Therapeutic efficacy of an antibiotic-loaded nanosheet in a murine burn-wound infection model
Graphical abstract
Introduction
Burn injuries render the host susceptible to bacterial infection because of the large skin defects that are created. Burn-wound infection often causes systemic sepsis and severe septicemia, resulting in an increase in mortality of burn-injured patients. Therefore, appropriate burn-wound care is important to prevent wound infection and improve patient outcome. It is currently estimated that ∼75% of mortality following burn injuries are related to infections, such as those caused by Pseudomonas aeruginosa (P. aeruginosa) or methicillin-resistant Staphylococcus aureus (MRSA), rather than osmotic shock and/or hypovolemia [1], [2]. Complication of burn-wound infection is thus considered as a serious problem in the care of severely burn-injured patients. Currently, such patients are treated by debridement to remove as much of the source of infection as possible, combined with wound dressing and, if required, tissue grafting [3]. However, it is difficult to heal “full-thickness burns”. In general, the current standard treatment of a full-thickness burn is tissue grafting. Thus, it is important to inhibit bacterial infection until tissue grafting can be performed.
There are three main factors that must be considered when choosing a suitable dressing material for burn wounds [4]: (i) controlling infection in the wound; (ii) generating a moist environment for healing; (iii) protection of the wound area from mechanical trauma. Traditionally, cotton gauze has been used as a wound-dressing material that can protect against bacteria. Unfortunately, these dressings adhere to the wound area, causing pain and further tissue damage [5]. Various wound dressings containing antimicrobial agents or silver ions have been clinically used for wound protection, such as creams (e.g. Geben cream®, Tanabe Mitsubishi Pharma Co.) [6], hydrocolloids (e.g. Dodder®, ConvaTec Co.) [7], [8], hydrofibers (e.g. Aquacel®, Convatec) [9], [10] and gauze containing antimicrobial agents (e.g. Acticoat®, Smith and Nephew Co.) [11]. However, because none of these materials is transparent, management of the wound-care (or infection) process is difficult to monitor visually through the overlaid dressings. Appropriate burn-wound dressings with high transparency and potent antibacterial activity will allow early detection and/or prevention of wound infections.
The recent development of nanotechnology has enabled the fabrication of two-dimensional freestanding polymeric ultra-thin films called “polymer nanosheets” (or simply ‘nanosheets’) [12], [13], [14], [15], [16]. The nanosheet has unique properties [17], [18], [19] including high transparency, due to its amorphous structure and low surface roughness (RMS value <1 nm), and high flexibility and non-covalent adhesive strength properties, due to the huge aspect ratio of over 106. A layer-by-layer (LbL) method has been reported for the fabrication of ultra-thin films [20], [21], [22]. The LbL method employs alternating electrostatic adsorption of polycations and polyanions on a substrate. We previously reported the biomedical application of nanosheets consisting of biocompatible polysaccharides such as chitosan and sodium alginate [19], [23], [24], [25], [26]. In addition, these nanosheets display excellent biological properties, e.g. only slight adhesion to other organs and reduced levels of cell adhesion on the surface compared with other materials [27]. We also demonstrated the fabrication of antibiotic-loaded nanosheets, which were found to be effective for the treatment of tissue defects in the murine gastrointestinal tract [26].
In this study, we investigate the clinical efficacy of nanosheets that were loaded with the antibiotic tetracycline (TC-nanosheets) as a wound-protective material for full-thickness burn-wound infection in a murine model. In the case of intra-abdominal lesions, it is necessary to close the puncture and inhibit bacterial infection. However, in the case of full-thickness burns, it is necessary to protect the wound site and inhibit bacterial infection until tissue grafting. Therefore, covering and inhibiting bacterial infection using a TC-nanosheet is required for the treatment of both intra-abdominal lesions and full thickness burns. Moreover, we reasoned that the high transparency of the TC-nanosheet would be an advantage for wound management. When the TC-nanosheet covers the burn-wound infection site, it is possible to inhibit bacterial infection locally, resulting in a significant improvement in mortality. The high flexibility of the TC-nanosheet enables the wound site to be effectively covered with the nanosheet. Moreover, the transparency of the nanosheet allows the wound to be inspected visually during the period of treatment, which is particularly important for the management of full-thickness burns.
Initially, we performed an in vitro study to evaluate the antimicrobial effect of the TC-nanosheets against P. aeruginosa, which is a common bacterium found in damaged skin. Next, we prepared a wound infection model following full-thickness burn-injury in mice, where the dorsal skin was artificially burnt and infected with P. aeruginosa. Thereafter, burn-wound lesions were treated with the TC-nanosheet in vivo. The TC-nanosheet was then evaluated in terms of murine survival, white blood cell (WBC) count, viable bacterial count at the wound site, histological analysis and determination of bacterial number in the liver.
Section snippets
Materials
The biodegradable polyelectrolytes, chitosan (Mw = 88,000) and sodium alginate (Mw = 106,000) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Poly(vinylalcohol) (PVA, Mw = 22,000) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Poly(vinyl acetate) (PVAc, Mw = 100,000) was purchased from Sigma–Aldrich Japan (Tokyo, Japan). An antimicrobial agent, tetracycline hydrochloride (TC), was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Silicon wafers (SiO2 substrates),
Preparation and characterization of a TC-nanosheet
The thickness of the TC-nanosheet was determined using a surface profiler to be 177 ± 9 nm (Table 1). Cross-sectional SEM images gave a similar thickness (Fig. 3a). The three-layered structure of the TC-nanosheet was confirmed by CLSM (Fig. 3b).
The antimicrobial effect of the TC-nanosheet was evaluated by the KB test. TC released from the nanosheet inhibited the growth of P. aeruginosa, giving a ZOI value of 6.6 mm (Table 1). In addition, we optimized the amount of TC on the nanosheet by varying
Discussion
The TC-nanosheet was composed of three layers: LbL as a bottom layer, TC as an antibiotic layer and PVAc as a hydrophobic barrier layer. The cross-sectional CLSM image indicated that the TC layer was specifically incorporated between LbL and PVAc. The autofluorescent properties of TC can be exploited to visualize the TC-nanosheet. Other advantages of TC include its broad antibacterial spectrum, affordability and comparatively minor side effects [29]. Therefore, TC is one of the most widely used
Conclusions
We investigated the antimicrobial effect of a TC-nanosheet as a wound-protecting material for burn-wound infection in a murine model. The TC-nanosheet can be easily fixed on the burn-injured skin without chemical glue. Moreover, the transparent properties of the dressing material allow observation of the wound care management until tissue grafting can be performed. The TC-nanosheet has a potent antibacterial activity, thereby inhibiting burn-wound infection. We conclude that the TC-nanosheet is
Acknowledgments
This work was supported in part by “High-Tech Research Center” Project for Waseda University: matching fund subsidy (S.T.) from MEXT; grant-in-aid for Scientific Research (B) 21300181 from MEXT (S.T.); Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) from JST, Japan (T.F. and S.T.); and a grant-in-aid for the Special Research Program from the National Defense Medical College (D.S.).
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