Elsevier

Acta Biomaterialia

Volume 73, June 2018, Pages 190-203
Acta Biomaterialia

Full length article
Electrofabrication of functional materials: Chloramine-based antimicrobial film for infectious wound treatment

https://doi.org/10.1016/j.actbio.2018.02.028Get rights and content

Abstract

Electrical signals can be imposed with exquisite spatiotemporal control and provide exciting opportunities to create structure and confer function. Here, we report the use of electrical signals to program the fabrication of a chloramine wound dressing with high antimicrobial activity. This method involves two electrofabrication steps: (i) a cathodic electrodeposition of an aminopolysaccharide chitosan triggered by a localized region of high pH; and (ii) an anodic chlorination of the deposited film in the presence of chloride. This electrofabrication process is completed within several minutes and the chlorinated chitosan can be peeled from the electrode to yield a free-standing film. The presence of active Nsingle bondCl species in this electrofabricated film was confirmed with chlorination occurring first on the amine groups and then on the amide groups when large anodic charges were used. Electrofabrication is quantitatively controllable as the cathodic input controls film growth during deposition and the anodic input controls film chlorination.

In vitro studies demonstrate that the chlorinated chitosan film has antimicrobial activities that depend on the chlorination degree. In vivo studies with a MRSA infected wound healing model indicate that the chlorinated chitosan film inhibited bacterial growth, induced less inflammation, developed reorganized epithelial and dermis structures, and thus promoted wound healing compared to a bare wound or wound treated with unmodified chitosan. These results demonstrate the fabrication of advanced functional materials (i.e., antimicrobial wound dressings) using controllable electrical signals to both organize structure through non-covalent interactions (i.e., induce chitosan’s reversible self-assembly) and to initiate function-conferring covalent modifications (i.e., generate chloramine bonds). Potentially, electrofabrication may provide a simple, low cost and sustainable alternative for materials fabrication.

Statement of Significance

We believe this work is novel because this is the first report (to our knowledge) that electronic signals enable the fabrication of advanced antimicrobial dressings with controlled structure and biological performance.

We believe this work is significant because electrofabrication enables rapid, controllable and sustainable materials construction with reduced adverse environmental impacts while generating high performance materials for healthcare applications. More specifically, we report an electrofbrication of antimicrobial film that can promote wound healing.

Introduction

Conventional methods to fabricate polymeric materials were developed in the last century and enlisted the remarkable advances in industrial organic chemistry. Often, the methods developed to create structure and confer function to polymers relied on processing steps and/or reactive reagents that subsequently were found to pose threats to worker safety, public health and the environment. There is a growing interest in employing electrical signals to programmably fabricate functional materials and devices [1], [2], [3], [4], [5], [6], [7], [8]. Electrical signals can be imposed with exquisite spatiotemporal control and provide exciting opportunities to create structure and confer function [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Here, we report the use of electrical inputs to program the fabrication of an antimicrobial chitosan film for a demanding medical application of chronic wound treatment.

With an aging population and increasing rates of diseases linked to ulcers and other skin wounds, such as diabetes, obesity and heart disease, the prevalence and incidence of chronic wounds are increasing [20]. Chronic wounds are defined as acute wounds that fail to heal in a normal physiological process. Bacterial infections are considered to be a major local factor resulting in chronic wounds, in which bacterial toxins stimulate inflammatory cells to secrete pro-inflammatory cytokines to induce exudate formation, delay wound healing, and facilitate improper collagen deposition [14], [20], [21]. Microbiological investigations have shown that Staphylococcus aureus (S. aureus) plays a major role in delaying the healing of nearly 25–30% of chronic wounds [22]. In fact, the contamination of chronic wounds with Methicillin-resistant Staphylococcus aureus (MRSA) represents an increasing worldwide problem. If MRSA infection progresses towards a systemic infection, options for antibiotic therapy become very limited [23].

Topical wound treatment using dressings with antimicrobial bioactivities is highly recommended to accelerate wound healing [24], [25], [26], [27], [28], [29]. Films are capable of providing a moist environment and enhancing the autolytic debridement of necrotic tissues at the wound site [30], [31]. Moreover, they can deliver antimicrobial agents locally which require smaller amounts of the antimicrobial and thus reduce the risk of systemic toxicity and the development of resistance [32]. Chitosan is a beta-1,4-linked polymer of glucosamine (2-amino-2-deoxy-beta-d-glucose) with a small amount of N-acetylglucosamine. Chitosan is often considered an appropriate material for wound dressings because it is reported to offer hemostatic, wound-healing, antimicrobial, nontoxic, biocompatible, and biodegradable properties [33], [34], [35], [36], [37], [38]. Typically, fabrication of desirable chitosan dressings involves both assembling chitosan into macro-scaled structures (i.e., matrix, fiber or film), as well as enhancing antimicrobial function. Chitosan-based macro-scaled structures are often obtained via chemical or photo cross-linking [39], [40], [41], macromolecular blending [42] solvent induced sol-gel processing [43], [44], [45], [46], or electrospinning [47]. A variety of chemical modification approaches have been considered to enhance chitosan’s antimicrobial functionality [48], [49], [50], [51], [52]. In this study, we focus on chloramine functionality which is increasingly considered as broad spectrum disinfectant active against a range of microorganisms such as bacteria, fungi and viruses, etc. [53], [54], [55], [56], [57]. A chloramine compound is defined as a compound containing one or more Nsingle bondCl covalent bonds that are usually formed by chlorination of amine, amide or imide functional groups using halogenating agents (e.g., hypochlorous acid, sodium hypochloride or dichlorisocyanurate sodium) [57], [58], [59], [60]. A chloramine has biocidal properties owing to the oxidation state chlorine atoms in chloramines. Chitosan, as an amino-polysaccharide, thus has the potential to be chlorinated and endowed with antibacterial property [52], [61], [62], [63].

In the present work, we report a two-step approach for enlisting electrode-imposed electrical signals for the facile fabrication of a chlorinated chitosan antimicrobial film. This approach is illustrated in Scheme 1A. In the first step, cathodic electrodeposition is used to organize chitosan (Chit) chains into a macroscopic film at the electrode surface. Mechanistically, the cathodic reduction reaction of hydrogen peroxide creates a localized region of high pH that induces chitosan chains to undergo pH-dependent self-assembly through reversible noncovalent interactions [64], [65], [66], [67], [68]. The second step is the anodic chlorination of the deposited chitosan to obtain chlorinated chitosan film (Chit-Cl). Chlorination is achieved by biasing the underlying electrode to an anodic voltage capable of oxidizing chloride ions for the in-situ generation of the reactive mediator HClO. As illustrated in Scheme 1A, we hypothesize that HClO chlorinates chitosan’s nitrogen groups to form Nsingle bondCl bonds [52]. This anodic chlorination step also likely includes a partial oxidation of chitosan leading to the formation of aldehyde and related derivatives, as shown in the dotted box in Scheme 1A [15], [69], [70], [71], [72]. As illustrated, this electrofabricated modified chitosan film can be peeled from electrode to obtain a free-standing chloramine film.

Here, we specifically report that electrical inputs control both the physical interactions required for macroscale structure formation by cathodic electrodeposition and the chemical reactions required for functionalization by anodic chlorination. We demonstrate that the chlorinated chitosan films confer antimicrobial activities in vitro and also promote wound healing in vivo using a MRSA infected wound model. Presumably, the antimicrobial mechanism involves the release of active chlorine from the Chit-Cl film as shown in Scheme 1B. We envision that electrofabrication provides a simple, rapid and controllable approach for the programmable preparation of antimicrobial chitosan films for wound management. More broadly, this study demonstrates the potential of electrochemistry to create novel structure and functions for achieving high performance polymeric materials.

Section snippets

Materials

Chitosan from crab shells (75–85% deacetylation and medium molecular weight) was purchased from Sigma-Aldrich. Hydrochloric acid, hydrogen peroxide, acetic acid, sodium thiosulfate, potassium iodide, glutaraldehyde, ethanol, paraformaldehyde, sodium phosphate dibasic, potassium phosphate monobasic, and sodium chloride were all purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Starch was purchased from Sinopharm Chemical Reagent Co., Ltd. Luria-Bertani broth and agar was purchased from

Characterization of Chit and Chit-Cl films

In this study, we program the fabrication of a chloramine chitosan film using a cathodic electrodeposition of chitosan and a subsequent anodic chlorination of the deposited material. The Chit-Cl1.70C film was generated with a total charge transferred during the anodic chlorination step of 1.70 C and this condition was used as a representative for chlorinated film characterization. The visual observations of the films in Fig. 1a show that chlorination converts the transparent chitosan film into

Conclusions

In this study, we report a rapid and controllable electrochemistry approach to construct functional biomaterials for wound treatment. This method involves a cathodic electrodeposition of pristine chitosan film and a subsequent anodic chlorination of this deposited material. We provide physical and chemical evidence for the occurrence of film chlorination. Chlorination can occur on the amino groups as well as on the amide groups in chitosan molecules. This electrofabrication approach is

Acknowledgements

The support from the National Natural Science Foundation of China(51621002, 51573047), the 111 project (B14018), the Fundamental Research Funds for the Central Universities (222201717002) and the United States National Science Foundation (CBET-1435957) and Defense Threat Reduction Agency (HDTRA1-13-0037).

References (91)

  • M.A. Kiechel et al.

    Non-covalent crosslinkers for electrospun chitosan fibers

    Carbohyd. Polym.

    (2013)
  • L.X. Lin et al.

    In situ cross-linking carbodiimide-modified chitosan hydrogel for postoperative adhesion prevention in a rat model

    Mat. Sci. Eng. C Mater.

    (2017)
  • C.Y. Tsai et al.

    Thermosensitive chitosan-based hydrogels for sustained release of ferulic acid on corneal wound healing

    Carbohyd. Polym.

    (2016)
  • Y. Xu et al.

    Fabrication and characterization of a self-crosslinking chitosan hydrogel under mild conditions without the use of strong bases

    Carbohyd. Polym.

    (2017)
  • A. Fiamingo et al.

    Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, physicochemical characterization, and biological evaluation

    Biomacromolecules

    (2016)
  • A. Montembault et al.

    Rheometric study of the gelation of chitosan in a hydroalcoholic medium

    Biomaterials

    (2005)
  • X. Zhao et al.

    Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing

    Biomaterials

    (2017)
  • O. Gutman et al.

    Characterization and antibacterial properties of N-halamine-derivatized cross-linked polymethacrylamide nanoparticles

    Biomaterials

    (2014)
  • R. Kaur et al.

    Antibacterial surface design – contact kill

    Prog. Surf. Sci.

    (2016)
  • R. Li et al.

    Antimicrobial N-halamine modified chitosan films

    Carbohyd. Polym.

    (2013)
  • H.K. Shin et al.

    Antimicrobial characteristics of N-halaminated chitosan salt/cotton knit composites

    J. Ind. Eng. Chem.

    (2014)
  • F. Pishbin et al.

    Single-step electrochemical deposition of antimicrobial orthopaedic coatings based on a bioactive glass/chitosan/nano-silver composite system

    Acta Biomateria.

    (2013)
  • X. Bao et al.

    A chitosan-graft-PEI-candesartan conjugate for targeted co-delivery of drug and gene in anti-angiogenesis cancer therapy

    Biomaterials

    (2014)
  • M. Ahmad et al.

    Preparation and characterization of antibacterial thiosemicarbazide chitosan as efficient Cu(II) adsorbent

    Carbohyd. Polym.

    (2015)
  • H. Amer et al.

    Synthesis and characterization of periodate-oxidized polysaccharides: Dialdehyde xylan (DAX)

    Biomacromolecules

    (2016)
  • A.K. Dutta et al.

    Facile preparation of surface N-halamine chitin nanofiber to endow antibacterial and antifungal activities

    Carbohyd. Polym.

    (2015)
  • S. Anjum et al.

    Development of antimicrobial and scar preventive chitosan hydrogel wound dressings

    Int. J. Pharm.

    (2016)
  • A. Dong et al.

    Bactericidal evaluation of N-halamine-functionalized silica nanoparticles based on barbituric acid

    Colloid. Surface. B

    (2014)
  • J. Park et al.

    Nitric oxide integrated polyethylenimine-based tri-block copolymer for efficient antibacterial activity

    Biomaterials

    (2013)
  • Q. Cai et al.

    Tailored synthesis of amine N-halamine copolymerized polystyrene with capability of killing bacteria

    J. Colloid Interf. Sci.

    (2015)
  • J.S. Boateng et al.

    Wound healing dressings and drug delivery systems: A review

    J. Pharm. Sci.

    (2008)
  • C.H. Dzudzevic et al.

    A novel acetylcholinesterase biosensor: core-shell magnetic nanoparticles incorporating a conjugated polymer for the detection of organophosphorus pesticides

    ACS Appl. Mater. Inter.

    (2016)
  • G. Rydzek et al.

    Simultaneous electropolymerization and electro-click functionalization for highly versatile surface platforms

    ACS Nano

    (2014)
  • K. Katarzyna et al.

    Advancing the delivery of anticancer drugs: conjugated polymer/triterpenoid composite

    Acta Biomater.

    (2015)
  • F.A. Plamper

    Changing polymer solvation by electrochemical means: basics and applications

  • C. Maerten et al.

    Review of electrochemically triggered macromolecular film buildup processes and their biomedical applications

    ACS Appl. Mater. Inter.

    (2017)
  • F. Ehret et al.

    Electrochemical control of rapid bioorthogonal tetrazine ligations for selective functionalization of microelectrodes

    J. Am. Chem. Soc.

    (2015)
  • E. Kim et al.

    Programmable “Semismart” Sensor: relevance to monitoring antipsychotics

    Adv. Funct. Mater.

    (2015)
  • G. Li et al.

    Enhanced osseointegration of hierarchical micro/nano-topographic titanium fabricated by micro-arc oxidation and electrochemical treatment

    ACS Appl. Mater. Inter.

    (2016)
  • S. Hirata et al.

    Reversible coloration enhanced by electrochemical deposition of an ultrathin zinc layer onto an anodic nanoporous alumina layer

    Adv. Funct. Mater.

    (2012)
  • L. Yi et al.

    Reversible electroaddressing of self-assembling amino-acid conjugates

    Adv. Funct. Mater.

    (2011)
  • X.L. Liu et al.

    Electrical signals triggered controllable formation of calcium-alginate film for wound treatment

    J. Mater. Sci. Mater. Med.

    (2017)
  • K.M. Gray et al.

    Electrodeposition of a biopolymeric hydrogel: potential for one-step protein electroaddressing

    Biomacromolecules

    (2012)
  • S. Gao et al.

    N-halamine coatings formed via the electroreduction of in situgenerated diazonium cations: toward antimicrobial surfaces

    Surf. Interface Anal.

    (2016)
  • A. Fakhry et al.

    Templateless electrogeneration of polypyrrole nanostructures: impact of the anionic composition and pH of the monomer solution

    J. Mater. Chem. A

    (2014)
  • Cited by (32)

    • Solid phase synthesis of oxidized sodium alginate-tobramycin conjugate and its application for infected wound healing

      2022, Carbohydrate Polymers
      Citation Excerpt :

      Skin injuries such as trauma, empyrosis or surgery may lead to infection providing proper measures are not taken in time (Ye et al., 2019). Medical research have shown that staphylococcus epidermidis, pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli etc. are the main pathogens existed in infected wounds (Woodford & Livermore, 2009), which may stimulate inflammatory cells to secrete pro-inflammatory cytokines, inducing the exudate and improper collagen deposition (Qu et al., 2018). Since the discovery of first antibiotics Penicillin in 1928, antibiotics have successfully cured various bacterial infections.

    • Graphene-silicon Schottky devices for operation in aqueous environments: Device performance and sensing application

      2022, Carbon
      Citation Excerpt :

      The presence of organic chlorine on graphene has already been detected by exposing the graphene to chlorine gas in graphene-based gas sensors. However, it has been suggested in the literature that the appearance of two new peaks at 198.78 eV (Cl 2p3/2 chloride) and (200.38 eV Cl 2p3/2 chloride) is due to chlorine that is covalently bound to the amine nitrogen of the pyrene (-N-Cl) (Fig. 6e) [65–67]. Further evidence of chlorination can be observed in N 1s spectra.

    View all citing articles on Scopus
    View full text