# Development of Chitosan-Assisted Fe3O4@SiO2 Magnetic Nanostructures Functionalized with Nisin as a Topical Combating System against Vancomycin-Intermediate Staphylococcus aureus (VISA) Skin Wound Infection in Mice

Authors: Mona Nasaj; Abbas Farmany; Leili Shokoohizadeh; Farid Aziz Jalilian; Reza Mahjoub; Ghodratollah Roshanaei; Alireza Nourian; Omid Heydari Shayesteh; Mohammad Reza Arabestani
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

The clinical significance of vancomycin-intermediateS. aureus (VISA) infections is intensified by its tendency to develop resistance to antimicrobials and persistent infections. The decreasing effectiveness of the antimicrobials available is now seriously compromised; thus, there is an emergent need to invent new classes of antimicrobial agents that can rapidly and efficiently eradicate infections. Fe3O4@SiO2@chitosan (CS) nanocomposites were successfully synthesized and then decorated with nisin to gain Fe3O4/SiO2/CS/nisin-based magnetic nanostructures (Fe3O4@SiO2@CS-NISIN). The nanomaterials were characterized comparatively via FTIR (Fourier transform infrared spectroscopy), XRD (X-ray diffraction), FE-SEM (field emission scanning electron microscopes), DLS (dynamic light scattering), and VSM (vibrating sample magnetometer) methods. The methyl thiazol tetrazolium (MTT) assay was performed to determine the inhibitory effects of antibacterial agents on the cell viability. The in vitro bactericidal effect of all compounds was characterized using the microdilution assay. Finally, the topical antibacterial efficacy of free nisin, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites against murine superficial wound infection models was determined. The functionalized nanocomposites were more efficient in suppressing bacterial growth in vitro and in vivo compared to the same quantities of untargeted nanocomposites. MTT results showed acceptable biocompatibility of all nanoformulations, and no substantial difference in the cell viability was recorded between treated cells and untreated control. These results suggest that Fe3O4@SiO2@CS-NISIN nanocomposites can be served as an alternative antimicrobial agent in innovative and emerging technologies to treat a variety of staphylococcal infections.


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## Body

## 1. Introduction

Staphylococcus aureus (S. aureus) is strongly connected with a broad range of clinical problems, including skin and soft tissue infections to life-threatening diseases such as endocarditis and sepsis [1]. Infections with strains of MRSA are often resistant to methicillin and nearly all B-lactam antibiotics. In the 1980s, the treatment of MRSA infections began to shift towards the use of glycopeptide antibiotics, in particular vancomycin. Unfortunately, the pressure to use glycopeptide antibiotics resulted in the appearance of vancomycin-intermediate-resistant S. aureus (VISA), first reported in Japan in 1997. Since then, VISA has been increasingly isolated from hospitals around the world [2]. The in vivo development of strains with the intermediate level resistance to vancomycin, known as VISA, has resulted in treatment failure and prolonged hospitalization. Although VISA strains are known to be related to persistent bacteremia, they do not generally lead to critical clinical complications or lethal sepsis, indicating that reduced susceptibility to vancomycin may damage the pathogenicity of S. aureus [1]. In addition to the vancomycin-intermediated phenotype, VISA strains appear to share some common characteristics such as thickened cell walls, reduced autolytic activity, and impaired virulence traits [3]. Infections caused by VISA are more often associated with failure of vancomycin therapy and lead to prolonged hospitalization, a higher risk of persistent infection, and increased treatment costs [4, 5]. Thus, the successful treatment of these infections has become more difficult and remains a major challenge; therefore, it is an imperative need to develop modern strategies to counter MRSA/VISA infections. Nisin, a prototype of the lantibiotic group of antimicrobial peptides, has been approved by the US Food and Drug Administration (FDA) for application in the food industry. Nisin is found to exert bactericidal effects towards a great amount of Gram-positive bacterial organisms, including crucial food-borne pathogens and various clinically important antibiotic-resistant bacteria, such as staphylococci, bacilli, mycobacteria, and clostridia; in addition, nisin is also known to be activating autolytic responses in the staphylococcal cell wall [6]. Although nisin has much low or no effect against Gram-negative bacteria and fungi, its conjunction with other compounds such as chelating agents can extend its spectrum of action to include Gram-negative bacteria [7]. Nisin and other lantibiotics have attracted great attention because they are characterized by potent and broad-spectrum antimicrobial effects, a low tendency to develop bacterial resistance, and relatively slight toxicity to mammalian cells at antimicrobial concentrations [8–11]. Chitosan (CS) is recognized as the second most abundant hydrophilic amino biopolymer naturally appearing on earth, next to cellulose, and is made from marine crustacean shells [12, 13]. CS and its derivatives are being widely investigated for application in various fields of biomedical and drug delivery [14, 15], due to some useful features such as nontoxicity, biodegradability, biocompatibility, immunological neutrality, hydrophilicity, antimicrobial capacity, anti-inflammatory, antitumor, and wound healing effects [16]. Magnetic-based nanoparticles (MNPs) have recently attracted special consideration due to their distinct physicochemical properties, especially ready to modify and biocompatibility [17, 18]. MNPs have the capacity to carry high dosages of pharmaceutical agents, thus achieving effective topical content and avoiding toxic and other undesirable effects resulting from large doses of drugs in other parts of the body [19]. The most attractive advantage of MNPs in the drug delivery involves controlled release of the therapeutics at the target site via external magnetic fields, enhancing the accumulation of drugs at the site of cutaneous lesions and potentially improving therapeutic efficacy [20, 21]. The MNPs have a large surface area-to-volume ratio that yields numerous chemically active sites. High surface free energy can trigger the formation of nanoparticle agglomerates and decrease the available active sites [22]. Coating magnetic nanoparticles with organic or inorganic materials can reduce the surface magnetic force and avoid particle agglomeration [23]. An inorganic capping agent, namely, silica (SiO2), with a low toxicity and the extraordinary physicochemical characteristics, is capable of improving stability and protecting the Fe3O4 MNPs from dissolution under acidic conditions, agglomeration, and chemical oxidation and also presents the suitable potential for modifying the surface [22, 24]. Some scientists found that naked Fe3O4-MNPs can trigger the generation of free hydroxyl radical species which interact with intracellular components such as endogenous DNA, resulting in impaired cellular function and proliferation [25, 26]. It has been documented that capping Fe3O4 MNPs by either inorganic materials (silica, gold) or synthetic/natural organic materials effectively prevents iron leaching in acidic intracellular degradation pathways and greatly improves the biocompatibility of metal particles for cellular applications, particularly at high magnetite loadings [27, 28]. In this work, we prepared Fe3O4 supermagnetic NP grafting with nisin to improve antibacterial property against VISA strain. Due to the ability to agglomerate, these nanoparticles are quickly removed from blood circulation through opsonization and phagocytosis by RES [29]. To prevent this issue, we should shield the surface of MNPs with biodegradable and biocompatible SiO2 layer leading to increase stability. To enhance the surface characteristics of MNPs, we also employed the polycationic chitosan crosslinked with glutaraldehyde.


## 2. Materials and Methodology

### 2.1. Materials

Ferrous chloride (FeCl2·4H2O), ferric chloride (FeCl3·6H2O), ammonium hydroxide (NH3·H2O, 25 wt%), hydrochloric acid (HCl, 35 wt%-37 wt%), sodium acetate (C2H3NaO2), tetraethyl orthosilicate (TEOS), glutaraldehyde (25 wt%), ethanol (96%), and vancomycin hydrochloride were purchased from Sigma Aldrich (MO, USA). Polyethylene glycol (PEG-400), medium molecular weight chitosan (MMWC, deacetylation degree of 75–85%), nisin from Lactococcus lactis 2.5% (balance sodium chloride), blood agar, and Mueller-Hinton broth (MHB) were obtained from Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco, USA. Male NMRI mice, 6–8 weeks old, were purchased from the Center of Experimental and Comparative Study (Iran University, Tehran). We have also used Gram-positive bacteria vancomycin-intermediate Staphylococcus aureus (VISA), from Hamadan University of Medical Sciences, Hamadan, IRAN.


### 2.2. Preparation of Fe3O4 Magnetic Nanoparticles

Monodispersed Fe3O4 magnetic nanoparticles (MNPs) were synthesized based on the chemical coprecipitation method as already described with some modifications: typically, 4.6 g FeCl3·6H2O, 2.3 g FeCl2·4H2O, and 0.6 g PEG-400 were dissolved in 100 mL of 1.2 mmol/L aqueous HCl in an ultrasonic bath for 30 min (Elma, Germany), then incubated with vigorous mechanical stirring (500 rpm) at 80°C (Hot Plate, Pole Ideal Tajhiz, Iran), and exposed to a high-purity nitrogen atmosphere. After continued stirring for 30 min, 20 mL NH3·H2O (25%) containing 0.3 g PEG-400 solution was slowly poured dropwise into the reaction mixture over 20 min until reaching pH to the alkaline state, at which the solution color changed from brown to dark brown and then became black. Upon lowering the temperature of the mixture, the ultrafine MNPs were repeatedly washed at first with deionized water and then ethanol until their pH became neutral, followed by drying in a vacuum oven (Bionics Scientific Technologies (P) Ltd., India) at 70°C for 10 h [30].


### 2.3. Synthesis of the Fe3O4@SiO2 Core/Shell Nanocomposites

The silica-coated Fe3O4 MNPs (Fe3O4@SiO2) were prepared based on the sol-gel approach according to the previously reported works with modifications [31]. 2 g of synthesized Fe3O4 particles was initially dispersed into a 200 mL mixture comprising ethanol and deionized water (160 : 40) then subjected to ultrasonic irradiation for 15 min using a probe sonicator (Vibra-Cell™ Jencons Scientific Ltd., USA). 30 min after the addition of ammonia solution (4 mL, 25 wt%), 2 mL of TEOS was dropped into the reaction solution, and the resulting suspension was vigorously stirred for 6 h. Silica was formed on the surface of the MNPs through hydrolysis and condensation of TEOS. Subsequently, the Fe3O4/SiO2 nanostructures were washed with ethanol (3×15mL) and dried under vacuum at 60°C for 12 h.


### 2.4. Preparation of Chitosan-Coated Fe3O4@SiO2 Nanocomposites

To prepare chitosan-modified Fe3O4@SiO2 MNPs, the nanostructures (0.5 gr) were first dispersed in distilled water (30 mL) under ultrasonication vibration for about 30 min. Then, 70 mL of acetic acid (CH3COOH) solution of CS (0.5 g CS in 100 mL 2% CH3COOH) was added in a dropwise manner into the nanocomposites aqueous solution under constant mechanical stirring at 50°C. The stirring of the reaction mixture was continued for about 3 h, followed by crosslinking of the nanocomposites with 2.5% glutaraldehyde at 40°C for 2 h to increase their mechanical and chemical stability. Finally, the chitosan/magnetite nanoparticles were recovered from the solution with a strong neodymium magnet; after that, the precipitated product was rinsed with ethanol several times, and the drying process was accomplished under vacuum at 60°C for 2 h [32].


### 2.5. Preparation of Calibration Curve of Nisin

Nisin solutions with different concentrations of 62.5, 125, 250, 500, 1000, and 2000μg/mL were prepared separately by dissolving 562.5 μg, 1.125 mg, 2.25 mg, 4.5 mg, 9, and 18 mg nisin in 9 mL of phosphate buffer saline (PBS, pH 7.4), 9 mL of Tris buffer (pH, 9.5), and 9 mL of sodium acetate buffer (pH, 9.5), followed by adding 1 mL of Coomassie Brilliant Blue solution as the chromogenic agent to each mixture solution; three mixtures containing 9 mL each of buffer solutions and 1 mL Brilliant Blue dye were considered as control. Subsequently, a UV-Vis spectrophotometer (SPECORD 205, Analytik Jena, China) was employed to determine the absorbance of each mixture solution at 595 nm [33].


### 2.6. Modification of Fe3O4@SiO2@CS Nanocomposites with Nisin

Covalent binding of nisin onto Fe3O4@SiO2@CS nanocomposites was achieved by dropping 50 μL of glutaraldehyde (25% v/v) into the particle suspensions (2000 μg of Fe3O4@SiO2@CS in 150 μL of sodium acetate buffer, 50 mM, pH 5.5), and the resulting suspension was allowed to proceed by shaking at 80 rpm for 1 h (Shaking Incubator (SI-100 R), Hanyang Scientific Equipment Co., Ltd., Korea). Thereafter, the samples were collected using twice washings with sodium acetate buffer (50 mM, pH 5.5), followed by rapid addition of a predetermined amount of nisin (2000 μg) to the activated Fe3O4@SiO2@CS suspensions. Shaking was continuous overnight at 25°C. At the end of the experiment, excessive nisin removal and triplicate washings were carried out using an acetate buffer solution and centrifugation (Centrifuge 5425, Eppendorf North America Co, US), at 13000×g for 20 min [34].


### 2.7. Determination of the Binding Efficiency of the Fe3O4@SiO2@CS Nanocomposites

The nisin-loaded Fe3O4@SiO2@CS composites were collected by ultrafiltration technique (Amicon, Ultracel-100 K, 100 kDa cutoff) after centrifuging at 2000×g and 4°C for 15 min to separate the uncoated peptides from the nanoparticles (Centrifuge 5920 R, Eppendorf North America Co, US). The residual nisin content in the outer tube was measured using Bradford protein assay with a UV-Vis microspectrophotometer (Synergy HTX, BioTek com, US) at a wavelength of 595 nm. Three repeats of all experiments were considered, and the average absorbance values were calculated; the final amount of loaded nisin was calculated indirectly with [35, 36]
(1)Loadingrate%=nisintotal−nisinsupernatantnisintotal×100.


### 2.8.In Vitro Release Profile of Nisin from the Nanocomposites

The nisin-loaded nanocomposites were separated from the aqueous suspension using the ultracentrifugation technique at2000×g for 20 min at 4°C; in the experiment, 20 mL of Fe3O4@SiO2@CS-NISIN composite solution in Tris buffer (pH 9.5, 4 mg/mL) and phosphate-buffered saline (PBS, pH: 7.4, 4 mg/mL) was separately poured into a conical flask containing 50 mL dissolution medium and incubated at 25°C under continuous stirring (100 rpm, 24 h). At appropriate intervals of 0, 30, 60, 120, 240, 300, 360, 420, and 520 min, 5 mL of the solution was used for withdrawal accompanied by simultaneous replacement of an equal volume of fresh medium, loaded into an ultrafiltration tube (M.W. cutoff of 30 kDa) and centrifuged at 2000×g for 20 min to remove possible insoluble constituents. After centrifugal separation, the nisin concentration in the supernatant was analyzed by the Bradford method, all release tests were performed in triplicate, and the mean value was reported [37, 38].


### 2.9. Characterization Methods

Chemical modification of Fe3O4 MNPs (Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2-CS-NISIN) was characterized using the Fourier transform infrared (FT-IR) spectra of the nanocomposites on an FTIR spectrometer (Bruker Co., Germany) with a resolution of 4 cm-1 in the range 4000–400 cm-1 using KBr pellets. The magnetic properties of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites were evaluated at room temperature (300 K) using a vibrating sample magnetometer (VSM, Quantum, USA). The X-ray diffraction (XRD) patterns of Fe3O4 and Fe3O4@SiO2@CS-NISIN composite nanoparticles were collected using an X-ray diffractometer (D8 ADVANCE, Bruker, Germany). A field emission scanning electronic microscope (FE-SEM, TSCAN, Czech Republic) was employed to evaluate the surface morphological characteristics and size of Fe3O4 and Fe3O4@SiO2 MNPs. Before measurements, the samples were covered with a conductive gold layer to avoid charging issues during examination. Hydrodynamic size and zeta potential measurements were detected via dynamic light scattering (DLS) technique using Zetasizer Nano ZS 3600 (Malvern, Worcestershire, United Kingdom).


### 2.10.In Vitro Antibacterial Behavior of Therapeutic Agents

#### 2.10.1. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

To measure the MIC and MBC values of Fe3O4@SiO2, Fe3O4@SiO2@CS, nisin, and Fe3O4@SiO2@CS-NISIN against the VISA strain, a standard broth microdilution susceptibility testing procedure was conducted using Mueller-Hinton broth (MHB) as recommended by the Clinical and Laboratory Standards Institute (CLSI) [39]. Briefly, the composites and nisin were diluted in 2-fold increments; for free nisin and Fe3O4@SiO2@CS-NISIN nanocomposites, different concentrations were prepared in the range of 768–12 μg/mL, and for Fe3O4@SiO2 and Fe3O4@SiO2@CS nanocomposites, concentrations ranged from 4000 to 125 μg/mL. A previously prepared culture of VISA (OD625 of 0.5) was diluted to a final concentration of 106 CFU/mL in MHB culture. The bacterial inocula and prepared antimicrobial compound dilutions were added in a 1 : 1 ratio to individual wells of a 96-well microplate and then incubated for 24 h at 37°C (Incubator KB, BINDER Inc., US). The lowest concentration of the antimicrobial substance at which by visual reading the microorganism growth was completely inhibited was determined as the MIC value. To estimate the MBC of the antimicrobials, a viable cell count was carried out: briefly, the bacterial suspensions (10 mL) of each well, considered as the MIC value, were spread on the MH agar plates, followed by bacterial colony count after incubation at 37°C for 24 h. The lowest antimicrobial concentration, where more than 99.9% of the total bacterial population was killed, was termed the MBC endpoint.


### 2.11. The Cell Viability Assessment

Thein vitro cytotoxicity effects of materials were identified by MTT assay in mouse L-929 fibroblast cells. Briefly, the cells were plated overnight into 96-well microtiter plates (Sigma, Germany) at a cell density of 1×105 cells per well with a volume of 200 μL complete DMEM, incubated at 37°C under an atmosphere of 5% CO2. Cells were then exposed to various treatments with the nanocarriers: 125, 250, 500, 1000, 1500, 2000, and 4000 μg/mL Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS; 12, 24, 48, 96, 192, 384, and 768 μg/mL free nisin and Fe3O4@SiO2@CS-NISIN composites. Following 24 hours of incubation at 37°C, each well was carefully resuspended in 10 μL of a 5 mg/mL MTT solution, and the plates were incubated for an additional four hours at 37°C, after which the MTT solution was removed from each well. The viable cells were exposed to 100 μL of dimethyl sulfoxide (DMSO) (Millipore Sigma) to solubilize the purple formazan crystals formed in the cells. After gently shaking the plate for 15 min with an orbital shaker (ES 20, Labortechnik, Germany), the absorbance of the wells was recorded with a microplate reader (680 XR, Bio-Rad, USA) at λmax=570nm to calculate cell viability.


### 2.12. Animal Studies

Thein vivo antibacterial efficacy of nisin, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites was evaluated in the healing of full-thickness wounds of mice according to the international guidelines’ principles of laboratory animal use and care [40]. Male NMRI mice, 6-8 weeks old, were divided into three groups (8 animals per group): nisin, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites. Animals were kept under sanitary circumstances in a laboratory with a temperature of 22–25°C, a humidity of 40–70%, a light/dark cycle of 12 h, and free access to standard rodent feed and water ad libitum. After 2 weeks of acclimation, the animals were anesthetized with ketamine (40 mg/kg, Woerden, Netherlands) and xylazine (20 mg/kg, Woerden, Netherlands) through intraperitoneal injection. Afterward, a shaved area of the skin on the animal back was disinfected with 70% ethanol, and full-thickness circular excisions (7 mm diameter) were created on the dorsal midline of the animal using a sterile 4 mm biopsy punch, after which 100 μL of a bacterial suspension at a concentration of 3×108CFU/mL was immediately introduced into each wound site. Treatment was performed according to the MIC dose for Fe3O4@SiO2@CS composites and at considerably greater dosages (4-fold higher than the MIC concentrations) of nisin (96 μg/mL) and Fe3O4@SiO2@CS-NISIN nanocomposites (500 μg/mL) to evaluate the antibacterial capacity to achieve a therapeutic effect in vivo. Nisin and Fe3O4@SiO2@CS composites were administered every 12 hours. However, Fe3O4@SiO2@CS-NISIN nanocomposites were used as a single daily dose. Subsequently, on days 4, 9, and 14, the mice were anesthetized and sacrificed, tissues from the wound area were harvested, and after homogenization, the bacterial cell count was determined.


### 2.13. Statistical Analysis

The variance (ANOVA) procedure and Tukey analysis are used for comparing the differences between relevant groups mean and to describe the possible relationship between variables. All tests were conducted in triplicate, and the differences of the mean findings had to be accepted at the significant levelp lower than 0.05.


## 2.1. Materials

Ferrous chloride (FeCl2·4H2O), ferric chloride (FeCl3·6H2O), ammonium hydroxide (NH3·H2O, 25 wt%), hydrochloric acid (HCl, 35 wt%-37 wt%), sodium acetate (C2H3NaO2), tetraethyl orthosilicate (TEOS), glutaraldehyde (25 wt%), ethanol (96%), and vancomycin hydrochloride were purchased from Sigma Aldrich (MO, USA). Polyethylene glycol (PEG-400), medium molecular weight chitosan (MMWC, deacetylation degree of 75–85%), nisin from Lactococcus lactis 2.5% (balance sodium chloride), blood agar, and Mueller-Hinton broth (MHB) were obtained from Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco, USA. Male NMRI mice, 6–8 weeks old, were purchased from the Center of Experimental and Comparative Study (Iran University, Tehran). We have also used Gram-positive bacteria vancomycin-intermediate Staphylococcus aureus (VISA), from Hamadan University of Medical Sciences, Hamadan, IRAN.


## 2.2. Preparation of Fe3O4 Magnetic Nanoparticles

Monodispersed Fe3O4 magnetic nanoparticles (MNPs) were synthesized based on the chemical coprecipitation method as already described with some modifications: typically, 4.6 g FeCl3·6H2O, 2.3 g FeCl2·4H2O, and 0.6 g PEG-400 were dissolved in 100 mL of 1.2 mmol/L aqueous HCl in an ultrasonic bath for 30 min (Elma, Germany), then incubated with vigorous mechanical stirring (500 rpm) at 80°C (Hot Plate, Pole Ideal Tajhiz, Iran), and exposed to a high-purity nitrogen atmosphere. After continued stirring for 30 min, 20 mL NH3·H2O (25%) containing 0.3 g PEG-400 solution was slowly poured dropwise into the reaction mixture over 20 min until reaching pH to the alkaline state, at which the solution color changed from brown to dark brown and then became black. Upon lowering the temperature of the mixture, the ultrafine MNPs were repeatedly washed at first with deionized water and then ethanol until their pH became neutral, followed by drying in a vacuum oven (Bionics Scientific Technologies (P) Ltd., India) at 70°C for 10 h [30].


## 2.3. Synthesis of the Fe3O4@SiO2 Core/Shell Nanocomposites

The silica-coated Fe3O4 MNPs (Fe3O4@SiO2) were prepared based on the sol-gel approach according to the previously reported works with modifications [31]. 2 g of synthesized Fe3O4 particles was initially dispersed into a 200 mL mixture comprising ethanol and deionized water (160 : 40) then subjected to ultrasonic irradiation for 15 min using a probe sonicator (Vibra-Cell™ Jencons Scientific Ltd., USA). 30 min after the addition of ammonia solution (4 mL, 25 wt%), 2 mL of TEOS was dropped into the reaction solution, and the resulting suspension was vigorously stirred for 6 h. Silica was formed on the surface of the MNPs through hydrolysis and condensation of TEOS. Subsequently, the Fe3O4/SiO2 nanostructures were washed with ethanol (3×15mL) and dried under vacuum at 60°C for 12 h.


## 2.4. Preparation of Chitosan-Coated Fe3O4@SiO2 Nanocomposites

To prepare chitosan-modified Fe3O4@SiO2 MNPs, the nanostructures (0.5 gr) were first dispersed in distilled water (30 mL) under ultrasonication vibration for about 30 min. Then, 70 mL of acetic acid (CH3COOH) solution of CS (0.5 g CS in 100 mL 2% CH3COOH) was added in a dropwise manner into the nanocomposites aqueous solution under constant mechanical stirring at 50°C. The stirring of the reaction mixture was continued for about 3 h, followed by crosslinking of the nanocomposites with 2.5% glutaraldehyde at 40°C for 2 h to increase their mechanical and chemical stability. Finally, the chitosan/magnetite nanoparticles were recovered from the solution with a strong neodymium magnet; after that, the precipitated product was rinsed with ethanol several times, and the drying process was accomplished under vacuum at 60°C for 2 h [32].


## 2.5. Preparation of Calibration Curve of Nisin

Nisin solutions with different concentrations of 62.5, 125, 250, 500, 1000, and 2000μg/mL were prepared separately by dissolving 562.5 μg, 1.125 mg, 2.25 mg, 4.5 mg, 9, and 18 mg nisin in 9 mL of phosphate buffer saline (PBS, pH 7.4), 9 mL of Tris buffer (pH, 9.5), and 9 mL of sodium acetate buffer (pH, 9.5), followed by adding 1 mL of Coomassie Brilliant Blue solution as the chromogenic agent to each mixture solution; three mixtures containing 9 mL each of buffer solutions and 1 mL Brilliant Blue dye were considered as control. Subsequently, a UV-Vis spectrophotometer (SPECORD 205, Analytik Jena, China) was employed to determine the absorbance of each mixture solution at 595 nm [33].


## 2.6. Modification of Fe3O4@SiO2@CS Nanocomposites with Nisin

Covalent binding of nisin onto Fe3O4@SiO2@CS nanocomposites was achieved by dropping 50 μL of glutaraldehyde (25% v/v) into the particle suspensions (2000 μg of Fe3O4@SiO2@CS in 150 μL of sodium acetate buffer, 50 mM, pH 5.5), and the resulting suspension was allowed to proceed by shaking at 80 rpm for 1 h (Shaking Incubator (SI-100 R), Hanyang Scientific Equipment Co., Ltd., Korea). Thereafter, the samples were collected using twice washings with sodium acetate buffer (50 mM, pH 5.5), followed by rapid addition of a predetermined amount of nisin (2000 μg) to the activated Fe3O4@SiO2@CS suspensions. Shaking was continuous overnight at 25°C. At the end of the experiment, excessive nisin removal and triplicate washings were carried out using an acetate buffer solution and centrifugation (Centrifuge 5425, Eppendorf North America Co, US), at 13000×g for 20 min [34].


## 2.7. Determination of the Binding Efficiency of the Fe3O4@SiO2@CS Nanocomposites

The nisin-loaded Fe3O4@SiO2@CS composites were collected by ultrafiltration technique (Amicon, Ultracel-100 K, 100 kDa cutoff) after centrifuging at 2000×g and 4°C for 15 min to separate the uncoated peptides from the nanoparticles (Centrifuge 5920 R, Eppendorf North America Co, US). The residual nisin content in the outer tube was measured using Bradford protein assay with a UV-Vis microspectrophotometer (Synergy HTX, BioTek com, US) at a wavelength of 595 nm. Three repeats of all experiments were considered, and the average absorbance values were calculated; the final amount of loaded nisin was calculated indirectly with [35, 36]
(1)Loadingrate%=nisintotal−nisinsupernatantnisintotal×100.


## 2.8.In Vitro Release Profile of Nisin from the Nanocomposites

The nisin-loaded nanocomposites were separated from the aqueous suspension using the ultracentrifugation technique at2000×g for 20 min at 4°C; in the experiment, 20 mL of Fe3O4@SiO2@CS-NISIN composite solution in Tris buffer (pH 9.5, 4 mg/mL) and phosphate-buffered saline (PBS, pH: 7.4, 4 mg/mL) was separately poured into a conical flask containing 50 mL dissolution medium and incubated at 25°C under continuous stirring (100 rpm, 24 h). At appropriate intervals of 0, 30, 60, 120, 240, 300, 360, 420, and 520 min, 5 mL of the solution was used for withdrawal accompanied by simultaneous replacement of an equal volume of fresh medium, loaded into an ultrafiltration tube (M.W. cutoff of 30 kDa) and centrifuged at 2000×g for 20 min to remove possible insoluble constituents. After centrifugal separation, the nisin concentration in the supernatant was analyzed by the Bradford method, all release tests were performed in triplicate, and the mean value was reported [37, 38].


## 2.9. Characterization Methods

Chemical modification of Fe3O4 MNPs (Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2-CS-NISIN) was characterized using the Fourier transform infrared (FT-IR) spectra of the nanocomposites on an FTIR spectrometer (Bruker Co., Germany) with a resolution of 4 cm-1 in the range 4000–400 cm-1 using KBr pellets. The magnetic properties of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites were evaluated at room temperature (300 K) using a vibrating sample magnetometer (VSM, Quantum, USA). The X-ray diffraction (XRD) patterns of Fe3O4 and Fe3O4@SiO2@CS-NISIN composite nanoparticles were collected using an X-ray diffractometer (D8 ADVANCE, Bruker, Germany). A field emission scanning electronic microscope (FE-SEM, TSCAN, Czech Republic) was employed to evaluate the surface morphological characteristics and size of Fe3O4 and Fe3O4@SiO2 MNPs. Before measurements, the samples were covered with a conductive gold layer to avoid charging issues during examination. Hydrodynamic size and zeta potential measurements were detected via dynamic light scattering (DLS) technique using Zetasizer Nano ZS 3600 (Malvern, Worcestershire, United Kingdom).


## 2.10.In Vitro Antibacterial Behavior of Therapeutic Agents

### 2.10.1. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

To measure the MIC and MBC values of Fe3O4@SiO2, Fe3O4@SiO2@CS, nisin, and Fe3O4@SiO2@CS-NISIN against the VISA strain, a standard broth microdilution susceptibility testing procedure was conducted using Mueller-Hinton broth (MHB) as recommended by the Clinical and Laboratory Standards Institute (CLSI) [39]. Briefly, the composites and nisin were diluted in 2-fold increments; for free nisin and Fe3O4@SiO2@CS-NISIN nanocomposites, different concentrations were prepared in the range of 768–12 μg/mL, and for Fe3O4@SiO2 and Fe3O4@SiO2@CS nanocomposites, concentrations ranged from 4000 to 125 μg/mL. A previously prepared culture of VISA (OD625 of 0.5) was diluted to a final concentration of 106 CFU/mL in MHB culture. The bacterial inocula and prepared antimicrobial compound dilutions were added in a 1 : 1 ratio to individual wells of a 96-well microplate and then incubated for 24 h at 37°C (Incubator KB, BINDER Inc., US). The lowest concentration of the antimicrobial substance at which by visual reading the microorganism growth was completely inhibited was determined as the MIC value. To estimate the MBC of the antimicrobials, a viable cell count was carried out: briefly, the bacterial suspensions (10 mL) of each well, considered as the MIC value, were spread on the MH agar plates, followed by bacterial colony count after incubation at 37°C for 24 h. The lowest antimicrobial concentration, where more than 99.9% of the total bacterial population was killed, was termed the MBC endpoint.


## 2.10.1. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

To measure the MIC and MBC values of Fe3O4@SiO2, Fe3O4@SiO2@CS, nisin, and Fe3O4@SiO2@CS-NISIN against the VISA strain, a standard broth microdilution susceptibility testing procedure was conducted using Mueller-Hinton broth (MHB) as recommended by the Clinical and Laboratory Standards Institute (CLSI) [39]. Briefly, the composites and nisin were diluted in 2-fold increments; for free nisin and Fe3O4@SiO2@CS-NISIN nanocomposites, different concentrations were prepared in the range of 768–12 μg/mL, and for Fe3O4@SiO2 and Fe3O4@SiO2@CS nanocomposites, concentrations ranged from 4000 to 125 μg/mL. A previously prepared culture of VISA (OD625 of 0.5) was diluted to a final concentration of 106 CFU/mL in MHB culture. The bacterial inocula and prepared antimicrobial compound dilutions were added in a 1 : 1 ratio to individual wells of a 96-well microplate and then incubated for 24 h at 37°C (Incubator KB, BINDER Inc., US). The lowest concentration of the antimicrobial substance at which by visual reading the microorganism growth was completely inhibited was determined as the MIC value. To estimate the MBC of the antimicrobials, a viable cell count was carried out: briefly, the bacterial suspensions (10 mL) of each well, considered as the MIC value, were spread on the MH agar plates, followed by bacterial colony count after incubation at 37°C for 24 h. The lowest antimicrobial concentration, where more than 99.9% of the total bacterial population was killed, was termed the MBC endpoint.


## 2.11. The Cell Viability Assessment

Thein vitro cytotoxicity effects of materials were identified by MTT assay in mouse L-929 fibroblast cells. Briefly, the cells were plated overnight into 96-well microtiter plates (Sigma, Germany) at a cell density of 1×105 cells per well with a volume of 200 μL complete DMEM, incubated at 37°C under an atmosphere of 5% CO2. Cells were then exposed to various treatments with the nanocarriers: 125, 250, 500, 1000, 1500, 2000, and 4000 μg/mL Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS; 12, 24, 48, 96, 192, 384, and 768 μg/mL free nisin and Fe3O4@SiO2@CS-NISIN composites. Following 24 hours of incubation at 37°C, each well was carefully resuspended in 10 μL of a 5 mg/mL MTT solution, and the plates were incubated for an additional four hours at 37°C, after which the MTT solution was removed from each well. The viable cells were exposed to 100 μL of dimethyl sulfoxide (DMSO) (Millipore Sigma) to solubilize the purple formazan crystals formed in the cells. After gently shaking the plate for 15 min with an orbital shaker (ES 20, Labortechnik, Germany), the absorbance of the wells was recorded with a microplate reader (680 XR, Bio-Rad, USA) at λmax=570nm to calculate cell viability.


## 2.12. Animal Studies

Thein vivo antibacterial efficacy of nisin, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites was evaluated in the healing of full-thickness wounds of mice according to the international guidelines’ principles of laboratory animal use and care [40]. Male NMRI mice, 6-8 weeks old, were divided into three groups (8 animals per group): nisin, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites. Animals were kept under sanitary circumstances in a laboratory with a temperature of 22–25°C, a humidity of 40–70%, a light/dark cycle of 12 h, and free access to standard rodent feed and water ad libitum. After 2 weeks of acclimation, the animals were anesthetized with ketamine (40 mg/kg, Woerden, Netherlands) and xylazine (20 mg/kg, Woerden, Netherlands) through intraperitoneal injection. Afterward, a shaved area of the skin on the animal back was disinfected with 70% ethanol, and full-thickness circular excisions (7 mm diameter) were created on the dorsal midline of the animal using a sterile 4 mm biopsy punch, after which 100 μL of a bacterial suspension at a concentration of 3×108CFU/mL was immediately introduced into each wound site. Treatment was performed according to the MIC dose for Fe3O4@SiO2@CS composites and at considerably greater dosages (4-fold higher than the MIC concentrations) of nisin (96 μg/mL) and Fe3O4@SiO2@CS-NISIN nanocomposites (500 μg/mL) to evaluate the antibacterial capacity to achieve a therapeutic effect in vivo. Nisin and Fe3O4@SiO2@CS composites were administered every 12 hours. However, Fe3O4@SiO2@CS-NISIN nanocomposites were used as a single daily dose. Subsequently, on days 4, 9, and 14, the mice were anesthetized and sacrificed, tissues from the wound area were harvested, and after homogenization, the bacterial cell count was determined.


## 2.13. Statistical Analysis

The variance (ANOVA) procedure and Tukey analysis are used for comparing the differences between relevant groups mean and to describe the possible relationship between variables. All tests were conducted in triplicate, and the differences of the mean findings had to be accepted at the significant levelp lower than 0.05.


## 3. Results

### 3.1. FE-SEM Analysis

The FE-SEM analysis was used to evaluate the particle size and microstructure of the samples, and the micrographs of each sample at different magnifications are shown in Figure1. As shown in Figure 1, quasispherical nanoparticles with different size ranges can be observed in all the samples. To determine the particle size of the samples, 100 particles were measured from each sample using ImageJ software, and histograms of the particle size distribution of these measurements are shown in Figure 2. According to the histograms shown in Figure 2, 25% of the Fe3O4 particles measured were in the range of 25-30 nm in diameter. The histogram of Fe3O4@SiO2@CS-NISIN nanocomposites showed that the greatest number of particles was concentrated in the size range of 40 to 45 nm, with a frequency of 20-24%. Statistical data obtained from these measurements are reported in Table 1. It revealed that Fe3O4@SiO2@CS-NISIN nanocomposites had a larger particle size, with an average particle size of 47.25 than Fe3O4 which had an average size of 28.55 nm. This may be due to the adsorption of organic agents on the surface of MNPs. It is also clear that the standard deviation in the particle size distribution for the samples of Fe3O4@SiO2@CS-NISIN and Fe3O4 was equal to 11.14 and 6.4 nm, respectively. The higher standard deviation values in the nisin-modified nanocomposites indicate a wider size distribution for these samples. In other words, the particle size values of the modified nanocomposites were further deviated from the average value compared to the Fe3O4 particles.Figure 1
SEM images of (a, b) Fe3O4 and (c, d) Fe3O4@SiO2@CS-NISIN at 60000 and 100,000 magnifications.
(a)(b)(c)(d)Figure 2
Particle size distribution histograms of (a) Fe3O4 and (b) Fe3O4@SiO2@CS-NISIN nanocomposites.
(a)(b)Table 1
Statistical results of histograms of the understudied samples.
SampleNumber of the measured particlesAverage particle size (nm)Standard deviation (nm)The smallest measured particle (nm)The measured particle with medium size (nm)The largest measured particle (nm)Fe3O410028.556.4017.4329.0747.90Fe3O4@SiO2@CS-NISIN10047.2511.1424.4348.8582.18


### 3.2. FTIR Analysis

Figure3 represents the FT-IR spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites. As shown in the spectra of Fe3O4, the typical absorption peaks concentrated at 468-571 cm−1 correspond to the stretching of the Fe–O or O-Fe-O associated with the magnetite phase of nanoparticles [41, 42]. The IR spectra of Fe3O4@SiO2 revealed an absorption band around 995 cm−1 associated with an asymmetric stretching vibration Si–O or Si–OH, suggesting the successful formation of a silica layer on the surface of Fe3O4 [43]. The spectra of the synthesized Fe3O4@SiO2@CS nanocomposites displayed a band concentrated at 1104 cm-1 that attributed to stretching vibrations of C-O and C-N bonds and a band at around 1411 cm-1, which can be regarded as stretching modes of C–H, confirming the presence of the –CH and –CH2 groups in the chitosan. The characteristic band at 1610 cm-1 is due to the O-H bending modes of water molecules and also to stretching vibrations of the N-H bond, confirming the existence of the –NH2 functional group in the chitosan. The absorption bands in the regions 2850 and 2923 cm-1 are attributed to the stretching vibrations of the C-H bond of the chitosan structure. As can be indicated in the corresponding spectra of Fe3O4@SiO2@CS-NISIN, the absorption bands detected between 1072 and 1238 cm-1 are described as C-O and C-N stretching modes of the chitosan structure. The absorption bands identified at 1454 cm-1, arising from the C-H stretching vibrations, indicate the -CH3 and -CH groups of chitosan and the aromatic C-C bond (Ar C-C) of the nisin structure. The band located at 1623 cm-1 refers to O-H bending vibrations of H2O molecules and also stretching vibrations deriving from the N-H bond of the -NH2 group. The emerging band at 1731 cm-1 is caused due to the vibrations of the C=O bond in the nisin structure, which confirm the appearance of nisin in the sample structure. The peak in the region 2923 cm-1 indicates stretching vibrations of the C-H group in chitosan and nisin. The peak stretching around 3428 cm-1 corresponds to O-H vibrational properties of H2O molecules absorbed into Fe3O4 and the O-H groups in chitosan and nisin.Figure 3
The FTIR spectra of synthesis materials Fe3O4, Fe3O4@SiO2, chitosan (CS), Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites.


### 3.3. XRD Analysis

Figure4 displays the X-ray diffraction (XRD) patterns of the Fe3O4 and Fe3O4@SiO2@CS-NISIN nanostructures. The six characteristic peaks of Fe3O4 at 2θ=30.3°, 35.7°, 43.4°, 53.9°, 57.3°, 62.8°, and 74.6° are related to their respective indices (220), (311), (400), (442), (511), (440), and (533), respectively, indicating the typical characteristics of Fe3O4 with a cubic spinel structure (JCPDS No. 00-075-0449). Meanwhile, following surface functionalization with the SiO2 layer, chitosan, and nisin, the intensity of the Fe3O4 corresponding peaks noticeably decreased, indicating that the content of Fe3O4 in these nanocomposites was decreasing. The crystalline size of the Fe3O4 and Fe3O4@SiO2@CS-NISIN particles was calculated using the Scherrer equation [44].
(2)D=KλFWHM×cosθ,where D is the crystalline size, K is the shape factor, λ is the X-ray wavelength used (1.54°A), FWHM is the bandwidth at half-height, and θ is the peak position. Given the values of cosθ and FWHM, as well as the constant values of λ (1.54°A) and K (0.9), the crystalline size is obtained according to the Scherrer equation. The crystalline size values are calculated to be 54.8 and 22.34 nm for Fe3O4 and Fe3O4@SiO2@CS@NISIN composites, respectively. The reduced crystalline size of nanomaterials containing organic compounds may be due to the capping of these compounds to the crystalline planes and preventing the growth of the crystal structure during the nucleation and the growth processes of the MNPs.Figure 4
The XRD patterns of (a) Fe3O4 and (b) Fe3O4@SiO2@CS-NISIN nanocomposites.


### 3.4. DLS Analysis

The surface electric charge (zeta potential) of particles dispersed in a colloidal media is an important indicator of the stability of the colloid [45]. It is generally argued that a lower absolute value of the zeta potential suggests a lower stability of colloidal systems, and absolute values of the potential above 30 mV indicate a stable suspension [46]. The zeta potential values for Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CS nanomaterials were around -14.25 mV, -7.19 mV, and 31.74 mV, respectively. The zeta potential values obtained suggest that, among the underexamined samples, only Fe3O4@SiO2@CS had an absolute zeta potential value of more than 30 mV. Therefore, it can be concluded that Fe3O4@SiO2@CS is a stable suspension. The hydrodynamic diameter (Dh) of all nanomaterials was recorded using the dynamic light scattering (DLS) method. The hydrodynamic diameters of Fe3O4 and Fe3O4@SiO2 nanostructures are calculated to be around 51.4 and 93.7 nm, respectively. The hydrodynamic diameters of the nanostructures were obtained to be larger than those calculated from SEM measurements due to the presence of hydrated layers on the surfaces in aqueous media. After surface modification with CS and nisin, due to the reduced dispersion of the nanostructures, the diameters of the Fe3O4@SiO2@CS and Fe3O4@SiO2@CS-NISIN nanocomposites increased up to 129.5 and 155.8 nm, respectively, confirming the successful modification of the silica shell using CS and nisin.


### 3.5. VSM Analysis

As represented in Figure5, magnetic hysteresis curves were calculated at 25°C to assess the magnetic behavior of the synthesized nanoparticles. The amount of saturated magnetization (Ms) of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN reached 66.5 emu/g, 47.3 emu/g, 36.4 emu/g, and 26.7 emu/g, respectively. A significant reduction in the saturation magnetization degree from Fe3O4 to Fe3O4@SiO2@CS-NISIN has been absolutely associated with the existence of nonmagnetic components, which are gradually added onto the surface of Fe3O4 magnetic nanoparticles such as SiO2, CS, and nisin, which was also in accordance with the DLS and FT-IR analyses. We found that the residual magnetic force in Fe3O4@SiO2@CS-NISIN nanocomposites was large enough to separate a valuable portion of nanovesicles under an external magnetic field, although the degree of saturation magnetization decreased. According to the above observation, Fe3O4@SiO2@CS-NISIN nanocomposites exhibited appropriate dispersibility and magnetic response in water, making them suitable for magnetically targeted applications. Following our results, the studied nanocarriers are isotropic and display superparamagnetic behaviors, and the coercivity and remanent magnetization of all nanomaterials are equal to zero.Figure 5
VSM magnetization curves of the obtained Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN, respectively.


### 3.6. Preparation of Calibration Curves of Nisin

The linear regression equations for the calibration curves were obtained as follows:Y=0.0001x+0.0176 (R2=0.998), which was prepared based on sodium acetate buffer to estimate loading nisin. Y=0.0001x+0.0267 (R2=0.998) and Y=0.0001x+0.0203 (R2=0.996) were obtained using PBS and Tris buffers, respectively, to determine the in vitro release profile of nisin, where X is nisin concentration (μg/mL) and Y is absorbance (Figure 6).Figure 6
The standard calibration curve for nisin dissolved in PBS buffer (pH, 7.4).


### 3.7. Release Profile and Covalent Binding Rate of Nisin onto Nanoparticles

The profiles of nisin release underin vitro conditions after 120 h incubation at the phosphate-buffered saline (PBS, pH 7.4) and Tris buffer (pH 9.5) are presented in Figure 7. The results confirmed the greater release rate of nisin over the first 20 h, after which it slowly reduced over time. Of interest to note is that during the initial and transitional release, more than 51% and 96% of the protein molecules were released into PBS and Tris buffer, respectively. After this phase, the release slowed down until it reached a steady state as expected (Figure 7). The results revealed that the release rate of nisin was greater in Tris buffer than that of PBS buffer. The higher release rate of nisin in Tris buffer (pH 9.5) can be ascribed to the high negative charge density of nisin when the pH is greater than the isoelectric point (PI, 8.5). Thus, nisin molecules with a net negative surface charge under such conditions are readily separated from surfaces bearing a negative charge in the Tris buffer. On the other hand, a slower release rate of nisin from Fe3O4@SiO2@CS composites was observed in PBS buffer (pH 7.5), which is due to the fact that nisin has a higher density of positive charge at this pH than at pH 9.5. The high level of positive charge leads to the slower release of nisin from the negatively charged nanocomposites in PBS buffer. In conclusion, the findings suggest that the nanocomposites are capable of controlling the release of conjugated peptides. The covalent binding rate was measured to be 73.2%-85.2%, which was determined using the equation y=0.0001x+0.0176 obtained through the preparation of a nisin calibration curve using sodium acetate buffer (50 mM, pH 5.5).Figure 7
Thein vitro nisin release profile of Fe3O4@SiO2@CS nanocomposites in PBS (pH 7.4) and Tris (pH 9.5) buffers at 37°C.


### 3.8. Antibacterial Assessment of Manufactured Nanoparticles

In our analyses, we found that the antibacterial efficiency of the CS-coated Fe3O4@SiO2 MNPs is partially greater (p≤0.05) than that of the unmodified Fe3O4@SiO2 particles (Table 2). The treatment with Fe3O4@SiO2@CS showed that the incorporation of chitosan onto the particles could further increase the sensitivity of the VISA strain against such nanostructures, as the chitosan-coated MNPs were capable of reducing both the MIC and MBC concentrations up to 2-fold when compared to free MNPs. The MIC and MBC ranges of Fe3O4@SiO2 MNPs were estimated to be around 500 and 1000 μg/mL, respectively, whereas Fe3O4@SiO2@CS composites exerted the bacteriostatic and bactericidal effects at the concentrations of 250 and 500 μg/mL, respectively. Nisin appeared to be extremely efficient against VISA strain as compared to two other compounds, with considerably lower MIC and MBC values (>24 μg/mL). It should be noted that the antibacterial capacity of the nisin-functionalized nanocomposites was greatly impaired when compared to free nisin, which can be explained due to the inactivation of some nisin molecules when covalently conjugated onto the Fe3O4@SiO2@CS nanocomposites, significantly limiting the antibacterial property of the peptide. Free nisin could reduce the MIC and MBC values about 8-fold over the nisin-decorated nanocomposites (MIC/MBC of Fe3O4@SiO2@CS-NISIN=125μg/mL, whereas free nisin had MIC/MBC>24μg/mL).Table 2
MIC and MBC of different concentrations of Fe3O4@SiO2, Fe3O4@SiO2@CS, nisin, Fe3O4@SiO2@CS-NISIN, and vancomycin.
Antibacterial agentsMicroorganismFe3O4@SiO2 (μg/mL)Fe3O4@SiO2@CS (μg/mL)Nisin (μg/mL)Fe3O4@SiO2@CS-NISIN (μg/mL)Vancomycin (μg/mL)MICMBCMICMBCMICMBCMICMBCMICMBCVISA5001000250500>24>2412512588


### 3.9. Cytotoxicity Activity of the Nanoparticles

We employed a standard MTT assay using various treatment concentrations of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CS (125-4000 μg/mL); nisin and Fe3O4@SiO2@CS-NISIN nanocomposites (12-768 μg/mL) to evaluate the in vitro cytotoxic effects of the as-synthesized materials. Figure 8 shows the viability profile of the L-929 mouse fibroblast cells treated with the as-prepared compounds after a 24 h incubation. It can be seen that Fe3O4 MNPs presented no noticeable cytotoxicity effects even until a high exposure level, such as 4000 μg/mL, at which the cell viability achieved 63%, indicating the excellent cytocompatibility of Fe3O4 MNPs. The dose-dependent toxicity of Fe-O-based nanoparticles may be related to the further release of iron ions into the intracellular space during particle degradation. Furthermore, the cell viability reduced when MNPs were decorated with a SiO2-coating layer, with a 56% reduction in the viability of cells exposed to a 4000 μg/mL suspension of Fe3O4@SiO2 particles. The cell viability percentages at the determined concentrations for MIC (500 μg/mL) and MBC (1000 μg/mL) were estimated to be 81% and 76%, respectively. It was also found that the cell viability reduced up to 63%, following an exposure to Fe3O4@SiO2 nanoparticles at concentrations about four- and twofold more remarkable than the MIC and MBC values, respectively. The cytotoxicity assay of free CS exhibited an exceptional biocompatibility even under exposure at the highest concentration (4000 μg/mL), with 80% of the cell viability and a superior biocompatibility behavior compared to other antimicrobial materials. The survey also demonstrated that modification of MNPs with chitosan could improve cytotoxicity compared to Fe3O4 and core/shell Fe3O4@SiO2 particles. The lower toxicity of Fe3O4@SiO2@CS nanocomposites may be due to the slower release of Fe+2 ions, which stimulates ROS-mediated cell death. The chitosan-modified Fe3O4@SiO2 MNPs had a slightly toxic effect on cell viability even after exposure to extremely high concentrations as 71% cell viability was achieved at 4000 μg/mL, which was much higher than the concentrations required to induce bacteriostatic and bactericidal effects. The results obtained in this work revealed that free nisin had relatively nontoxic effects on the viability of mouse fibroblast cells. These findings also indicated that the nisin-functionalized nanocomposites could not suppress the growth of the fibroblast cells even after treatment to high nisin concentrations (12-768 μg/mL). These findings suggest a favorable biocompatibility of nisin and the modified nanostructures under in vivo conditions, with the viability values of 88% and 78% following exposure to a concentration of 768 μg/mL, respectively.Figure 8
Representation of the cell viability rate of the L-929 cells after 24 hours following exposure with different treatments of (a) Fe3O4, Fe3O4@SiO2, chitosan (CS), and Fe3O4@SiO2@CS nanocarriers and (b) nisin and Fe3O4@SiO2@CS-NISIN.
(a)(b)


### 3.10. Animal Studies

Therapeutic efficacy of the Fe3O4@SiO2@CS nanocomposites, nisin, and nisin-incorporated nanocomposites was assessed using male NMRI mice (6-8 weeks old) with VISA-infected wounds as models (Table 3). All formulations exhibited a considerable capacity in lowering the bacterial loads at excision sites, and in agreement with in vitro studies, nisin had the greatest efficiency, followed by nisin-modified nanostructures and then Fe3O4@SiO2@CS. It was discovered that 9 days after the initial wound treatment, the nisin-treated groups had a bacterial burden of 7.8×104±0.8×104 in the surgical incisions compared to those exposed to the nisin-loaded Fe3O4@SiO2@CS and Fe3O4@SiO2@CS nanocomposites, which contained the bacterial loads of about 2.2×105±1.3×105 and 4.3×105±1.2×105, respectively. According to Table 2, the bacterial load in the wound site was remarkably reduced after 14 days of exposure to all formulations, in comparison with the initial days of treatment as follows: 266±18CFU/mL was the VISA population of the groups treated with free nisin, the bacterial load amounted to 305±25CFU/mL in the groups of mice that received Fe3O4@SiO2@CS-NISIN nanocomposites, and around 360±14CFU/mL in the groups treated with the Fe3O4@SiO2@CS nanomaterials alone was observed. The superior performance of free nisin in suppressing the bacterial growth and wound healing process over other materials was also evident from the wound photographs (Figure 9), followed by nanocomposites containing nisin and Fe3O4@SiO2@CS. In this regard, the lower efficiency of nisin-decorated Fe3O4@SiO2@CS composites than free nisin (p<0.05) can be explained by the fact that some nisin molecules lose their function during the conjugation process. The results of the in vivo survey also revealed that the Fe3O4@SiO2@CS-NISIN nanocomposites and free nisin were more potent than the unmodified Fe3O4@SiO2@CS composites in reducing the number of colonies during different time points (p<0.05). The in vivo and in vitro antimicrobial efficiency of polycationic chitosan encapsulated with Fe3O4@SiO2 can be attributed to the improvement of the mechanical strength and enhanced antibacterial properties and biocompatibility of the nanocomposites. Some researchers showed that a combination of the polymeric nanofibers with MNPs could enhance complementary mechanical and antibacterial properties to accelerate the wound healing process. In this regard, Cai et al. demonstrated that the introduction of Fe3O4 NPs into chitosan-gelatin (CS-GE) nanofiber matrix yielded a significant enhancement in the mechanical and antibacterial characteristics. It showed that an improvement in the loading content of Fe3O4 NPs from 1 to 4 w % led to an enhancement of Young’s modulus and tensile strength values by 37% and 22%, respectively. It has been extensively reported that Young’s modulus of materials can profoundly promote cell proliferation and differentiation, which are strongly correlated with the improved wound healing [47]. Wei et al. indicated that the efficient loading of rigid magnetic NPs (5%) into the composite polyvinyl alcohol-chitosan nanofibers (Fe3O4-PVA-CS) could contribute to the enhancement of Young’s modulus from 48% to 57.4% and also the improvement in dynamics of cell attachment and growth due to the effective incorporation of Fe3O4 MNPs [48]. Some scientists reported that the TiO2 NPs embedded into composite chitosan-pectin nanofibers and PVP- (poly (N-vinylpyrrolidone)-) chitosan nanofibers resulted in better antimicrobial efficiency and mechanical strength. It also observed a higher wound healing rate in comparison with chitosan-treated groups [49].Table 3
Bacterial counts (CFU/g) of a mouse skin abrasion lesion on the 4th, 9th, and 14th days after treatment.
DayVISAFree nisinFe3O4@SiO2@CSFe3O4@SiO2-CS-NISIN03×1075.5×1075.6×1075.5×1074Fatal5.7×106±1.5×1065.8×107±2.4×1074.8×107±1.6×1079Fatal7.8×104±0.8×1044.3×105±1.2×1052.2×105±1.3×10514Fatal266±18360±14305±25Figure 9
Photographs of wound healing process in the groups treated with free nisin, Fe3O4@SiO2@CS-NISIN, and Fe3O4@SiO2@CS groups on days 0, 4, 9, and 14 after skin injury.


## 3.1. FE-SEM Analysis

The FE-SEM analysis was used to evaluate the particle size and microstructure of the samples, and the micrographs of each sample at different magnifications are shown in Figure1. As shown in Figure 1, quasispherical nanoparticles with different size ranges can be observed in all the samples. To determine the particle size of the samples, 100 particles were measured from each sample using ImageJ software, and histograms of the particle size distribution of these measurements are shown in Figure 2. According to the histograms shown in Figure 2, 25% of the Fe3O4 particles measured were in the range of 25-30 nm in diameter. The histogram of Fe3O4@SiO2@CS-NISIN nanocomposites showed that the greatest number of particles was concentrated in the size range of 40 to 45 nm, with a frequency of 20-24%. Statistical data obtained from these measurements are reported in Table 1. It revealed that Fe3O4@SiO2@CS-NISIN nanocomposites had a larger particle size, with an average particle size of 47.25 than Fe3O4 which had an average size of 28.55 nm. This may be due to the adsorption of organic agents on the surface of MNPs. It is also clear that the standard deviation in the particle size distribution for the samples of Fe3O4@SiO2@CS-NISIN and Fe3O4 was equal to 11.14 and 6.4 nm, respectively. The higher standard deviation values in the nisin-modified nanocomposites indicate a wider size distribution for these samples. In other words, the particle size values of the modified nanocomposites were further deviated from the average value compared to the Fe3O4 particles.Figure 1
SEM images of (a, b) Fe3O4 and (c, d) Fe3O4@SiO2@CS-NISIN at 60000 and 100,000 magnifications.
(a)(b)(c)(d)Figure 2
Particle size distribution histograms of (a) Fe3O4 and (b) Fe3O4@SiO2@CS-NISIN nanocomposites.
(a)(b)Table 1
Statistical results of histograms of the understudied samples.
SampleNumber of the measured particlesAverage particle size (nm)Standard deviation (nm)The smallest measured particle (nm)The measured particle with medium size (nm)The largest measured particle (nm)Fe3O410028.556.4017.4329.0747.90Fe3O4@SiO2@CS-NISIN10047.2511.1424.4348.8582.18


## 3.2. FTIR Analysis

Figure3 represents the FT-IR spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites. As shown in the spectra of Fe3O4, the typical absorption peaks concentrated at 468-571 cm−1 correspond to the stretching of the Fe–O or O-Fe-O associated with the magnetite phase of nanoparticles [41, 42]. The IR spectra of Fe3O4@SiO2 revealed an absorption band around 995 cm−1 associated with an asymmetric stretching vibration Si–O or Si–OH, suggesting the successful formation of a silica layer on the surface of Fe3O4 [43]. The spectra of the synthesized Fe3O4@SiO2@CS nanocomposites displayed a band concentrated at 1104 cm-1 that attributed to stretching vibrations of C-O and C-N bonds and a band at around 1411 cm-1, which can be regarded as stretching modes of C–H, confirming the presence of the –CH and –CH2 groups in the chitosan. The characteristic band at 1610 cm-1 is due to the O-H bending modes of water molecules and also to stretching vibrations of the N-H bond, confirming the existence of the –NH2 functional group in the chitosan. The absorption bands in the regions 2850 and 2923 cm-1 are attributed to the stretching vibrations of the C-H bond of the chitosan structure. As can be indicated in the corresponding spectra of Fe3O4@SiO2@CS-NISIN, the absorption bands detected between 1072 and 1238 cm-1 are described as C-O and C-N stretching modes of the chitosan structure. The absorption bands identified at 1454 cm-1, arising from the C-H stretching vibrations, indicate the -CH3 and -CH groups of chitosan and the aromatic C-C bond (Ar C-C) of the nisin structure. The band located at 1623 cm-1 refers to O-H bending vibrations of H2O molecules and also stretching vibrations deriving from the N-H bond of the -NH2 group. The emerging band at 1731 cm-1 is caused due to the vibrations of the C=O bond in the nisin structure, which confirm the appearance of nisin in the sample structure. The peak in the region 2923 cm-1 indicates stretching vibrations of the C-H group in chitosan and nisin. The peak stretching around 3428 cm-1 corresponds to O-H vibrational properties of H2O molecules absorbed into Fe3O4 and the O-H groups in chitosan and nisin.Figure 3
The FTIR spectra of synthesis materials Fe3O4, Fe3O4@SiO2, chitosan (CS), Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN nanocomposites.


## 3.3. XRD Analysis

Figure4 displays the X-ray diffraction (XRD) patterns of the Fe3O4 and Fe3O4@SiO2@CS-NISIN nanostructures. The six characteristic peaks of Fe3O4 at 2θ=30.3°, 35.7°, 43.4°, 53.9°, 57.3°, 62.8°, and 74.6° are related to their respective indices (220), (311), (400), (442), (511), (440), and (533), respectively, indicating the typical characteristics of Fe3O4 with a cubic spinel structure (JCPDS No. 00-075-0449). Meanwhile, following surface functionalization with the SiO2 layer, chitosan, and nisin, the intensity of the Fe3O4 corresponding peaks noticeably decreased, indicating that the content of Fe3O4 in these nanocomposites was decreasing. The crystalline size of the Fe3O4 and Fe3O4@SiO2@CS-NISIN particles was calculated using the Scherrer equation [44].
(2)D=KλFWHM×cosθ,where D is the crystalline size, K is the shape factor, λ is the X-ray wavelength used (1.54°A), FWHM is the bandwidth at half-height, and θ is the peak position. Given the values of cosθ and FWHM, as well as the constant values of λ (1.54°A) and K (0.9), the crystalline size is obtained according to the Scherrer equation. The crystalline size values are calculated to be 54.8 and 22.34 nm for Fe3O4 and Fe3O4@SiO2@CS@NISIN composites, respectively. The reduced crystalline size of nanomaterials containing organic compounds may be due to the capping of these compounds to the crystalline planes and preventing the growth of the crystal structure during the nucleation and the growth processes of the MNPs.Figure 4
The XRD patterns of (a) Fe3O4 and (b) Fe3O4@SiO2@CS-NISIN nanocomposites.


## 3.4. DLS Analysis

The surface electric charge (zeta potential) of particles dispersed in a colloidal media is an important indicator of the stability of the colloid [45]. It is generally argued that a lower absolute value of the zeta potential suggests a lower stability of colloidal systems, and absolute values of the potential above 30 mV indicate a stable suspension [46]. The zeta potential values for Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CS nanomaterials were around -14.25 mV, -7.19 mV, and 31.74 mV, respectively. The zeta potential values obtained suggest that, among the underexamined samples, only Fe3O4@SiO2@CS had an absolute zeta potential value of more than 30 mV. Therefore, it can be concluded that Fe3O4@SiO2@CS is a stable suspension. The hydrodynamic diameter (Dh) of all nanomaterials was recorded using the dynamic light scattering (DLS) method. The hydrodynamic diameters of Fe3O4 and Fe3O4@SiO2 nanostructures are calculated to be around 51.4 and 93.7 nm, respectively. The hydrodynamic diameters of the nanostructures were obtained to be larger than those calculated from SEM measurements due to the presence of hydrated layers on the surfaces in aqueous media. After surface modification with CS and nisin, due to the reduced dispersion of the nanostructures, the diameters of the Fe3O4@SiO2@CS and Fe3O4@SiO2@CS-NISIN nanocomposites increased up to 129.5 and 155.8 nm, respectively, confirming the successful modification of the silica shell using CS and nisin.


## 3.5. VSM Analysis

As represented in Figure5, magnetic hysteresis curves were calculated at 25°C to assess the magnetic behavior of the synthesized nanoparticles. The amount of saturated magnetization (Ms) of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN reached 66.5 emu/g, 47.3 emu/g, 36.4 emu/g, and 26.7 emu/g, respectively. A significant reduction in the saturation magnetization degree from Fe3O4 to Fe3O4@SiO2@CS-NISIN has been absolutely associated with the existence of nonmagnetic components, which are gradually added onto the surface of Fe3O4 magnetic nanoparticles such as SiO2, CS, and nisin, which was also in accordance with the DLS and FT-IR analyses. We found that the residual magnetic force in Fe3O4@SiO2@CS-NISIN nanocomposites was large enough to separate a valuable portion of nanovesicles under an external magnetic field, although the degree of saturation magnetization decreased. According to the above observation, Fe3O4@SiO2@CS-NISIN nanocomposites exhibited appropriate dispersibility and magnetic response in water, making them suitable for magnetically targeted applications. Following our results, the studied nanocarriers are isotropic and display superparamagnetic behaviors, and the coercivity and remanent magnetization of all nanomaterials are equal to zero.Figure 5
VSM magnetization curves of the obtained Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS-NISIN, respectively.


## 3.6. Preparation of Calibration Curves of Nisin

The linear regression equations for the calibration curves were obtained as follows:Y=0.0001x+0.0176 (R2=0.998), which was prepared based on sodium acetate buffer to estimate loading nisin. Y=0.0001x+0.0267 (R2=0.998) and Y=0.0001x+0.0203 (R2=0.996) were obtained using PBS and Tris buffers, respectively, to determine the in vitro release profile of nisin, where X is nisin concentration (μg/mL) and Y is absorbance (Figure 6).Figure 6
The standard calibration curve for nisin dissolved in PBS buffer (pH, 7.4).


## 3.7. Release Profile and Covalent Binding Rate of Nisin onto Nanoparticles

The profiles of nisin release underin vitro conditions after 120 h incubation at the phosphate-buffered saline (PBS, pH 7.4) and Tris buffer (pH 9.5) are presented in Figure 7. The results confirmed the greater release rate of nisin over the first 20 h, after which it slowly reduced over time. Of interest to note is that during the initial and transitional release, more than 51% and 96% of the protein molecules were released into PBS and Tris buffer, respectively. After this phase, the release slowed down until it reached a steady state as expected (Figure 7). The results revealed that the release rate of nisin was greater in Tris buffer than that of PBS buffer. The higher release rate of nisin in Tris buffer (pH 9.5) can be ascribed to the high negative charge density of nisin when the pH is greater than the isoelectric point (PI, 8.5). Thus, nisin molecules with a net negative surface charge under such conditions are readily separated from surfaces bearing a negative charge in the Tris buffer. On the other hand, a slower release rate of nisin from Fe3O4@SiO2@CS composites was observed in PBS buffer (pH 7.5), which is due to the fact that nisin has a higher density of positive charge at this pH than at pH 9.5. The high level of positive charge leads to the slower release of nisin from the negatively charged nanocomposites in PBS buffer. In conclusion, the findings suggest that the nanocomposites are capable of controlling the release of conjugated peptides. The covalent binding rate was measured to be 73.2%-85.2%, which was determined using the equation y=0.0001x+0.0176 obtained through the preparation of a nisin calibration curve using sodium acetate buffer (50 mM, pH 5.5).Figure 7
Thein vitro nisin release profile of Fe3O4@SiO2@CS nanocomposites in PBS (pH 7.4) and Tris (pH 9.5) buffers at 37°C.


## 3.8. Antibacterial Assessment of Manufactured Nanoparticles

In our analyses, we found that the antibacterial efficiency of the CS-coated Fe3O4@SiO2 MNPs is partially greater (p≤0.05) than that of the unmodified Fe3O4@SiO2 particles (Table 2). The treatment with Fe3O4@SiO2@CS showed that the incorporation of chitosan onto the particles could further increase the sensitivity of the VISA strain against such nanostructures, as the chitosan-coated MNPs were capable of reducing both the MIC and MBC concentrations up to 2-fold when compared to free MNPs. The MIC and MBC ranges of Fe3O4@SiO2 MNPs were estimated to be around 500 and 1000 μg/mL, respectively, whereas Fe3O4@SiO2@CS composites exerted the bacteriostatic and bactericidal effects at the concentrations of 250 and 500 μg/mL, respectively. Nisin appeared to be extremely efficient against VISA strain as compared to two other compounds, with considerably lower MIC and MBC values (>24 μg/mL). It should be noted that the antibacterial capacity of the nisin-functionalized nanocomposites was greatly impaired when compared to free nisin, which can be explained due to the inactivation of some nisin molecules when covalently conjugated onto the Fe3O4@SiO2@CS nanocomposites, significantly limiting the antibacterial property of the peptide. Free nisin could reduce the MIC and MBC values about 8-fold over the nisin-decorated nanocomposites (MIC/MBC of Fe3O4@SiO2@CS-NISIN=125μg/mL, whereas free nisin had MIC/MBC>24μg/mL).Table 2
MIC and MBC of different concentrations of Fe3O4@SiO2, Fe3O4@SiO2@CS, nisin, Fe3O4@SiO2@CS-NISIN, and vancomycin.
Antibacterial agentsMicroorganismFe3O4@SiO2 (μg/mL)Fe3O4@SiO2@CS (μg/mL)Nisin (μg/mL)Fe3O4@SiO2@CS-NISIN (μg/mL)Vancomycin (μg/mL)MICMBCMICMBCMICMBCMICMBCMICMBCVISA5001000250500>24>2412512588


## 3.9. Cytotoxicity Activity of the Nanoparticles

We employed a standard MTT assay using various treatment concentrations of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CS (125-4000 μg/mL); nisin and Fe3O4@SiO2@CS-NISIN nanocomposites (12-768 μg/mL) to evaluate the in vitro cytotoxic effects of the as-synthesized materials. Figure 8 shows the viability profile of the L-929 mouse fibroblast cells treated with the as-prepared compounds after a 24 h incubation. It can be seen that Fe3O4 MNPs presented no noticeable cytotoxicity effects even until a high exposure level, such as 4000 μg/mL, at which the cell viability achieved 63%, indicating the excellent cytocompatibility of Fe3O4 MNPs. The dose-dependent toxicity of Fe-O-based nanoparticles may be related to the further release of iron ions into the intracellular space during particle degradation. Furthermore, the cell viability reduced when MNPs were decorated with a SiO2-coating layer, with a 56% reduction in the viability of cells exposed to a 4000 μg/mL suspension of Fe3O4@SiO2 particles. The cell viability percentages at the determined concentrations for MIC (500 μg/mL) and MBC (1000 μg/mL) were estimated to be 81% and 76%, respectively. It was also found that the cell viability reduced up to 63%, following an exposure to Fe3O4@SiO2 nanoparticles at concentrations about four- and twofold more remarkable than the MIC and MBC values, respectively. The cytotoxicity assay of free CS exhibited an exceptional biocompatibility even under exposure at the highest concentration (4000 μg/mL), with 80% of the cell viability and a superior biocompatibility behavior compared to other antimicrobial materials. The survey also demonstrated that modification of MNPs with chitosan could improve cytotoxicity compared to Fe3O4 and core/shell Fe3O4@SiO2 particles. The lower toxicity of Fe3O4@SiO2@CS nanocomposites may be due to the slower release of Fe+2 ions, which stimulates ROS-mediated cell death. The chitosan-modified Fe3O4@SiO2 MNPs had a slightly toxic effect on cell viability even after exposure to extremely high concentrations as 71% cell viability was achieved at 4000 μg/mL, which was much higher than the concentrations required to induce bacteriostatic and bactericidal effects. The results obtained in this work revealed that free nisin had relatively nontoxic effects on the viability of mouse fibroblast cells. These findings also indicated that the nisin-functionalized nanocomposites could not suppress the growth of the fibroblast cells even after treatment to high nisin concentrations (12-768 μg/mL). These findings suggest a favorable biocompatibility of nisin and the modified nanostructures under in vivo conditions, with the viability values of 88% and 78% following exposure to a concentration of 768 μg/mL, respectively.Figure 8
Representation of the cell viability rate of the L-929 cells after 24 hours following exposure with different treatments of (a) Fe3O4, Fe3O4@SiO2, chitosan (CS), and Fe3O4@SiO2@CS nanocarriers and (b) nisin and Fe3O4@SiO2@CS-NISIN.
(a)(b)


## 3.10. Animal Studies

Therapeutic efficacy of the Fe3O4@SiO2@CS nanocomposites, nisin, and nisin-incorporated nanocomposites was assessed using male NMRI mice (6-8 weeks old) with VISA-infected wounds as models (Table 3). All formulations exhibited a considerable capacity in lowering the bacterial loads at excision sites, and in agreement with in vitro studies, nisin had the greatest efficiency, followed by nisin-modified nanostructures and then Fe3O4@SiO2@CS. It was discovered that 9 days after the initial wound treatment, the nisin-treated groups had a bacterial burden of 7.8×104±0.8×104 in the surgical incisions compared to those exposed to the nisin-loaded Fe3O4@SiO2@CS and Fe3O4@SiO2@CS nanocomposites, which contained the bacterial loads of about 2.2×105±1.3×105 and 4.3×105±1.2×105, respectively. According to Table 2, the bacterial load in the wound site was remarkably reduced after 14 days of exposure to all formulations, in comparison with the initial days of treatment as follows: 266±18CFU/mL was the VISA population of the groups treated with free nisin, the bacterial load amounted to 305±25CFU/mL in the groups of mice that received Fe3O4@SiO2@CS-NISIN nanocomposites, and around 360±14CFU/mL in the groups treated with the Fe3O4@SiO2@CS nanomaterials alone was observed. The superior performance of free nisin in suppressing the bacterial growth and wound healing process over other materials was also evident from the wound photographs (Figure 9), followed by nanocomposites containing nisin and Fe3O4@SiO2@CS. In this regard, the lower efficiency of nisin-decorated Fe3O4@SiO2@CS composites than free nisin (p<0.05) can be explained by the fact that some nisin molecules lose their function during the conjugation process. The results of the in vivo survey also revealed that the Fe3O4@SiO2@CS-NISIN nanocomposites and free nisin were more potent than the unmodified Fe3O4@SiO2@CS composites in reducing the number of colonies during different time points (p<0.05). The in vivo and in vitro antimicrobial efficiency of polycationic chitosan encapsulated with Fe3O4@SiO2 can be attributed to the improvement of the mechanical strength and enhanced antibacterial properties and biocompatibility of the nanocomposites. Some researchers showed that a combination of the polymeric nanofibers with MNPs could enhance complementary mechanical and antibacterial properties to accelerate the wound healing process. In this regard, Cai et al. demonstrated that the introduction of Fe3O4 NPs into chitosan-gelatin (CS-GE) nanofiber matrix yielded a significant enhancement in the mechanical and antibacterial characteristics. It showed that an improvement in the loading content of Fe3O4 NPs from 1 to 4 w % led to an enhancement of Young’s modulus and tensile strength values by 37% and 22%, respectively. It has been extensively reported that Young’s modulus of materials can profoundly promote cell proliferation and differentiation, which are strongly correlated with the improved wound healing [47]. Wei et al. indicated that the efficient loading of rigid magnetic NPs (5%) into the composite polyvinyl alcohol-chitosan nanofibers (Fe3O4-PVA-CS) could contribute to the enhancement of Young’s modulus from 48% to 57.4% and also the improvement in dynamics of cell attachment and growth due to the effective incorporation of Fe3O4 MNPs [48]. Some scientists reported that the TiO2 NPs embedded into composite chitosan-pectin nanofibers and PVP- (poly (N-vinylpyrrolidone)-) chitosan nanofibers resulted in better antimicrobial efficiency and mechanical strength. It also observed a higher wound healing rate in comparison with chitosan-treated groups [49].Table 3
Bacterial counts (CFU/g) of a mouse skin abrasion lesion on the 4th, 9th, and 14th days after treatment.
DayVISAFree nisinFe3O4@SiO2@CSFe3O4@SiO2-CS-NISIN03×1075.5×1075.6×1075.5×1074Fatal5.7×106±1.5×1065.8×107±2.4×1074.8×107±1.6×1079Fatal7.8×104±0.8×1044.3×105±1.2×1052.2×105±1.3×10514Fatal266±18360±14305±25Figure 9
Photographs of wound healing process in the groups treated with free nisin, Fe3O4@SiO2@CS-NISIN, and Fe3O4@SiO2@CS groups on days 0, 4, 9, and 14 after skin injury.


## 4. Discussion

The antibacterial property of Fe-O-based NPs is of special interest since the incidence and dissemination of strains developing resistance to antibiotics are turning into a critical issue for public health threats throughout the world [50]. The potent inhibitory effect of Fe3O4 magnetic nanostructures has been confirmed on some Gram-positive and Gram-negative species, including S. aureus, Xanthomonas, Escherichia coli, and Proteus vulgaris [51–53]. Furthermore, Fe-O-based NPs are an economically beneficial biocide for various applications [54]. According to our findings, Fe3O4@SiO2 MNPs exhibited good bacteriostatic and bactericidal effects at MIC and MBC values of 500 and 1000 μg/mL towards the VISA strain. The antibacterial effects of iron oxide NPs can stem from the release of Fe3+ ions, which are transported into the cell and interact with macromolecules such as proteins and DNA, impairing enzymatic activity, altering protein structures, and interrupting DNA strands. The Fe3+ ions also can effectively eliminate different bacterial species by the destruction of both cell walls and membranes due to their potent reducing effects [55]. The attachment of metal NPs to the bacterial cell surface triggers the generation of ROS such as superoxide (O-2) or hydroxyl radical (-OH), hydrogen peroxide (H2O2), and 1[O2], which can ultimately lead to polysaccharide depolymerization, DNA strand breaks, lipid peroxidation, and enzyme inactivation [56, 57]. Nonoxidative mechanisms involve direct interaction and alteration of the integrity of the bacterial cell wall, allowing foreign molecules to penetrate and then covalently bind to proteins and cellular constituents, disrupting the normal function of the bacterial cell [58, 59]. The hydrodynamic diameters of the prepared nanostructures in this work were determined to be in the size ranges from 51 to 155 nm, with an obvious difference between DLS and SEM methods. We got smaller MNP sizes using SEM than those calculated by DLS. The MNPs with high volumes or surface areas allow delivery of a sufficient amount of drug molecules while preventing the recognition and immediate removal by RES systems. The nanostructures smaller than 5 nm may be fast eliminated from the bloodstream through renal filtration or liver excretion, whereas nanoparticles with sizes greater than 200 nm in diameter are efficiently cleared by the spleen or RES [60, 61]. Therefore, nanocarriers with diameters ranging from 20 to 200 nm can remain in the bloodstream for longer times because they can evade detection by the body’s scavenger system [62]. A long-term systematic circulation period predicts a higher distribution of nanoparticles in the vicinity of target organs and accumulation to greater concentration [63]. The dependence of the antibacterial property of IONPs on the synthesis procedure was confirmed in several studies. We constructed Fe3O4 MNPs with a size of 20 nm in SEM using PEG-400 as an antibacterial agent, FeCl3, and FeCl2, when modified with a SiO2 layer resulted in an effective inhibitory effect against VISA at MIC and MBC concentrations corresponding to 500 and 1000 μg/mL, respectively. In this regard, Fe3O4 MNPs prepared with the novel surfactant TWEEN80 and urea fuel with a size of about 35 nm displayed excellent inhibitory effects against both Gram-positive and Gram-negative bacterial strains [64]. In another work, the incorporation of Malva sylvestris in the synthesis of Fe3O4 NPs greatly improved the bacteriostatic and bactericidal effects against S. aureus, Corynebacterium sp., Pseudomonas aeruginosa, and Klebsiella pneumonia as compared to unmodified Fe3O4 MNPs [65]. Saqib et al. demonstrated the bactericidal performance of IONPs (25-40 nm) in the presence of predefined concentrations (50 and 100 μg/mL) against S. aureus and Gram-negative bacteria Shigella dysenteriae and E. coli with an inhibition zone of about 13 mm [66]. Al-Shabib et al. demonstrated that synthesized IONPs using PEG, FeCl3, and Fe2(SO4)3 oversize ranging of 6 to 9 nm had a high significant antibacterial property against a diverse array of Gram-negative and Gram-positive bacteria at different concentrations of MICs between 32 and 128 μg/mL, with a capacity to degrade the formation of biofilms and to destroy preformed biofilm structures [67]. The difference in antibacterial properties of IONPs can be attributed to the type of susceptible microorganisms, morphology and physicochemical characteristics of the nanocrystals, and the mechanism of antimicrobials action. Surface functionalization can also be an effective procedure for the improvement of the antibacterial capacity of Fe-O NPs. In this work, chitosan-coated MNPs had more pronounced antibacterial effects compared to Fe3O4@SiO2, suggesting that the presence of chitosan on the surface of MNPs can improve the antibacterial properties of as-prepared MNPs. Incompatible with our findings, Arakha et al. showed that chitosan-coated IONPs could lead to the enhanced bactericidal activity of IONPs against Bacillus subtilis and E. coli, which correlated with the generation of ROS [68]. In another work, coating with chitosan significantly affected the antimicrobial propensity of Fe3O4 NPs against Candida albicans, Aspergillus niger, and Fusarium solani [69]. The most predominant proposed antibacterial effect of chitosan is based on its potential to attach with the negatively charged constituents of the bacterial cell wall and induce cell disruption, resulting in a change in membrane permeability [70], after which it attaches to DNA, leading to inhibition of DNA replication and protein synthesis and subsequent cell death [71]. Another possible mechanism is that chitosan molecules are capable of binding trace elements due to their chelating properties, thereby inhibiting the production of toxins and microbial growth [70]. In the present investigation, nisin exhibited a significantly high antibacterial efficacy when used at a concentration>24μg/mL. However, after exposure to nisin-modified Fe3O4@SiO2@CS nanocomposites, the MIC and MBC values were approximately 5-fold lower than those of nisin alone (125 μg/mL), suggesting that several nisin molecules fail to exhibit antibacterial effects because of impaired functional activity during the conjugation process. Numerous investigations have indicated the prospects of antimicrobial peptides (AMPs) such as nisin in the treatment of bacterial infectious diseases [72–74]. The targeting mechanism of nisin involves a tight interaction with the negatively charged components in the bacterial surface structures and the formation of pores on cell membranes, which causes a change in cell membrane permeability and the efflux of small metabolites such as amino acids, nucleotides, ions, and other cytoplasm-solubilized substances [75] and also leads to the inhibition of peptidoglycan (PG) biosynthesis through the interaction with lipid II specific, resulting in cell death [76]. Jensen et al. demonstrated the antimicrobial effects of nisin in the MIC range 6.4-12.8 μg/mL by damaging the cell membrane and disrupting chromosome replication or segregation in S. aureus [77]. Severina et al. found that nisin had a notable efficacy in the killing of a wide array of Gram-positive species, including MRSA, VRE, and Streptococcus pneumonia [78]. Additionally, a strain of L. lactis capable of nisin production was characterized as leading to reduced intestinal localization by VRE in a mouse infection model [79]. According to in vivo findings, the bacterial burden of wounds treated with nisin-coated Fe3O4@SiO2@CS nanocomposites was 1074.8±1.6, 1052.2±1.3, and 305±25CFU/mL, whereas wounds exposed to free nisin had the bacterial loads of 1065.7±1.5, 1047.8±0.8, and 266±18CFU/mL, on the 4th, 9th, and 14th days of the trial, respectively, which reduced inhibitory effects of nisin-modified nanocomposites relative to free nisin, indicating that the antibacterial effects of some nisin molecules were inhibited during the conjugation process (p<0.05). The presence of glutaraldehyde molecules on the surface of the nanocomposites may have interfered with the binding of some nisin molecules to the VISA cell wall, or they may have blocked the nisin active sites. In line with our study, some researchers have also documented the in vivo antimicrobial and wound healing efficacy of nisin. Heunis et al. demonstrated the wound healing properties of nisin in a mouse model, where a nisin-containing nanofiber dressing remarkably affected wound closure and reduced the bacterial burden in the wound site, such that the number of surviving S. aureus cells was declined to 4.3×102CFU/wound on day 7 following the exposure of nisin incorporated into nanofiber dressings, compared to 2.2×107 in wounds treated with control nanofiber wound dressings [80]. Following previous studies, Mouritzen et al. proposed nisin as a potential therapeutic tool to promote wound healing at a concentration of 25 μg/mL because of its immunomodulatory effect, robust functional efficiency against a variety of Gram-positive bacteria, and low cytotoxicity [81]. To determine the effect of compounds on cell viability, the in vitro cytotoxicity of nisin and nanocomposites on L-929 fibroblast cells was evaluated using a traditional MTT assay. The results showed extremely high cell viability (more than 85% even at a high concentration of 1 mg/mL) of L-929 cells treated with Fe3O4@SiO2@CS nanocomposites, indicating remarkable cellular compatibility. Our findings reveal that the chitosan oligosaccharide coating onto Fe3O4 nanoparticles improves the viability of cells exposed to Fe3O4@SiO2@CS magnetic nanocomposites compared to naked MNP treatment due to moderate ROS production. The lower cytotoxicity of Fe3O4@SiO2@CS may correlate with the sustained release of Fe2+ ions, which trigger ROS-mediated cell death [82]. Similar results were also reported by Guo et al. who showed that Fe3O4@SiO2 particles modified with DMSA can improve the cytotoxicity compared to Fe3O4 and Fe3O4@SiO2 magnetic particles. Furthermore, in line with this study, they found that cell cytotoxicity was enhanced when Fe3O4 MNPs were modified with a SiO2 coating layer [83]. We displayed that free nisin and nisin-modified-Fe3O4@SiO2@CS nanocomposites tested at concentrations ranging from 12 to 768 μg/mL had significantly favorable biocompatibility and can be safely used as the wound dressing.


## 5. Conclusions

Fe3O4@SiO2 MNPs have been successfully embedded in a chitosan matrix to obtain Fe3O4@SiO2@CS nanocomposites and then functionalized with nisin as an antimicrobial peptide (AMP), using a relatively efficient covalent linkage. All materials with particle sizes small sufficient and spherical shapes retained the magnetization properties. When chitosan-coated MNPs were applied, the antibacterial efficacy of the prepared nanocomposites exhibited a greater improvement than that of Fe3O4@SiO2 alone, due to the high density of polycationic NH+3 groups in the chitosan structure and the strengthened interaction between the negatively charged components of the bacterial cells and the nanoparticles. Under both in vivo and in vitro settings, it was found that free nisin exerted a significantly higher inhibitory potency compared to Fe3O4@SiO2@CS nanocomposites decorated with nisin against the VISA strain. This could be attributed to the inactivation of some nisin molecules during the conjugation process. The cytotoxicity results indicated that all the formulations had slightly significant effects on cell viability even at high concentrations, which may be greatly favorable for applying these magnetic nanocomposite materials in various biomedical fields.



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*Source: 2914210-2022-04-28.xml*