of a freeze dried SC / CMC 3 hydrogel, the chemically

9

crosslinked (grey) and not crosslinked (black)

10 11

The cross sectional morphology of the hydrogel was studied by SEM of gels in the equilibrium

12

swelling state (in water). A significant difference in the pore size was observed comparing the

13

cross linked hydrogel (figure 2b) with its unreacted counterpart (figure 2a), whereby a much

14

higher pore size was obtained after cross linking. A homogeneous distribution of the pore size

15

was found along the gel proving that the cross linking worked out homogeneously.

16 ACS Paragon Plus Environment


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1

A critical but often favored aspect of polysaccharide-based hydrogels is their moderate swelling

2

in contact with aqueous media. The swelling behavior of the stable SC /CMC 3 hydrogel was

3

measured in different media including AWF to simulate the water take up of wound fluids. The

4

influence of enzyme incorporation was evaluated but did not result in different swelling

5

behaviors. After 24 h, a steady state of Si around 1.7 was observed in AWF (figure 3), which was

6

slightly increased compared to the take up of buffer with around 1.3 (data not shown). Since the

7

pH of buffer and AWF were unified, effects of the pH were excluded which supposes the fluid

8

composition influencing fluid take up of the hydrogel. The Si values were considerably lower

9

compared to published SC – alginate and CMC-based hydrogels

30

. The super-absorbing nature

10

of CMC seemed to be lowered by the moderate cross linking degree of 68% which is known to

11

greatly influence the swelling properties of chitosan hydrogels 31.

12 13 14

Figure 3: Evolution of the swelling of SC / CMC hydrogels containing CDH for H2O2 production. The swelling ratio Si was measured over 4 days incubation in AWF.

15 16

Rheological characterization

17

Since hydrogels of this composition were not reported in literature before, a thorough rheological

18

characterization was performed and is a crucial aspect evaluating the applicability of the

19

hydrogel as wound dressing. In a first step, the gelation time of the hydrogels was analyzed by

20

determining the gelation point. The gelation point is the point when the elastic modulus (G’) is

21

exceeding the viscous modulus (G’’)

22

demonstrated in figure 4A, indicating a fast gel formation within 150 sec. The gel characteristics

23

changes from a more viscous to an elastic behavior and generates a solid-like gel state, also

32

. The gelation speed and the gel intensity are


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33

1

suggesting a three-dimensional (3D) network

. A 3D network can be established by covalent

2

binding, as true for the present study, as well as due to crystallization, and molecular secondary

3

forces. The mechanism of network formation is influencing the viscoelastic behavior of

4

hydrogels. At the gelation point, the hydrogel 3D network is not fully established, since there are

5

many non-interacting segments available as manifested by G’’. After finished gelation of the

6

hydrogels, G’ values are above 10 Pa (figure 4A) describing an adequate strength of the

7

hydrogels. This characteristic is underlined by the dominant difference between G’ and G’’

34

8

The fast gel formation of less than 3 min was also seen by genipin cross-linked hydrogels

35

9

Without the chemical cross-linker the solid-like gel behavior is not formed (data not shown).

. .

10

A strain sweep was performed to determine the linear viscoelastic regime (LVR) of the hydrogels

11

(figure 4B). The LVR range is demonstrating the deformation range where G’ is independent of

12

the deformation (strain in %). The critical strain can be identified by a sharp change in the slope

13

of G’ that is decreasing as a function of the deformation applied to the hydrogel. This drop is

14

indicative for a breakdown of the microstructure of the hydrogel

15

before the critical strain is a quite unusual phenomenon that can be explained due to a strain

16

hardening

17

increase of G’ due to a formation of microcrystalline of chitosan under shear that might act as

18

additional cross-links. Further studies regarding the unusual behavior of G’ around 1% strain are

19

required to elucidate the mechanism.

20

In the present study, the hydrogels can bear up to a strain of 1.5%, which is typical for a weaker

21

cross-linking if compared to a chitosan-PEG hydrogel that shows critical strains of 200%

22

When the strain is above 1.5%, the hydrogel underwent gel-sol transition and started to behave

23

like a liquid. There are several studies with critical strains under 10% like for example ovalbumin

24

hydrogels and also enzymatically cross-linked chitosan hydrogels 22,36.

25

The recovery of hydrogels is also an important parameter

26

the G’ values are much higher than G’’, which is typical for an ideal gel. After increasing the

27

strain above the critical strain the gel is breaking. The G’ values decreased to very low values

28

below 10 Pa, indicating a loose network of the hydrogel

29

when the amplitude was decreased again to a strain of 0.1% at the same frequency, the G’

30

values rose again near to the initial values and the gel is recovering.

31

Frequency sweep experiments ranging from 0.01 to 100 Hz were carried out to investigate the

32

stability of the cross-linked network in a fully cured hydrogel (figure 4C). After performing a strain

33

sweep to determine the LVR range, a strain under the critical strain was chosen (0.1%). The

34

hydrogels exhibit an independency of frequency up to 25 Hz, while G’’ is increasing slowly, as

35

typical for hydrogel materials

36

. The increase of the strain

37,38,39

. A similar phenomenon was shown by a study of He et al

40

, explaining the

41

.

42

. Therefore, the gel is formed and

43

34

as shown in figure 4D. However,

. Above 25 Hz, G’ is rising, which is indicating a structure break ACS Paragon Plus Environment


Page 14 of 27

32

1

by mechanical shear

. The viscoelastic response of hydrogel materials is first affected by the

2

length of the flexible polymer chains within the gel and second by the nature of the imposed

3

motion. Longer polymeric chains are known to exhibit longer relaxation times

4

increase of G’ is therefore related to the failure of long polymeric chains to rearrange themselves

5

in the time scale of the imposed motion. Hence, the chains are getting stiff, assuming a more

6

“solid-like” behavior of the hydrogels 35.

35

. The sharp

7

8 9

Figure 4: Rheological characterization of the SC / CMC3 hydrogel with the elastic (G’) and viscous (G’’)

10

modulus is presented. The dynamics of G’ and G’’ next to the gelation point is presented in the presence

11

of the cross-linking reagent (A). The evolution of G’ and G’’ over a frequency range of 0.01 to 100 Hz (B)

12

and a strain range from 0.01 to 1000% (C) is given. The recovery of the hydrogels was tested by

13

continuous step strain measurements with a frequency of 1 Hz (D).

14 15

Generation of H2O2 of the hydrogel


Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

After the thorough characterization, the ability of the dual-enzyme system to produce H2O2 in the

2

hydrogel was evaluated. The enzyme loaded hydrogel was incubated in a buffered solution and

3

H2O2 was quantified in the solution. Within short incubation times, a concentration around 30 µM

4

was detected, which was in accordance with the previous results applying the enzyme system in

5

solution (figure 5). Thus, the H2O2 generation system is not impeded by the enzyme covalently

6

incorporated into the hydrogel and by crosslinking of CMC. The dotted line in figure 5 depicts the

7

H2O2 generation of the same hydrogel but only loaded with CDH, whereby the hydrogel was

8

incubated in a buffered cellobiose solution. This control system was assumed to produce the

9

maximum possible amount of H2O2 by using CDH within a hydrogel. Around 40 µM H2O2 were

10

produced by this system. Thus only 10 µM H2O2 more were produced by directly supplying the

11

CDH substrate compared to the in situ generation of a suitable substrate by TrlCel from CMC.

12

These experiments proved the concept to be successful for the production in-situ of clinically

13

relevant levels of H2O2 for wound healing applications. The supply of a small molecule substrate

14

was circumvented by the addition of TrlCel as missing link converting the hydrogel matrix to a

15

suitable CDH substrate. Compared to previous studies in our lab immobilizing CDH on the

16

surface of chitosan particles, a decrease in the H2O2 production was observed

17

clearly explained by the significant differences in the diffusion of substrate to the enzyme within

18

a hydrogel compared to CDH located on the surface of a particle.

13

. This can be

19 20

Figure 5: H2O2 production of a SC/ CMC 3 hydrogel loaded with TrlCel and CDH. As control sample a

21

hydrogel solely with CDH was incubated in a buffered cellobiose solution.

22



Page 16 of 27

1

Although the H2O2 production was proved at neutral pH, the workability of both enzymes was

2

already previously proven under varying conditions

3

premise to synthesize a robust bioactive system generating H2O2 under varying conditions. As

4

conditions in culture media of antimicrobial assay vary due to metabolite secretion, so do the

5

conditions of wounds and wound fluids regarding pH changes and varying composition of

6

metabolites 48.

15,44–47

. This promiscuity depicts an important

7 8

Antimicrobial activity

9

The produced H2O2 was tested upon its antimicrobial properties against E. coli and S. aureus as

10

Gram negative and Gram positive bacterial representatives, respectively. The ZOI assay was

11

chosen for that purpose because it is best simulating the application as dressing material and

12

outlines if inhibitory levels of the antimicrobial were eluted (figure 6). For both bacteria, a ZOI

13

was detected as depicted in figure 6A and 6C, whereby E. coli was more susceptible towards

14

released H2O2. The ZOI after 24 h was measured 23±2 mm for E. coli and 14.5±0.5 mm for S.

15

aureus. The control samples (hydrogel without TrlCel but with CDH) did not inhibit bacterial

16

growth around the hydrogel (figure 6B and 6C) indicating that the hydrogel itself did not show

17

any antimicrobial effects. Although antimicrobial activity of chitosan is described, N-acyl

18

derivatives do not show any inhibitory effects which is confirmed by our results with the SC

19

/CMC hydrogel

20

H2O2 are produced also in the absence of the substrate generated by TrlCel. Previous studies

21

state that H2O2 is produced by CDH in presence of CMC in solution

22

H2O2 within the hydrogels was not detected neither were antimicrobial effects observed in

23

course of the ZOI assay. This is most probably caused by the different conditions CMC is

24

provided to CDH in the hydrogel compared to the supply as solution. Another factor may be the

25

decreased amount of CMC used within the hydrogels, which could be too low for CDH.

26

The obtained results proved that the generated H2O2 indeed led to the inhibition of bacterial

27

growth as described in literature

28

This phenomenon cannot be generalized to all gram positive bacteria occurring in the wound

29

environment but the assay gave a liable outlook on the efficiency of this system.

30

The hydrogels were also tested in liquid antimicrobial assays, whereby the hydrogels were fully

31

covered with inoculated culture medium and the CFUs per mL were determined allowing a

32

quantitative assessment of the antimicrobial properties. The hydrogels inhibited growth of E. coli

33

to 24% (8.1*106 CFU/mL) and S. aureus to 43% (4.6*105 CFU/mL) after 8 h incubation and

34

compared to the respective positive control (figure S2). The influence of immobilized CDH in the

35

hydrogels was investigated adding the 4-fold amount of the enzyme but a significant difference

29,49

. The control hydrogels contained CDH to test whether relevant amounts of

11

12

. However, a production of

whereby the gram positive representative was less inhibited.


Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

in growth inhibition compared to the initial hydrogels was not observed. Thus the amount of used

2

CDH was not the rate limiting component in the antimicrobial system. Interestingly the hydrogels

3

lacking of enzymes showed slight inhibitory effects to both E. coli and S. aureus which was not

4

observed in the ZOI assays. This effect may be a reason of the increased contact area of

5

hydrogels to the culture medium in the liquid assay compared to the ZOI assay.

6

Another aspect strengthening the applicability of the antimicrobial SC /CMC hydrogel is that

7

H2O2 is continuously produced in-situ over a long time. The continuous on site production is a

8

great improvement in drug delivery from hydrogels since commonly loaded antimicrobials are

9

usually released in higher dose after short time, a known disadvantage of topical antimicrobial

10

wound therapies 5.

11

Another concern was that the accumulated H2O2 and TrlCel destructs the hydrogel but a

12

destruction of the gel was not observed as depicted in figure 6 and proved by the obtained

13

rheology data (figure 4).

14

15 16 17

Figure 6: Determination of the antimicrobial activity of the SC /CMC 3 hydrogel by the ZOI assay after

18

24 h. As negative control a hydrogel lacking of TrlCel was chosen. (A) against E. coli (ZOI 23 mm) (B)

19

negative control against E. coli (C) against S. aureus (ZOI 14 mm) (D) negative control against S. aureus.

20 21

The effect of the enzymes loaded hydrogel towards biofilms of E. coli and S. aureus was

22

additionally investigated since biofilms are a critical aspect influencing wound healing.

23

Microorganisms in biofilms are commonly more resistant to antimicrobial treatment and difficult



50

1

to remove once they are formed

. A microtiter plate biofilm assay was performed monitoring

2

biofilm formation in presence of hydrogels (table S1). The hydrogel lacking of enzymes already

3

inhibited biofilm formation to 35% (E. coli) and 49% (S. aureus), respectively. No significant

4

difference between enzyme loaded hydrogels and one lacking enzymes was observed,

5

indicating that biofilm formation is just impeded by the hydrogel matrix rather than by the H2O2

6

generation system. CLSM observation of biofilm grown on cut out microtiter plates confirmed

7

these results whereby a decreased biofilm density was observed when grown in the presence of

8

the hydrogels (data not shown).

9

Additionally the bactericidal effect of the hydrogel / H2O2 system on mature biofilms was

10

investigated by incubating existing biofilms with the hydrogels. Life-dead staining and

11

subsequent CLSM observation did not indicate any bactericidal activity on the fully grown

12

biofilms which may be a reason of insufficient diffusion of produced H2O2 in the biofilm matrix.

13 14

Biocompatibility

15

Hydrogels for dressing applications have to be biocompatible since they are in contact with body

16

organs

17

of the hydrogels. The indirect cytotoxicity method was performed with the help of ViCell (Fa.

18

Beckmann Coulter, USA) exploring the growth rate and the viability of the NIH 3T3 mouse

19

fibroblast cell line after treatment with media, where hydrogels were incubated before (leachate

20

media). The data in figure 7 demonstrate no relevant cytotoxicity of the hydrogels with a NIH 3T3

21

fibroblast cell line. The cells were highly viable after 3 and 6 days of incubation with leachate

22

media as well as compared to the control that was treated with fresh cell media. All viabilities

23

were above 95%, indicating no cytotoxic effect of the cross-linked hydrogels. The results

24

underline the fact, that SC is not cytotoxic to a mouse fibroblast cell line. The growth rate of the

25

SC / CMC 3 hydrogels with and without the enzyme was reduced within 3 and also 6 days as

26

illustrated in figure 7. Compared to the hydrogel sample with a growth rate of 80.98% after 6

27

days, the enzymes did not have an effect on the growth rate as expressed by a growth rate of

28

82.61%.

51

and therefore cytotoxicity tests were conducted to evaluate in vitro the biocompatibility

29


Page 18 of 27

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 7: Growth rate [%] (bars) with control set to 100% and viability [%] (lines) of NIH 3T3 mouse

3

fibroblast cell line is shown. The results were determined in triplicates and are presented with the

4

according standard deviation.

5 6

Although

the

hydrogel

system

proved

biocompatible,

further

studies

are

required

7

comprehensively describing the interference of the developed system with the biological

8

processes of tissue cells. Especially the influence of immobilized enzymes and generated H2O2

9

on inflammatory processes cannot be forecasted and deserve separate investigations

52,53

.

10

Preliminary experiments of anti-inflammatory assays outlined inhibitory effects on human

11

leukocyte elastase activity, a known inflammation biomarker commonly found in wound fluids. A

12

dose dependent inhibition between 40-60% elastase activity was observed compared to the

13

positive control (figure S3). However, this initial result requires further verification considering

14

further inflammation biomarker, which was outside the scope of this work.

15 16 17

Storage stability

18

The in situ generation of H2O2 also diminishes the risk that the antimicrobial agent degrades

19

during storage of the functionalized hydrogel. However, applying biological matter like enzymes

20

always poses the risk that eg. the activity of the enzyme decreases over time. The first remedy

21

to overcome this issue is increasing the stability of the enzyme by covalent immobilization on a

22

carrier material. The preparation method of the hydrogel including the incorporation of the

23

enzymes assures a covalent immobilization of CDH and TrlCel, which was previously proved

24

No enzymes were detected in the buffered solutions wherein the hydrogels were incubated,


13

.


1

which indirectly proved covalent crosslinking since no leaching off the hydrogels was observed.

2

The change in activity of the combined enzyme system over time was tested by storing the

3

hydrogels over 1 month in the fridge and regularly testing the H2O2 production (figure 8). For

4

every measurement a new gel was taken. There was no significant loss of activity within 7 days

5

of storage considering the H2O2 concentrations after 24 h incubation. However, a constantly

6

decreasing H2O2 production was observed after 1 month storage, nevertheless generating over

7

25 µM H2O2. These levels of the antimicrobial agent are still known to show inhibitory effects

8

towards bacterial growth which rendered the antimicrobial hydrogels pretty robust already in the

9

proof-of-concept phase.

10

Succinyl chitosan makes a part of hydrogel, thus the gel´s susceptibility towards lysozyme was

11

also investigated. This enzyme occurs in wound fluids and is known to degrade succinyl

12

chitosan. When incubating the hydrogels in buffered lysozyme solution of 5000 U/mL (equal to

13

the lysozyme activity in infected wounds), a loss of the gel network structure was observed after

14

48 h. This experiment illustrates that lysozyme was able to degrade the hydrogel when fully

15

covered with enzyme solution. However, the hydrogel degradation rate by lysozyme is supposed

16

to be strongly decreased when just one side of the gel is in contact with lysozyme secreting

17

tissue.

18

Since the enzyme machinery is inherently active, a temperature mediated inhibition of the H2O2

19

production system was evaluated. To do so, the hydrogel was incubated in buffered solution at

20

4°C for 24 h and the H2O2 concentration was measured, whereby no H2O2 could be detected.

21

Thus just temperature control (eg. storing the gels in the fridge) is necessary to provide an on/off

22

control of the antimicrobial system.

23


Page 20 of 27

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1 2 3

Figure 8: Evaluation of the H2O2 production after storing the hydrogel in the fridge. After distinct storage times hydrogels were incubation in buffer and the H2O2 generation over time was measured.

4

Conclusion

5

In this proof-of-concept study, an antimicrobial dual-enzyme based polysaccharide-based

6

hydrogel was developed for wound dressing applications. The hydrogel continuously released

7

H2O2 which was in situ generated by an enzyme system consisting of CDH and TrlCel. TrlCel

8

was found able to hydrolyze crosslinked CMC in the hydrogel and produced oligomers which

9

were oxidized by CDH and led to the production of H2O2. The resulting H2O2 production

10

machinery provided the antimicrobial agent in concentrations around 30 µM which is known to

11

inhibit bacterial growth and also stimulates the proliferation of fibroblasts. The thoroughly

12

characterized hydrogels showed antimicrobial activity against E. coli and S. aureus and

13

biocompatibility tests proved that the cell viability of mouse fibroblasts is not influenced by the

14

hydrogels. Temperature control by storing the gels at cool room conditions was found a simple

15

on/off trigger for the H2O2 generation system. The presented SC / CMC hydrogels loaded with

16

the enzyme machinery proved to be a promising wound dressing material which elegantly

17

eradicates some critical issues of topical antimicrobial wound treatment and gives rise for further

18

investigations.

19 20

Acknowledgements

21

Framework Programme (FP7/ 2007-2013) under grant agreement no. 604278 and the Austrian

22

Centre of Industrial Biotechnology (ACIB).

The research leading to these results has received funding from the European Union Seventh



1 2

Associated Content

3

Supporting Information available:

4

1

5

performing a liquid antimicrobial assay, Anti-inflammatory assay based on inhibition of elastase,

6

Microtiter plate biofilm assay;

7

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A Dual-Enzyme Hydrogen Peroxide Generation Machinery in

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