Regular Article
Comparative study of novel in situ decorated porous chitosan-selenium scaffolds and porous chitosan-silver scaffolds towards antimicrobial wound dressing application

https://doi.org/10.1016/j.jcis.2018.01.007Get rights and content

Abstract

Dermal defects caused by trauma or disease are challenging to treat due to difficult-to-treat infections that impair wound healing. Due to the widespread emergence of drug-resistant bacteria and dwindling discoveries of new antibiotics, there is currently an urgent need to introduce novel antimicrobials effective against antibiotic-resistant bacteria without causing damage to host tissues. As selenium (Se) and silver (Ag) are known for their antimicrobial properties, we investigated the separate loading of these materials into porous chitosan/PVA (CS) scaffolds through a simple in situ deposition method to create two distinct wound dressing materials (CS-Se and CS-Ag). Scaffolds with Se nanostructures and scaffolds containing Ag nanostructures were characterized and their activities against S. aureus – (a Gram-positive bacterium), E. coli – (a Gram-negative bacterium) and Methicillin-Resistant S. aureus (MRSA) – (a multi-drug resistant bacterium) were compared. The release of Ag and Se in vitro was shown to depend strongly on the release medium used (deionised water, mammalian or bacterial culture media). Ag-loaded scaffolds showed a significant reduction in CFUs and cytotoxicity towards fibroblasts while Se-loaded scaffolds showed abilities to damage bacterial cell membrane and non-toxicity to fibroblast. Overall, in this study we have demonstrated simple, in situ immobilization porous CS scaffolds with either Se or Ag nanostructures which could be used to suit different wound healing applications.

Introduction

Bacteria have been able to develop antibiotic resistance faster than the introduction of new antibiotics [1]. Due to diminishing economic returns for new antibiotic drugs, long drug development times and strict regulatory guidelines, very few new antibiotic drugs are being introduced into the market [2]. Worryingly, no new classes of antibiotics have been introduced since 2003 [3]. Hence drug-resistant bacterial infections are becoming more prevalent and can once again pose the same threat as infections before the advent of modern antibiotics [4].

Currently, obesity-related diseases, such as diabetes and vascular diseases, are resulting in increasing incidences of debilitating medical conditions, including chronic wounds [5]. Treatment of chronic wounds is time intensive and results in a significant economic burden of approximately 31 billion USD worldwide [6]. Some studies have estimated that microbial biofilms can be present in more than 70% of chronic wounds [7]. The exopolysaccharides in biofilms provide significant barrier towards antibiotics and can, in turn, be breeding grounds for the development or exchange of antibiotic resistance genes in microbes, such as Staphylococcus aureus and Escherichia coli [8]. Thus, there is a current and pressing need for the introduction of new antimicrobials that can tackle microbial infection without causing significant cytotoxicity to the surrounding host tissue [9].

Ag nanoparticles have gained significant acclaim in the past two decades for their antimicrobial properties [10]. Ag-based nanoparticles and composites have been shown in a large number of studies to display broad spectrum antibiotic properties towards a large number of known pathogenic bacteria, including drug-resistant strains [11]. The exact mechanism of Ag’s antibiotic properties is still debated; however, it is generally accepted that Ag ions from Ag nanoparticles can bind to thiol residues present ubiquitously on membrane proteins and respiratory enzymes, resulting in reactive oxygen species (ROS) generation, which is toxic towards cells [9]. Furthermore, they are also known to bind to DNA, most likely via binding to various heat shock proteins, which results in irreversible coiling of DNA, inhibiting its replication [12].

Significantly, the mechanisms of Ag’s effects on bacteria can also impact mammalian cells [13]. A number of studies have shown that Ag nanoparticles can result in cytotoxicity and genotoxicity towards mammalian cells [14]. However, the widespread use of Ag nanoparticles in applications including dressings [15], medical implants [16], fabrics [17], and water treatment [18] indicates a focus on the antimicrobial properties of Ag, with perhaps less regard for its potentially negative effects on human cells. Several meta-analyses of clinical trials involving comparison of Ag-containing dressings to other dressings have failed to come to any conclusion regarding enhanced wound healing with Ag based dressings [19]. This may be in part due to the presence of co-morbidities in the patients. These studies also demonstrate a lack of emphasis towards the potential fate of Ag nanoparticles (embedded within various substrates such as dressings and medical devices) in in vivo conditions. To improve understanding of Ag nanoparticles released from substrates similar to Ag dressings, we have fabricated in situ and loaded Ag nanoparticles into chitosan/PVA scaffolds and studied their release in various complex media in an attempt to better model their potential fate under conditions routinely utilised in various in vitro assays. Furthermore, we have also probed the cytotoxicity (towards fibroblasts) and antimicrobial effect (on Gram-negative and Gram-positive bacteria) of Ag species released from chitosan/PVA scaffolds in vitro to provide better insights into how Ag (nanoparticles and ions) may behave under various controlled in vitro conditions. Ultimately, such controlled tests could help guide clinicians, researchers and regulatory bodies in better understanding effects of Ag-coated medical devices and dressings on mammalian cells and pathogenic bacteria.

Recently, Se nanoparticles have been gaining more attention as potential antimicrobial [20] and anticancer [21] agents. Se is an essential nutrient present as a trace element in humans and forms a group of selenoproteins which have important antioxidant properties [22]. Interestingly, Se nanoparticles have shown selective toxicity towards bacteria, leaving mammalian cells unharmed in a dose-dependent manner [23]. Given that Se is already present as a trace element in humans, Se-based nanoparticles may be better tolerated by human cells, while providing the beneficial antimicrobial properties that are currently needed for tackling resistant pathogenic bacteria [20].

Previously we have developed a chitosan/PVA hydrogel via mechanical foaming and thermally induced phase separation (TIPS) [24], which can potentially be utilised in chronic wound healing. The technique utilised here is relatively simple and is amenable to scale-up. Compared to other methods such as porogen leaching, pressurised gas foaming and freeze drying, the combined gas-foaming/TIPS used in our work has the versatility that can be explored to create different scaffold’s pore architecture, pore size by changing parameters such as mixing speed, surfactant concentration, mixing time [24]. Chitosan was chosen due to its structural similarities towards native glycosaminoglycans and its reported ability as a wound healing agent [25]. In this study, we investigated the in situ formation and loading of Ag and Se nanostructures into foamed TIPS chitosan/PVA scaffolds. The resultant scaffolds were assessed in vitro under various culture conditions to compare the release of Se and Ag. We also studied whether they had cytotoxic effects towards fibroblast cells, essential components of the dermis [26]. The mechanisms of the effect of extracts from the scaffolds on two Gram positive bacteria (Staphylococcus aureus (S. aureus) and Methicillin-resistant Staphylococcus aureus (MRSA)) and one Gram-negative bacterium (Escherichia coli) [27] were then investigated. These three bacterial species were chosen as they frequently contaminate chronic wounds and are implicated in device related and hospital acquired infections [28].

Section snippets

Materials

Medium molecular weight chitosan (180–310 kDa, degree of deacetylation 85%) and polyvinyl alcohol (PVA) (10,000 Da, 80% hydrolysed) (Sigma-Aldrich, Australia) were used for foamed hydrogel formulation. All solutions were made with ultrapure water, purified to a resistivity of 18.2 MΩ in a Millipore water filtration system (Millipore, Victoria, Australia) and will be referred to as deionised water in this discourse. Chitosan and PVA were dissolved in 0.88 M acetic acid (Chem supply, Victoria,

Synthesis of scaffolds

In this study, we utilised wet synthesis methods for in situ fabrication of Se chitosan/PVA and Ag chitosan/PVA scaffolds. Chitosan/PVA scaffolds were decorated in situ post-fabrication with Se nanostructures via reduction of absorbed sodium selenite solution by GSH (reduced form, Eq. (1) [29])Na2SeO3(aq)+GSHreducedform(aq)alkalinepHGS-Se-SG(aq)GSSG(aq)+Se(s)where GS-Se-SG represents the intermediate species and GSSG the oxidised glutathione. With increasing sodium selenite precursor

Conclusions

Treatment of dermal wounds requires advanced biomaterials that cover the wounds and provide protection against bacterial colonization and infection. The focus of this study was developing such a material taking into consideration the current antibiotic resistance crisis and the need for reduced material’s cytotoxicity to help wound healing [1], [2], [3], [4], [5]. We used an in situ solution-based synthesis method to form and immobilize nanostructures of antimicrobial Se and Ag on chitosan

Acknowledgements

The authors thank Roger Curtain from the Bio21 Advanced Microscopy Facility (the University of Melbourne) for help with SEM imaging. We would also like to thank the Particulate Fluids Processing Centre (PFPC) and the Oral Health CRC at the Melbourne Dental School for access to infrastructure and equipment. DB gratefully acknowledges the Australian Government for an Australian Postgraduate Award. PT was the recipient of the University of Melbourne’s McKenzie fellowship and the Seed Funding from

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