Synergistic Antibacterial Activity and Wound Healing Properties of Selenium-Chitosan-Mupirocin Nanohybrid System: An in Vivo Study on Rat Diabetic Staphylococcus aureus Wound Infection Model

The current study aimed to formulate Selenium-Chitosan-Mupirocin (M-SeNPs-CCH) complex. The nanohybrid system was prepared using chitosan-cetyltrimethylammonium bromide (CTAB)-based hydrogel (CCH) that entrapped mupirocin (M) and selenium nanoparticles (SeNPs). The in vitro studies were performed by evaluation of the antibacterial activity and toxicity on L929 mouse fibroblast cell line. The in vivo study was conducted on rat diabetic wound infection model that was infected by mupirocin-methicillin-resistant Staphylococcus aureus (MMRSA). The wounds were treated by M-SeNPs-CCH nanohybrid system with concentrations of M; 20 mg/ml, CCH; 2 mg/ml and SeNPs; 512 μg/ml in two times/day for 21 days. The therapeutic effect of this nanohybrid system was evaluated by monitoring wound contraction and histopathological changes. Evaluation of the average wound healing time showed a significant difference between the treatment and control groups (P≤0.05). The histopathological study indicated that the amount of wound healing was considerable in M-SeNPs-CCH nanohybrid system groups compared to the control and M groups. The M-SeNPs-CCH nanohybrid system formulated in this study was able to reduce 3-fold MIC of mupirocin with synergistic antibacterial activity as well as to play a significant role in wound contraction, angiogenesis, fibroblastosis, collagenesis, proliferation of hair follicle, and epidermis growth compared to the control group (P ≤ 0.05). This research suggests that this nanohybrid system might be a development for the treatment of diabetic wound infection at mild stage.


Material and Methods
Selenium nanoparticles (Senps) synthesis. A stock of aqueous solution of 16 mM (4.208 mg/ml) sodium selenite pentahydrate (Na 2 SeO 3 .5H2O) and 80 mM (14.089 mg/ml) ascorbic acid (Merck, Germany) were prepared. 24 ml deionized water was added to 1 ml of Na 2 SeO 3 stock solution 22 . Ascorbic acid stock solution (1 ml) and tween 20 (20 μl) were added dropwise to Na 2 SeO 3 solution under magnetic stirring at 1200 rpm at room temperature 22,23 . After changing the solution color to the light orange at 40 min, the mixture was diluted to 25 ml with deionized water. The nanoparticles were characterized by UV-Vis Spectrophotometer (Lambda25, USA), Dynamic Light Scattering (DLS), Zeta Potential (Malvern Zeta sizer, UK) and Transmission Electron Microscopy (TEM; Zeiss -EM10C −80 KV, Germany). The nanoparticles were purified by centrifugation at 10,000 rpm for 10 min for use in in vitro and in vivo studies 24 .
Selenium content of the mixture was determined by Atomic Absorption Spectroscopy (AAS) 25 . ImageJ 1.52i software was used to calculate the average diameter of the nanoparticles observed in TEM micrographs. chitosan-n-cetyl-n,n,n-trimethylammonium bromide-based hydrogel synthesis (ccH). The hydrogel was synthetized based on a study previously described by de Oliveira Pedro R et al. In the present study CTAB was used instead of alkyltrimethylammonium bromide 17 . Deacetylated low molecular weight chitosan (1.5 g) (Sigma, Germany) was dissolved in 1% acetic acid solution in the final volume of 20 ml and pH was adjusted to 9.0 by adding NaOH (0.5 M) under magnetic stirring (1200 rpm). 20 ml N-cetyl-N,N,N-trimethylammonium bromide solution (1.0 g, 137.1 mmol) was added while stirring. The process was continued at 60 °C for 72 hours and pH was monitored during the reaction time. The mixture was then dialyzed (MWCO 12KD, Sigma, Germany) to remove the unreacted CTAB in the following steps: against water for 2 days, then aqueous NaOH (0.05 M) for 1 day, and finally water for 2 days. The product was lyophilized and then characterized by Brunauer-Emmett-Teller (BET) surface area and Scanning Electron Microscopy (SEM) 26 .
After completing the synthesis and characterization of SeNPs and CCH, mupirocin powder (Syngen Biotech Co, LTD, Taiwan) was dissolved in polyethylene glycol 400 (Scharlau, Spain) to entrap in the SeNPs-CCH, and formulate Selenium-Chitosan-Mupirocin (M-SeNPs-CCH) as the nanohybrid system. Antimicrobial susceptibility testing. The antimicrobial susceptibility testing was performed by disc diffusion and minimum inhibitory concentration (MIC) by broth dilution 27 method. Double checkerboard assay was used to determine M-CCH and M-SeNp fractional inhibitory concentration (FIC) 28,29 . toxicity assay. Dulbecco's Modified Eagle Medium-F12 (DMEM-F12, Sigma-Aldrich, USA) in 5 ml, 1 ml (10%) fetal bovine serum (FBS, Sigma-Aldrich, USA) and 1% Penicillin-Streptomycin (Aria CELL; Gibco/Sigma Source, Iran) were mixed and transferred to T-25 cell culture flask. The number of 1×10 4 cells were seeded to the flask and incubated at 37 °C in a humidified atmosphere of 5% CO 2 up to 7 days. After the third passage, the cells were harvested by Trypsin-EDTA 0.25 (Aria CELL; Sigma Source, Iran) 30,31 . Toxicity assay of M, SeNPs, CCH, along with their dual and triple combinations were tested in four folds higher than MIC concentrations using MTT cell viability assay kit (DNA Biotech Co, Iran) on mouse fibroblast connective tissue cell lines (L929, Iran Biological Resource Center), according to the manufacturer's protocol. The cells of each well were incubated with 5 mg/ml of MTT solution for 4 hours. Afterwards, formazan crystals were dissolved by dimethyl sulfoxide (DMSO) solution (DNA Biotech Co, Iran). The absorbance was measured at 570 nm by ELISA reader (Labsystem MultisKan, USA), and the cell viability percentage was calculated according to company protocol (DNA Biotech Co, Iran).
In vivo study. Diabetes induction: Approval of Tarbiat Modares University Ethics Committee was obtained before beginning the In vivo study [IR.TMU.REC.1395.414]. We declare that all methods were performed in accordance with the relevant guidelines and regulations. A total of 30 adult male Wistar rats weighing 225 ± 15 gr were fed with a high-fat diet containing 25% lipids (10% cholesterol). Two weeks later, the single low-dose of streptozotocin (STZ; Sigma) at a concentration of 40 mg/kg body weight was injected intraperitoneally to induce type 2 diabetes 32,33 . The rats' blood glucose levels were measured after 72 hour by blood glucose monitoring systems (Medisign, Korea).
Surgery, experimental infection and treatment: Three days after the induction of diabetes, the diabetic rats were anesthetized with 30 mg/kg ketamine (Alfasan, Holland) and 4 mg/kg xylazine (Alfasan, Holland). Their dorsal hair was shaved and disinfected with iodine, and full thickness round wound was created in the interscapular region of the upper back of each rat and the skin was excised using a punch biopsy (with a diameter of 8 mm) and iris scissors [34][35][36] . All of the wounds were inoculated with freshly 10 8 CFU mupirocin-resistant MRSA strains (30 μl). Immediately, the Comfeel Transparent (Coloplast, Denmark) were placed on the wounds and dressed with bundling. Three days after inoculation, all the wounds infections were confirmed by observation of infectious secretions and the presence of bacteria (gram staining and cultivation) 35,36 . The infectious wounds were divided into five groups including group 1: control/not treated group (Infectious Wound: IW), group 2; M, group 3; M-CCH, group 4; M-SeNPs, and group 5; M-SeNPs-CCH. Depending on the treatment groups, the wounds were treated by mupirocin (20 mg/ml) 36 , CCH (2 mg/ml) and SeNPs (512 μg/ml) twice a day for 21 days. Four of 30 rats (13.3%) died during the period of induction of diabetes, surgery and treatment.
Macroscopic and microscopic evaluation: The images were taken from wounds on days 1, 3, 7, 10, 14, 17, and 21. The average area of wounds was measured by ImageJ 1.52i software. The percentage of wound contraction was calculated using the following equation: Percentage of wound contraction Initial wound size Specific day wound size Initial wound size = − Tissue sampling was carried out for each group on days 3, 7, 14, and 21. All tissue samples were placed in a 10% formalin solution for 24 hours, dehydrated, and paraffin embedded using a tissue processor (DS 2080\H; Did Sabz Co, Iran). The tissue sections were stained with hematoxylin and eosin (H & E) and Trichrome-Masson staining was used for histopathological studies. The angiogenesis, fibroblastosis, hair follicles proliferation, epidermis growth, inflammation and collagenesis were evaluated in all tissue sections [37][38][39] .
Statistical analysis: Data was analyzed by SPSS 16 software. Non-parametric Kruskal-Wallis test was used because of the low sample size in each group for comparing the target groups and Mann-Whitney test was used for comparing the groups. P-value less than 0.05 was considered as statistically significant. toxicity assay results. Results of the toxicity assay revealed that the highest viability level at the same concentration (1024 μg/ml) was related to M (85%) in comparison with SeNPs (71.6%) and CCH (61.8%). The viability percentage of M, SeNPs, CCH, as well as their dual and triple combinations are shown in Fig. 3. The results were the same based on the independent third test (P ≤ 0.05).
In vivo study. Macroscopic evaluation: The macroscopic evaluation results showed that the average wound healing of treated groups (M-SeNPs-CCH, M-SeNPs, M-CCH and M) were higher than infectious wound (IW) group on days 7, 10, 14, 17 and 21 (Fig. 4). The M-SeNPs-CCH-treated group showed considerable healing of 80% in 10 days. The M-SeNPs and M-SeNPs-CCH-treated groups showed 90 and 92% in 21 days, respectively. In addition, the wound contraction in M-SeNPs-CCH and M-SeNPs-treated groups was higher than M and M-CCH-treated groups. The percentage of wound healing is shown in Fig. 5 Table 1). The results were obtained based on three independent tests (P ≤ 0.05).

Discussion
In this study, SeNPs and CCH carrier systems were synthesized, characterized and optimized. Then, by entrapping mupirocin in the nanocarrier system, M-SeNPs-CCH was prepared. The SeNPs characterization result by UV/VIS spectrophotometer, DLS (PDI = 0.170, mean diameter = 66.48), zeta potential measurement (−34 mV) and TEM (mean diameter = 66.48) results indicated favorable characteristic features. BET and Langmuir surface area measurements of CCH were 0.9680 and 2.3074 m 2 /g, respectively. Taking the available reports on the antibacterial properties of individual selenium nanoparticles and chitosan into consideration, the M-SeNPs-CCH nanohybrid system in this study was designed to investigate its possible synergistic effects on mupirocin MIC reduction and wound healing in the rat diabetic infection model. To increase the antibacterial properties of chitosan, cetyltrimethylammonium bromide (CTAB) was used during the preparation of chitosan hydrogel. The study conducted by de Oliveira Pedro et al., reported that propyl and pentyl trimethylammonium bromides induce greater antimicrobial activities on chitosan 17,18 . In the current study, we used CTAB to increase the antimicrobial activity of chitosan.
There are some studies on chitosan hydrogel in relation to this study. Verma et al., reported antimicrobial and wound healing activity for sericin-chitosan-capped silver nanoparticle (S/C-SNP)-loaded hydrogel 19 . Masood et al., treated diabetic wounds with chitosan-PEG-Silver Nitrate-based hydrogel, and confirmed improvement in antibacterial activity and diabetic wound healing 40 . Xie et al., found that entrapment of silver nanoparticles into chitosan hydrogels could increase its antibacterial activity. In addition to wound healing properties of hydrogel, this study also showed that the hydrogel significantly improved the re-epithelialization and collagen deposition 41 . Another study by Kumar et al. on chitosan hydrogel/nano zinc oxide composite bandages (CZBs) indicated that combination of zinc oxide nanoparticles with chitosan hydrogel improved antibacterial activity and wound healing. Moreover, the nanocomposite accelerated re-epithelialization and collagen deposition. Kumar et al. also suggested application of this system in treatment of diabetic wounds 42 .
The M-SeNPs-CCH system formulated in this study was able to reduce mupirocin MIC 3-fold by macrobroth dilution. Furthermore, M-SeNPs and M-CCH reduced the MIC value by microbroth dilution checkerboard assay www.nature.com/scientificreports www.nature.com/scientificreports/   www.nature.com/scientificreports www.nature.com/scientificreports/ one and two fold, respectively. Also, the checkerboard results of M-CCH represented an additive effect. Due to the difficulty of aggregating nature of M-SeNPs-CCH, the triple checkerboard was not performed, and the disc diffusion test was used instead. Based on the latter assay, increase in the diameter of inhibition zone of M-SeNPs, M-CCH and M-SeNPs-CCH (22, 23 and 24 mm, respectively) compared to the M (19 mm), revealed an increase in antibacterial activity.
Some studies have reported that SeNPs inhibit S. aureus growth [43][44][45] . In this study, although the SeNPs reduced the growth of bacteria by one fold, there was no total inhibition of bacteria by this nanoparticle. Therefore, to obtain complete inhibition by this nanoparticle, further investigations are required. Considering other properties of SeNPs, including immunomodulatory 20 , anti-inflammatory 46 , and anti-oxidant 47 properties, it seems that these nanoparticles can be promising candidates for combination with other antibiotics.
The toxicity assay result of this study revealed that the highest viability level at the same concentration (1024 μg/ml) is related to M (85%); in comparison with SeNPs (71.6%) and CCH (61.8%). According to the previous studies, toxicity of SeNPs is less than that of Se 15,20 . In this research, reduction in the viability percentage of CCH (71.6%) compared to chitosan can be due to the presence of CTAB.
According to the macroscopic evaluation results, the amount of wound healing is as follow: M-SeNPs-CCH> M-SeNPs> M-CCH> M> IW. This indicates that SeNPs has a more efficient effect on wound contraction than that of CCH. Also, the wound contraction in M-SeNPs-CCH, M-SeNPs and M-CCH was more than M group, with a remarkable healing effect compared to the control group (IW). The wound contraction was notable in the M-SeNPs-CCH group for 21 days. The average wound healing time of the treated groups were significant compared to the IW group on days 7, 10, 14, 17, and 21 (P ≤ 0.05).
The microscopic results of H & E and Trichrome-Masson staining showed increment in the amount of collagenization and epidermization in all three groups of M-SeNPs, M-CCH and M-SeNPs-CCH during 21 days. Based on angiogenesis results, the M-CCH group was significant compared to the M-SeNPs group in all days (P ≤ 0.05). Also, the M-SeNPs and M-CCH groups were highly significant compared to the other groups in all days (P ≤ 0.05). In fibroblastosis, epidermis growth and hair follicle proliferation investigations, the M-CCH group was highly significant compared to the other groups (P ≤ 0.05). In collagenesis study, the M-SeNPs group was highly significant, and in inflammation results, it was significant compared to the other groups (P ≤ 0.05).
The two groups of M-CCH and M-SeNPs-CCH showed the highest levels of fibroblasts and optimum vascularization on day 14, showing their significant effect on wound healing. On the other hand, the amount of inflammation decreased sharply during 21 days. Histopathology evaluation showed that the acceleration of wound healing in two groups of M-CCH and M-SeNPs-CCH were much higher than the other treatment groups during 21 days. This indicated that the presence of chitosan hydrogel in M-CCH and the SeNPs-chitosan hydrogel in the nanohybrid system played a significant role in controlling the infection and accelerating the wound healing as well as forming the main layers of the skin during 21 days.
An overall comparison of the macroscopic and microscopic evaluation results indicated that the presence of SeNPs had a significant role in wound contraction and collagenesis, and the presence of CCH was effective in angiogenesis, fibroblastosis and proliferation of hair follicle and epidermis (P ≤ 0.05). Considering the role of SeNPs and CCH in this study, these substances appear to be essential in such types of formulations.
conclusion. The M-SeNPs-CCH nanohybrid system formulated in this study was able to reduce 3-fold MIC of mupirocin. The system also played a significant role in wound contraction, angiogenesis, fibroblastosis, collagenesis, and proliferation of hair follicle and epidermis compared to the control groups (P ≤ 0.05). In addition to antimicrobial activity against MRSA strain and reduction of MIC value of mupirocin, the SeNPs and CCH could play a vital role in the wound healing process in rat model of diabetic wound infection. Thus, it seems that the presence of SeNPs and CCH to be essential in this formulated system. Therefore, this research suggests that SeNPs and CCH can be considered as promising candidates in developing of mupirucin-based drugs for the treatment of diabetic wound infection at the mild stage.

Data availability
The data sets and analysis of current study are available from the corresponding author upon reasonable request.