Protein-reduced gold nanoparticles mixed with gentamicin sulfate and loaded into konjac/gelatin sponge heal wounds and kill drug-resistant bacteria

Highlights

• Gold nanoparticles with high fluorescence were prepared in an environmentally friendly way.

• A composite sponge of konjac and gelatin can provide and maintain moisture for the wound.

• The combination of nano-gold and gentamicin sulfate provided a new method for killing superbacteria.

• Konjac gelatin composite sponge loaded with Au NPs/GS can be used as a new dressing to promote wound healing.

Abstract

Timely antibacterial treatment of wounds reduces the probability of wound infection and promotes wound healing. However, the materials used to treat wounds often fail to provide both sterilization (especially for super bacteria) and moisture, and some may even cause secondary injury to the wound. In this study, gold nanoparticles (Au NPs) of average grain diameter of 3 ± 1 nm were prepared using egg white as the reductant. These particles showed no aggregation and pink fluorescence. Au NPs were mixed with gentamicin sulfate (GS) and loaded into a mixture of konjac glucomannan (KGM) and gelatin as wound dressing (KGM/Gelatin@Au NPs/GS). Antibacterial experiments showed that the Au NPs amplified the antibacterial activity of GS; Au NPs/GS efficiently eliminated bacteria, especially super bacteria. Cytotoxicity tests indicated that KGM/Gelatin@Au NPs/GS showed basically no cytotoxicity to L929 cells. In addition, KGM/Gelatin@Au NPs/GS possesses good water absorption, water retention, and enhanced mechanical properties, which can provide a moist environment for wounds and promote healing. In conclusion, our study showed that the antibacterial activity of KGM/Gelatin@Au NPs/GS is better than that of only GS and that it efficiently eliminated super bacteria. Therefore, KGM/Gelatin@Au NPs/GS can be used for killing superbugs, inhibiting bacterial growth, and promoting wound healing.

Introduction

Skin trauma is one of the most common injuries in real life that occurs mostly due to accidents [1]. Injured skin can be easily infected by various pathogens, which can cause inflammation and infection, and retard wound healing [2]. Wound dressings can relieve the symptoms of wound infection. A good wound dressing should be non-toxic, non-allergenic, and biocompatible [3]. In addition, a certain extent of moisture is conducive for wound healing; hence, a wound dressing should be capable of maintaining a moist environment in the vicinity of the wound [4,5]. Furthermore, wound dressings should also possess excellent antibacterial properties to prevent bacterial invasion [6,7].

The overuse of antibiotics has led to the emergence of superbugs and antibiotic resistance, thereby weakening the bactericidal effects of antibiotics at concentrations at which they were previously effective [8]. Multidrug-resistant pathogens can lead to the re-emergence of many lethal but incurable diseases, and also pose a threat to human life and health [9]. To circumvent this problem, certain inorganic nanoparticles (NPs), such as Ag NPs, Cu NPs, and ZnO NPs have been used for developing antibacterials. Studies have shown that these NPs not only possess excellent antibacterial ability, but are also refractory to the development of antibiotic resistance [[10], [11], [12]]. However, several reports have demonstrated the cytotoxicity of Ag NPs, Cu NPs, and ZnO NPs toward different mammalian cells [[13], [14], [15], [16]]. Another alternative involves elimination of superbugs by amplifying the antibacterial effect of antibiotics. Studies have shown that Au NPs can enhance the bactericidal ability of antibiotics, such as carbapenems [17], levofloxacin [18], and streptomycin [19]. NPs are used as drug carriers that improve dissolution and enhance absorption of drugs, increase drug targeted performance, control drug release, and reduce side effects [[20], [21], [22]]. In addition, studies have also shown that the higher the surface-area-to-volume ratio of Au NPs, the stronger the amplification effect on antibiotics [23]. Furthermore, Au NPs themselves are non-cytotoxic and antibacterial in nature [24,25]. Au NPs can also be efficiently produced by the reduction of auric chloride acid (HAuCl₄) by various chemical reagents, such as sodium citrate, sodium borohydride, and ascorbic acid [[26], [27], [28]]. However, these reductants have some disadvantages of polluting the environment, harming the human body, and being expensive [29]. Therefore, it is of great significance to find a simple, inexpensive, and green Au NP synthesis method. Proteins can act as reductants and stabilizers for high-fluorescence Au NPs. [30]. Egg white, which is a rich source of proteins, is biocompatible and possesses foaming, emulsifying, and adhesive abilities, enabling it to improve water retention of dressings and maintain moisture content in wounds [31,32]. Furthermore, it is inexpensive and abundant. Therefore, use of egg white as a green reduction system for preparing small-size Au NPs that can amplify the antibacterial effect of antibiotics is an effective method for eliminating superbugs. Gentamycin sulfate (GS), an aminoglycoside antibiotic, possesses many advantages such as good water solubility and wide antimicrobial spectrum, can be used to treat gram-negative as well as gram-positive bacterial infections, is less costly, and elicits low side effects [33,34]. The amine groups (NH₂) in GS and Au NPs are connected by hydrogen bonds, and their combination improves the antibacterial efficiency of GS. Therefore, Au NPs amplify the antibacterial activity of GS and may effectively kill superbugs [35].

In the wound dressing area, in addition to good antibacterial properties, dressings provide a moist environment for the wound. Gelatin is a natural polymer, which has been widely used in the pharmaceutical industry [36] and has good biocompatibility. However, the mechanical properties of gelatin deteriorate after freeze-drying, and its water absorption capacity is weak. KGM is a natural polysaccharide of high molecular weight, which has good biodegradability and biocompatibility [37]. KGM is rich in hydroxyl and carbonyl groups; hence, it can utilize hydrogen bonds and van der Waals forces to attract water molecules and maintain moisture content [38,39]. The addition of KGM to gelatin can increase the mechanical and water retention properties of materials [40]. Furthermore, KGM/gelatin does not cause secondary damage to the wound, and effectively improves the material's water absorption, water retention, and mechanical properties.

In this study, we used two steps to prepare KGM/Gelatin@Au NPs/GS for treating skin trauma. The egg white reducer was used to prepare Au NP solution, followed by dissolution of KGM and gelatin, and finally freeze-drying. Fig. 1 clearly shows the KGM/gelatin @ preparation process of the Au NPs/GS and the sterilization principle. KGM/gelatin@Au NPs/GS was studied using X-ray diffraction spectrometry (XRD) and scanning electron microscopy (SEM), and its mechanical properties were assessed. We used Escherichia coli and Staphylococcus aureus to determine the antibacterial activity of KGM/Gelatin@Au NPs/GS. Further, we used animal wound healing experiments and histopathological examination to evaluate the healing of wounds treated with KGM/Gelatin@Au NPs/GS.