In situ reduction of silver nanoparticles by chitosan-l-glutamic acid/hyaluronic acid: Enhancing antimicrobial and wound-healing activity

Highlights

• AgNPs loaded Chitosan-l-glutamic acid/hyaluronic acid sponge dressings were successfully prepared by an in-situ reduction.

• The dressing showed good antibacterial efficacy and cytotoxicity.

• The increas of AgNP content improved antibacterial property and mechanical properties of the sponge dressings.

• The dressings prove effective for accelerating the healing rate of cutaneous wounds in rabbits.

Abstract

Spongy composites with silver nanoparticles (AgNPs) were synthesized by freeze-drying a mixture of silver nitrate (AgNO₃) and chitosan-l-glutamic acid (CG) derivative loaded with hyaluronic acid (HA) solution. CG/AgNP spongy composites had an interconnected porous structure and rough surfaces. When AgNPs (5–20 nm) were immobilized on these spongy composites, AgNP aggregation was dependent on AgNO₃ concentration. The spongy composites exhibited good mechanical properties, swelling, and water retention capacity. In vitro antibacterial activity showed that the CG/AgNP spongy composites effectively inhibited bacterial (Escherichia coli and Staphylococcus aureus) growth and penetration. Spongy composites containing low concentrations of AgNP were non-toxic to L929 cells, while CG/HA/AgNP spongy composites promoted wound healing, as determined by in vivo tests, wound contraction ratio, average healing time, and histological examination. These results indicate that the spongy composites can serve as effective antibacterial wound dressings.

Introduction

Human skin is an important component of the immune system since it acts as a barrier against bacteria. Damage to the skin can result in pathogens entering the underlying tissue, leading to inflammation and infection (Chen et al., 2017, Chopra et al., 2016, Chuang et al., 2012) and potentially causing scar formation and maturation of the epidermis and dermis (Chopra et al., 2016, Jiang et al., 2016, Romić et al., 2015). Maintaining a moist environment around a wound can accelerate recovery and decrease the risk of systemic infection (Chuang et al., 2012; Siafaka, Zisi, Exindari, Karantas & Bikiaris, 2016; Zeng, Mccarthy, Deletic & Zhang, 2015). This can be achieved with wound dressings that have antibacterial and swelling properties and can inhibit bacterial growth and absorb exudate from the wound (Kozicki et al., 2016, Liu et al., 2013).

Chitosan is composed of N-glucosamine and N-acetylglucosamine units and is a natural polysaccharide that is widely used in wound dressings owing to its antibacterial and hemostatic properties and low toxicity (Boiteux, Boullanger, Cassagnau, Fulchiron & Seytre, 2006; Doganay, Coskun, Kaynak & Unalan, 2016; Ozcelik et al., 2014). Chitosan-based sponge-like wound dressings have been developed that are suitable for use with full-thickness skin or areas of missing flesh, acting as a substrate for cell adhesion and providing a moist environment for wound repair (Lv, Wang, Zhu & Zhang, 2014; Phaechamud, Yodkhum, Charoenteeraboon & Tabata, 2015). In addition, spongy chitosan composites have high liquid absorption capacity, biocompatibility, antibacterial properties, and high permeability to gas (Siafaka et al., 2016, Ye et al., 2016). However, it is also brittle and forms tight connections to tissue, which limits their application.

l-Glutamic acid (l-GA) is widely used for human and animal nutrition and as an ingredient in pharmaceutical products (Phaechamud et al., 2015). Only a few studies have reported the fabrication of chitosan/GA spongy composites. A chitosan acetic acid solution was combined with GA to yield a soluble aerogel derivative with a concentration of up to 8% (w/v) in water, acetic acid, and alkali solvents (Shao et al., 2016). l-GA may therefore be an effective additive for reducing the dosage of acetic acid and improving the physiological activity of chitosan.

Hyaluronic acid (HA) plays an important role in human skin as a key component of the skin extracellular matrix (Anisha, Biswas, Chennazhi & Jayakumar, 2013), and has many advantageous properties such as hydrophilicity, biodegradability, and a unique viscoelastic nature (Hancı and Altun, 2015). Additionally, HA/chitosan has been used in different forms to enhance wound healing (Lewandowska, Sionkowska, Grabska, Kaczmarek & Michalska, 2016). Recently, HA was used as a cross-linking agent to prepare collagen/hyaluronan/chitosan three-dimensional (3D) porous structures (Sionkowska et al., 2016), which was suggested as an environmentally safe process, as it did not require toxic cross-linking agents.

Silver nanoparticles (AgNPs) have been widely used in biological materials and in optical, magnetic, catalytic, and sensing technologies owing to their favorable properties such as antibacterial activity and electrical conductivity (El-Nahrawy, Ali, Abou Hammad & Youssef, 2016; Gu et al., 2016, Lu et al., 2016a, Lu et al., 2016b, Shao et al., 2016, Siafaka et al., 2016). The fabrication of Ag/chitosan composite materials has been previously reported. For example, chitosan was mixed with HA and nano-Ag to generate a porous structure; the compound consisting of chitosan and tripolyphosphate was loaded with nano-Ag and used for drug release (Ong, Wu, Moochhala, Tan & Lu, 2008). Typically, reducing agents such as NaBH₄, citrate, glucose, hydrazine, and ascorbate are used to prepared AgNPs while stabilizing agents are also needed to prevent AgNP aggregation (Moharram, Khalil, Sherif & Khalil, 2014). Thus, so-called green synthesis methods are becoming a priority (Marambio-Jones & Hoek, 2010). Chitosan—both a reducing and stabilizing agent (Moharram et al., 2014)—can be used to produce AgNPs. Hydrogel has been formed by reacting silver nitrate (AgNO₃) with chitosan (Kozicki et al., 2016), and are thickened and made homogeneous by the chitosan solution, while nano-Ag is immobilized within the hydrogel structure (El-Nahrawy et al., 2016, Madhumathi et al., 2010, Poon and Burd, 2004a, Roberto et al., 1997; Sarhan, Azzazy & Elsherbiny, 2016; Tshukudu, Van & Wessels, 2010; Wang, Zhu, Xue & Wu, 2012). In addition, HA is an environmentally friendly and efficient reducing and stabilizing agent (Abdel-Mohsen et al., 2017).

In this study, we developed a simple method for preparing a novel sponge-like dressing composed of chitosan-l-GA/HA solution mixed with AgNO₃. l-GA can be reacted with chitosan to obtain a chitosan-l-GA derivative, which reduces the dosage of acetic acid and improves the physiological activity of chitosan (Singh et al., 2009). l-GA was first used to dissolve a low concentration acetic acid solution before chitosan was added. HA was used as a cross-linking agent for reaction with chitosan, and Ag⁺ was then added to the mixture and reduced with chitosan-l-GA/HA solution to form AgNPs. The spongy composites were prepared by freeze-drying the mixture. We then evaluated the antibacterial, mechanical, and swelling properties of the spongy composite and its ability to promote wound healing in vivo (Fig. 1).