Mussel-inspired, antibacterial, conductive, antioxidant, injectable composite hydrogel wound dressing to promote the regeneration of infected skin

https://doi.org/10.1016/j.jcis.2019.08.083Get rights and content

Abstract

Infection is a major obstacle to wound healing. To enhance the healing of infected wounds, dressings with antibacterial activities and multifunctional properties to promote wound healing are highly desirable. Herein, gelatin-grafted-dopamine (GT-DA) and polydopamine-coated carbon nanotubes (CNT-PDA) were used to engineer antibacterial, adhesive, antioxidant and conductive GT-DA/chitosan/CNT composite hydrogels through the oxidative coupling of catechol groups using a H2O2/HRP (horseradish peroxidase) catalytic system. The addition of the antibiotic doxycycline endowed the hydrogels with antimicrobial activity to treat infected full-thickness defect wounds. Additionally, CNT-PDA endowed these hydrogels with an excellent photothermal effect, leading to good in vitro and in vivo antibacterial activities against Gram-positive and Gram-negative bacteria. The catechol group and polydopamine imparted tissue adhesiveness, and the hemostatic and antioxidant abilities of these hydrogels were also investigated. The porosity, degradability, swelling, rheological, mechanical, and conductive behaviors of these hydrogels were finely regulated by changing the concentration of CNT-PDA. Hemolysis and cytocompatibility tests using L929 fibroblast cells confirmed the good biocompatibility of these hydrogels. The wound closure, collagen deposition, histomorphological examination and immunofluorescence staining results demonstrated the excellent effects of these hydrogels in an infected full-thickness mouse skin defect wound. In summary, the adhesive antibacterial and conductive GT-DA/chitosan/CNT hydrogels showed great potential as multifunctional bioactive dressings for the treatment of infected wounds.

Introduction

It is well-known that infection is a major obstacle to wound healing [1], and it has become a continuously growing cause of death among patients with serious illnesses. In addition, the treatment of infection places a substantial burden on the medical system and even society overall. Currently, antibiotics are still the main strategy used in the clinical treatment of infection. Therefore, developing proper antibacterial drug delivery systems that can not only effectively and accurately deliver antibiotics to the wound site but also control the release behaviors for active wound healing are highly desirable [2]. Thus, many types of modern wound dressings including semipermeable films, semipermeable foams, hydrocolloids and hydrogels with sustained drug release properties have been developed in an attempt to not only avoid the infection of defect wounds but also to promote the wound repair process [3], [4], [5], [6]. Among these dressings, due to their hydrophilic nature, hydrogels can reduce the risk of wound infection by absorbing wound exudate and maintaining a moist environment, characteristics which present good potential for improving the repair of damaged tissue.

As a protein derivative, gelatin (GT) is considered beneficial for wound healing due to its non-immunogenicity, cell adhesion behavior and blood coagulation characteristics. GT has been used in combination with a large number of synthetic or natural macromolecules to fabricate wound dressings. For example, the addition of chitosan (CS) can overcome the shortcomings (weak mechanical strength and fast degradation rate) of gelatin-based hydrogels. In addition, CS itself can also trigger hemostasis and accelerate tissue regeneration due to the migration of inflammatory cells and the activation of fibroblasts that produce various cytokines [7]. Moreover, previous works on polyelectrolyte GT-CS scaffolds/films [8], [9] have demonstrated their excellent potential for use in skin tissue engineering applications. On the other hand, excessive reactive oxygen species (ROS) during the wound repair process often affect the repair by changing or degrading extracellular matrix (ECM) proteins, damaging dermal fibroblasts and reducing the function of keratinocytes [10]. Therefore, controlling the levels of ROS has been demonstrated to be an effective way to promote wound healing [11]. However, hydrogels based on GT/CS have no antioxidant capacity. Fortunately, the structure of catechol has been proven to be widely present in a variety of natural antioxidants and plays an important role in scavenging ROS [12]. Thus, grafting dopamine to GT (gelatin-grafted-dopamine (GT-DA)) will endow GT with good antioxidant properties [13], [14]. Moreover, the addition of catechol groups to GT will also enhance the adhesiveness of GT-DA/CS-based hydrogels due to the physical bonding and chemical crosslinking between the catechol or polydopamine group and the wounded tissue [15]. Additionally, the good adhesion properties also allow the hydrogel to seal the wound, achieving a good hemostatic effect [16]. Therefore, grafting dopamine onto GT will give the GT-DA/CS hydrogel good adhesiveness and hemostatic and antioxidant properties, making it an excellent candidate for use in wound dressings.

In addition, researchers have found that the human body has endogenous bioelectric systems. The surface of intact human skin carries a more negative charge than do the deeper skin layers [17]. However, when a defect or wound occurs in the skin, the deeper cells of the epidermis and the cells of the wound are positively charged. The combination of a positively charged wound and negatively charged surrounding intact skin creates what is called a skin battery [18]. This bioelectric current facilitates wound healing best when the wound tissue is moistened. The roles of conductive materials in promoting wound healing have also been demonstrated in previous studies [19], [20], [21], [22], [23], [24], [25]. Due to their excellent mechanical, thermal and electronic properties, carbon nanotubes (CNTs) have led the boom in nanotechnology research in the past decades [26], [27], [28]. Meanwhile, CNTs-embedded scaffolds [29], [30] have been demonstrated to enhance electrical properties and facilitate signal propagation, consequently improving cell-cell coupling [31]. However, the strong hydrophobic interaction between individual CNTs makes it difficult for the original CNTs to disperse in water [32]. Surface coating has been demonstrated to be an efficient way to improve the CNTs dispersion and to reduce their cytotoxicity [33], [34]. Dopamine coating is an easy and effective way to modify the surface of CNTs [35], as dopamine will self-polymerize in alkaline solution and form a layer of polydopamine (PDA) coating on the surfaces of CNTs [31], which avoids the disadvantages of conventional oxidation modification strategies. Additionally, CNTs can strongly absorb near‐infrared (NIR) energy and efficiently convert it into thermal energy, and they also exhibit good photothermal antibacterial effects, which will benefit the healing of infected wounds [36], [37]. Thus, developing CNT-PDA and GT-DA/CS-based antibacterial adhesive antioxidative conductive hydrogels is highly desirable to heal infected wounds.

Here, chitosan, gelatin-grafted-dopamine and polydopamine-coated CNTs were used as building blocks to engineer antibacterial adhesive conductive GT-DA/CS/CNT composite hydrogels by the self-polymerization of dopamine using a H2O2/HRP catalytic system, and their good therapeutic effect in the treatment of infected full-thickness defect wounds was also demonstrated. Chitosan was added into the system to improve the hydrogels’ mechanical properties and compensate for the disadvantage of rapid degradation. The use of the H2O2/HRP catalytic system not only reduced the biological safety problems observed in the traditional oxidative methods but also produced similar or comparatively better mechanical properties than the traditional chemical methods. The swelling ratio, degradation behaviors, morphology and rheology properties of the engineered GT-DA/CS/CNT hydrogels were characterized. The properties of tissue adhesiveness, hemostatic and antioxidant activities, conductivity, photothermal effects and sustained drug release were also studied. Additionally, to endow antibacterial ability to the composite hydrogels, doxycycline was loaded into the polymeric network, and the antimicrobial properties of the GT-DA/CS/CNT/Doxy hydrogels were evaluated against Gram-(+) Staphylococcus aureus (S. aureus) and Gram-(−) Escherichia coli (E. coli). Furthermore, in vitro cytocompatibility and blood compatibility testing of the GT-DA/CS/CNT hydrogels was also conducted. Last, an infected mouse full-thickness skin defect wound model was used to evaluate the therapeutic effects of these hydrogels. These GT-DA/CS/CNT hydrogels’ multifunctional properties, including antibacterial activity, adhesiveness and conductivity, make them excellent candidates for the treatment of infected skin wounds.

Section snippets

Materials

Gelatin (Type A, gel strength ∼ 300 g Bloom, Sigma-Aldrich), dopamine hydrochloride (DA, Sigma-Aldrich, 98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, J&K Chemical, >98.5%), N-Hydroxysuccinimide (NHS, Sigma-Aldrich, 98%), carbon nanotube (CNTs, XFNANO, Materials Tech CO. Ltd.), 2,2-Diphenyl-1-(2, 4,6-trinitrophenyl)-hydrazyl (DPPH, J&K Chemical, 97%), horseradish peroxidase (HRP, J&K Chemical, 98%), and doxycycline hydrochloride (J&K Chemical, 98%) were used as received.

Preparation of GT-DA/CS/CNT hydrogel

Herein, we present antimicrobial, adhesive, antioxidative and conductive hydrogels to treat infected skin defect wounds. These designed hydrogels were based on gelatin-grafted-dopamine and polydopamine-coated CNTs. Gelatin, a highly regarded polymer that provides structural integrity and mimics the native composition of the ECM to modulate cell function in human skin, is suitable for skin regeneration applications. Because of its good tissue adhesion characteristics, dopamine was chosen and

Conclusions

We present a series of antibacterial adhesive antioxidant and conductive GT-DA/CS/CNT composite hydrogels produced through the oxidative coupling of catechol groups between gelatin-grafted-dopamine and polydopamine-coated CNTs. The addition of the antibiotic doxycycline endowed the hydrogel with antimicrobial activity, and these multifunctional hydrogels showed great potential for promoting the repair of infected wounds [78], [79]. The porosity, rheology, mechanical properties, conductivity,

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgement

This work was jointly supported by the National Natural Science Foundation of China (grant number: 51673155), the State Key Laboratory for Mechanical Behavior of Materials, the Fundamental Research Funds for the Central Universities, the World-Class Universities (Disciplines), the Characteristic Development Guidance Funds for the Central Universities, the Natural Science Foundation of Shaanxi Province (Nos. 2018JM5026, and 2019TD-020), and the Opening Project of Key Laboratory of Shaanxi

References (82)

  • M. Tarfaoui et al.

    Mechanical properties of carbon nanotubes based polymer composites

    Compos. Part B-Eng.

    (2016)
  • M. Kharaziha et al.

    Tough and flexible CNT–polymeric hybrid scaffolds for engineering cardiac constructs

    Biomaterials

    (2014)
  • Q. Wan et al.

    Surface modification of carbon nanotubes by combination of mussel inspired chemistry and SET-LRP

    Polym. Chem.

    (2015)
  • O. Meincke et al.

    Mechanical properties and electrical conductivity of carbon-nanotube filled polyamide-6 and its blends with acrylonitrile/butadiene/styrene

    Polymer

    (2004)
  • 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)
  • J. Qu et al.

    pH-responsive self-healing injectable hydrogel based on N-carboxyethyl chitosan for hepatocellular carcinoma therapy

    Acta Biomater.

    (2017)
  • Y. Liang et al.

    pH-responsive injectable hydrogels with mucosal adhesiveness based on chitosan-grafted-dihydrocaffeic acid and oxidized pullulan for localized drug delivery

    J. Colloid Interf. sci.

    (2019)
  • J. Qu et al.

    Antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing

    Biomaterials

    (2018)
  • J. Qu et al.

    Injectable antibacterial conductive hydrogels with dual response to an electric field and pH for localized “smart” drug release

    Acta Biomater.

    (2018)
  • S.J. Ryu et al.

    Layered double hydroxide as novel antibacterial drug delivery system

    J. Phys. Chem. Solids

    (2010)
  • X. Zhao et al.

    Antibacterial and conductive injectable hydrogels based on quaternized chitosan-graft-polyaniline/oxidized dextran for tissue engineering

    Acta Biomater.

    (2015)
  • J. Qu et al.

    Degradable conductive injectable hydrogels as novel antibacterial, anti-oxidant wound dressings for wound healing

    Chem. Eng. J.

    (2019)
  • B. Guo et al.

    Degradable conductive self-healing hydrogels based on dextran-graft-tetraaniline and N-carboxyethyl chitosan as injectable carriers for myoblast cell therapy and muscle regeneration

    Acta Biomater.

    (2019)
  • L. Wang et al.

    Electrospun conductive nanofibrous scaffolds for engineering cardiac tissue and 3D bioactuators

    Acta Biomater.

    (2017)
  • D. Gopinath et al.

    Dermal wound healing processes with curcumin incorporated collagen films

    Biomaterials

    (2004)
  • M. Li et al.

    Electroactive anti-oxidant polyurethane elastomers with shape memory property as non-adherent wound dressing to enhance wound healing

    Chem. Eng. J.

    (2019)
  • S. Sanjabi et al.

    Anti-inflammatory and pro-inflammatory roles of TGF-β, IL-10, and IL-22 in immunity and autoimmunity

    Curr. Opin. Pharmacol.

    (2009)
  • M. Chen et al.

    Dynamic covalent constructed self-healing hydrogel for sequential delivery of antibacterial agent and growth factor in wound healing

    Chem. Eng. J.

    (2019)
  • F. Zehtabi et al.

    Chitosan-doxycycline hydrogel: an MMP inhibitor/sclerosing embolizing agent as a new approach to endoleak prevention and treatment after endovascular aneurysm repair

    Acta Biomater.

    (2017)
  • J. Zhou et al.

    Bacteria-responsive intelligent wound dressing: simultaneous In situ detection and inhibition of bacterial infection for accelerated wound healing

    Biomaterials

    (2018)
  • Q. Shi et al.

    Cobalt-mediated multi-functional dressings promote bacteria-infected wound healing

    Acta Biomater.

    (2019)
  • G. Han et al.

    Chronic wound healing: a review of current management and treatments

    Adv. Ther.

    (2017)
  • H. Liu et al.

    A functional chitosan-based hydrogel as a wound dressing and drug delivery system in the treatment of wound healing

    RSC Adv.

    (2018)
  • M.L. Mangoni et al.

    Antimicrobial peptides and wound healing: biological and therapeutic considerations

    Exp. Dermatol.

    (2016)
  • S. Dhivya et al.

    Wound dressings–a review

    BioMedicine

    (2015)
  • N. Zhao et al.

    Assembly of bifunctional aptamer-fibrinogen macromer for VEGF delivery and skin wound healing

    Chem. Mater.

    (2019)
  • J. Zhao et al.

    Injectable alginate microsphere/PLGA–PEG–PLGA composite hydrogels for sustained drug release

    RSC Adv.

    (2014)
  • R. Moseley et al.

    Extracellular matrix metabolites as potential biomarkers of disease activity in wound fluid: lessons learned from other inflammatory diseases?

    Brit. J. Dermatol.

    (2004)
  • C. Dunnill et al.

    Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process

    Int. Wound J.

    (2017)
  • A.I. Neto et al.

    Nanostructured polymeric coatings based on chitosan and dopamine-modified hyaluronic acid for biomedical applications

    Small

    (2014)
  • S. Hong et al.

    Hyaluronic acid catechol: a biopolymer exhibiting a pH-dependent adhesive or cohesive property for human neural stem cell engineering

    Adv. Funct. Mater.

    (2013)
  • Cited by (430)

    View all citing articles on Scopus
    View full text