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Applied Biological Chemistry
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The green approach of chitosan/Fe2O3/ZnO-nanocomposite synthesis with an evaluation of its biological activities

Applied Biological Chemistry volume 67, Article number: 75 (2024) Cite this article

Abstract

Biopolymers embedded with nanoparticles of metal oxides (MOs) demonstrate a wide range of bio-functions. Chitosan-incorporated MOs are an interesting class of support matrices for enhancing the biological function, compared to other support matrices. Therefore, the importance of this study lies in exploiting chitosan as a carrier not of one metal as in previous studies, but of two metals in the form of a nanocomposite to carry out several biological functions. The coprecipitation approach was employed to synthesize chitosan/Fe2O3/ZnO-nanocomposite in the present research. The characterization of chitosan/Fe2O3/ZnO-nanocomposite was performed to find out the morphology and dispersion properties of chitosan/Fe2O3/ZnO-nanocomposite. The X-ray diffraction (XRD) investigation revealed that these were crystalline. Fourier transforms infrared (FTIR) spectrum bands were viewed at 400/cm and 900/cm, due to the stretching vibration of Fe and Zn oxygen bond. TEM showed that chitosan/Fe2O3/ZnO-nanocomposite was of 20–95 nm in size. chitosan/Fe2O3/ZnO-nanocomposite exhibited inhibitory potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Candida albicans with inhibition zones of 25 ± 0.1, 28 ± 0.2, 27 ± 0.1, and 27 ± 0.2 mm, respectively while didn’t inhibited Aspergillus niger. MIC value of nanocomposite was 15.62 ± 0.33 µg/mL for C. albicans, B. subtilis and E. coli, while it was 62.50 ± 0.66 µg/mL for Pseudomonas aeruginosa. Ranged values of nanocomposite MBC (15.62 ± 0.33 to 125 ± 1 µg/mL) were attributed to all tested bacteria. Different concentrations of chitosan/Fe2O3/ZnO-nanocomposite MBC (25, 50, and 75%) reflected anti-biofilm activity against E. coli (85.0, 93.2, and 96.0%), B. subtilis (84.88, 92.21, and 96.99%), S. aureus 81.64, 90.52, and 94.64%) and P. aurogenosa (90.11, 94.43, and 98.24%), respectively. The differences in the levels of antimicrobial activities may depend on the type of examined microbes. Antioxidant activity of chitosan/Fe2O3/ZnO-nanocomposite was recorded with excellent IC50 values of 16.06 and 32.6 µg/mL using DPPH and ABTS scavenging, respectively. Wound heal by chitosan/Fe2O3/ZnO-nanocomposite was achieved with 100% compared to the untreated cells (76.75% of wound closer). The cytotoxicity outcomes showed that the IC50 of the chitosan/Fe2O3/ZnO-nanocomposite was 564.32 ± 1.46 µg/mL normal WI-38 cells. Based on the achieved findings, the chitosan/Fe2O3/ZnO-nanocomposite is a very promising agent for perform pharmacological activities.

Introduction

The development of biological applications has been greatly influenced recently by green nanotechnology. Researchers' interest in metallic oxide nanoparticles coated with bio-organic polymers has grown recently because of their numerous uses in the pharmacological and biomedical sectors [1,2,3]. The surface of metallic oxide nanoparticles (NPs) can be changed using organic polymers to improve their chemical, biological, and physical characteristics [4,5,6]. Chitosan has gained a lot of attention among the different polymers because of its special qualities, which include low levels of toxicity, biocompatibility, biodegradability, and antibacterial capabilities [7,8,9]. Chitosan surface modification of metallic oxide NPs improves their adaptability and biological characteristics [10]. According to several investigators, the integration of chitosan–inorganic nanocomposites enhances its antibacterial properties, weak mechanical strength, adsorption, and characteristics of drug-delivery [11]. Because of its numerous uses in medical science, catalytic processes, and breakdown, different metallic oxide NPs and their surface covering of chitosan on them have drawn a lot of interest [12]. A new generation of biopolymer nanocomposites is produced by combining metallic oxide NPs with chitosan in nanocomposites. Chitosan ZnO NPs demonstrated inhibitor potential versus Escherichia coli [13]. Numerous investigations indicated that chitosan improve the antimicrobial activities of NPs through interaction among negatively charged and positively charged of plasma membrane of bacteria and amino groups of glucosamine, respectively [14]. In the current decade, Chitosan-ZnO nanocomposite attracted abundant importance for their potential utilize as UV protector and biological activity [15].

In medicine, magnetic materials are used for a variety of purposes, such as tissue repair, hyperthermia, cell separation, and continuous drug delivery to specific targeted organs or cells. The phenomenon known as super para-magnetism is exhibited by magnetic NPs, which lose their magnetism when exposed to a magnetic field, thereby posing a risk of particle aggregation. The magnetic particles synthesized from magnetic transition metals such as iron, zinc, cobalt and nickel oxidized readily, whereas iron oxides, such as magnetite, are more stable against oxidation. Particles of nanomagnetite exhibit robust ferrimagnetic properties and reduced susceptibility to oxidation. Due to the presence of iron ions, magnetite particles belong to a class of materials that are both biologically compatible and non-toxic, this has attracted a lot of attention to them [16].

Iron oxide NPs represent one of the promising constituent with different types and derivatives comprising hematite (α-Fe2O3), magnetite (Fe3O4), and maghemite (γ-Fe2O3). These are extensively applied in medicinal [17]. Because of its special qualities, iron oxide is desirable NPs for use in biological applications. In particular, Fe2O3 NPs can increase the therapeutic agents' permeability and stability through tissues, resulting in a prolonged circulation time [18]. As a result, using Fe2O3-based nanocarriers offers a successful treatment with a lower dosage requirement for medication. According to Raisi et al. [19], incorporation of Fe2O3 NPs with carboxymethyl chitosan possess good properties of wound healing. Zinc oxide's ability to modify the iron oxide NPs surface is gaining more and more interest in biomedical studies. Essential metal oxide NPs, iron oxide NPs have a variety of therapeutic uses [20].

Several investigators studied the interaction among nanocomposites of metallic oxides and biological activities. For instance, Gamboa-Solana et al. [21] reported the antibacterial activity of zinc NPs but their activity enhanced via doped with chitosan. Also, in recent study, anti-diabetic, antibacterial, and wound healing properties of zinc oxide NPs and chitosan/zinc oxide NPs were investigated; a promising result was recorded using NPs and chitosan/zinc oxide NPs compared to zinc oxide NPs Gracias et al. [22]. The excellent antibacterial activity of metals NPs loaded with chitosan as mentioned by Yue et al. [23] was attributed to various features of the surface of bacteria and chitosan i.e., the positive charge of chitosan and negative charge on the bacterial cell surface increased attraction between them. Because addition of chitosan to zinc oxide NPs to creating nanocomposite recently [24], the wound closure and antioxidant were enhanced not compared only to zinc oxide NPs without chitosan or standard drug. Also, the previous results of Bharathi et al. [25] indicated that chitosan loaded with iron oxide NPs as nanocomposite possess higher antimicrobial and antioxidant potential than that obtained by unloaded iron oxide NPs.

Co-precipitation is the greatest ubiquitous process for fabrication of iron oxide NPs. Moreover, it is more convenient and facile as compared with other creation methods. Co-precipitation is a proficient approach to create the bimetallic NPs with chitosan coating [17]. In our study, preliminary experiments were performed on Zinc NPs and Iron NPs individually including antimicrobial, antioxidant, and wound healing activities but giving unpromising, therefor we decided to develop the these NPs. Aim of the current paper was done to synthesis of chitosan/Fe2O3/ZnO-nanocomposite via the coprecipitation method with studying the biological activities of the synthesized nanocomposite.

Materials and methods

Preparation of chitosan/Fe2O3/ZnO-nanocomposite

First, a real solution of 0.5% chitosan was prepared by mixing it well with 0.3% acetic acid, which was then filtered to obtain a well-homogeneous solution. Secondly, 4 mM ferric nitrate and 4 mM zinc nitrate solutions were added to the solution of chitosan, and then this mixture was heated under magnetic stirring at 80 ℃ for 2 h. Drops of 2 M NaOH were gradually added during this procedure to vary the pH value of the solution until a discernible color shift took place, resulting in the development of a brown precipitate. A centrifuge was used to separate the nanocomposite precipitate, and it ran for 5 min at a speed of 3000 rpm. To get rid of contaminants, the precipitate was then repeatedly washed with distilled H2O. The moist powder was then dried by leaving it in an oven overnight at 90 °C. The final chitosan/Fe2O3/ZnO-nanocomposite was produced by annealing the dry powder for three hours at 200 °C in an oven. This procedure made it easier for the intended chitosan/Fe2O3/ZnO-nanocomposite structure to develop and stabilise.

Characterization of chitosan/Fe2O3/ZnO-nanocomposite

X-ray diffraction (XRD) was achieved by Shimadzu XRD-6000. The Cu Kα radiation was set as 40 kV, 20 mA. The rate of scan was 1o/min starting at 10° to 80° (2θ). The functional groups that were responsible for stabilization of chitosan/Fe2O3/ZnO-nanocomposite were detected by Fourier transform infrared spectroscope (JASCO, FT-IR 6100). Using TEM- JEOL 1010, the NPs' dimensions and shape were evaluated. The NPs mixture was added to the carbon/copper TEM grid until complete adsorbed took place and examination. SEM examination was performed to examine the surface form, boundary region size, and dispersion of the chitosan/Fe2O3/ZnO nanocomposite. The elemental makeup, utmost purity and spatial distribution of the chitosan/Fe2O3/ZnO nanocomposite elements were studied using an EDX-BRUKER from Germany.

Antibacterial activity of nanocomposite

Employing the agar-well diffusion procedure, the antibacterial activity of nanocomposite was assessed against tested bacteria and fungi. The examined tested microorganisms included Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), and Pseudomonas aeruginosa (ATCC 90274) which provided from was obtained from Ain Shams University Hospital, Cairo, Egypt as well as Aspergillus niger, and Candida albicans which provided from center of mycology at Assiut University, Egypt. By dispersing nanocomposite in sterile water (2 mg/mL), stock solution was created. After 18/72 h of bacterial/fungal growth on nutrient/potato dextrose broth at 37/30 °C, pure cultures of bacteria/fungi were swapped out uniformly across each plate using sterile cotton swabs. Utilising inverted micro tips (6 mm), wells were created in agar layer. Each well was filled with dispersed nanocomposite, and Gentamycin/ketoconazoledrugs (positive control), and Dimethyl sulfoxide (DMSO) (negative control). The diameter of inhibition zone was used to measure the antimicrobial activities of nanocomposite in the seeded plates after they were incubated for 24/96 h at 37/30 °C [26].

Estimation of the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC)

The microdilution strategy was utilized, per the CLSI, to decide the MIC. To begin with, Müeller-Hinton broth was included to each well on the microdilution plates. While later, nanocomposite was added at different concentrations. The suspensions of bacteria were balanced to 0.5 on the McFarland scale, then diluted, and added a last concentration of 2 × 105 CFU/mL in wells. Next, the plates were incubated (37 °C for 24 h). Employing a spectrophotometric examination at 620 nm, the standard medication's least inhibitory concentration (MIC) that might hinder microbial development was found. Based on the MIC information, MBC was made. Ten µL of aliquot was taken from each well that appeared no signs of bacterial development. The aliquot was at that point aseptically excluded and put on Müeller-Hinton agar. At optimum growth temperature (37 °C), the plates were incubated for 24 h. Following this incubation, during which no microorganisms grew, MBC had the lowest concentration [27].

Assessment of composite against biofilm formation of studied bacteria

Using a microtiter plate test, anti-biofilm activity of nanocomposite was performed in plates (96-well polystyrene flat-bottom) that were supplemented with sub-lethal doses of nanocomposite (75, 50, and 25% of MBC) and fresh broth of trypticase soy yeast (TSY) (300 μL/well). A bacterial inoculation of 106 CFU/mL was applied to the plates followed by incubation (48 h at 37 ℃). After that, the broth was separated, and sterile distilled water (SDW) was used to remove any remaining free-floating bacterial cells. Following a 30-min air drying period, the plates were stained for 17 min at 25°Cemploying 0.1% solution of crystal violet (CV) dissolved in SDW. After removing the extra CV, the plates underwent three SDW washes. To solubilize the linked dye to bacterial cells, 250 μL of 95% ethanol was added to each well. Via a microplate reader, the absorbance (Ab) was recorded at 570 nm after the incubation period of 15 min. The subsequent equation was utilized to compute the inhibition of bacterial film formation (IBF):

$${\% }{\text{I}}{\text{BF}} =1-\frac{{\text{Treated}} \, \text{Ab } - \text{Blank Ab}}{{\text{Control}} \, \text{Ab } - \text{Blank Ab}} {\times 100}$$

Blank indicated the Ab of the media without any composite treatment; the Ab of bacteria from the treatment by composite was represented by treatment with nanocomposite. On the other hand, the control group showed the level of bacteria absorption when not treated with nanocomposites [28].

Antioxidant potential of nanocomposite via DPPH radical scavenging assay

The antioxidant capacity of the nanocomposite was assessed spectrophotometrically at OD517 nm using the 1,1-diphenylpicrylhydrazyl (DPPH) analysis, with ascorbic acid serving as a standard. The nanocomposite was distributed in methanol at varying doses (10 to 100 μg/mL), with ascorbic acid serving as the standard drug [29]. The next equation was used to decide each sample's capacity to scavenge DPPH free radicals:

$$\text{Percentage of DPPH activity }(\% \text{ scavenging}) =\frac{\text{Control OD }-\text{ Sample OD}}{\text{Control OD }}\times 100$$

Scavenging activity of ABTS radical

With minor adjustments, the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging potential of nanocomposite was calculated [30]. ABTS was dissolve in water for concentration of 7 mM. ABTS stock solution was reacted with 2.45 mM potassium persulfate (final concentration), and letting the mixture withstand for 15 h in the dark at 25 ℃ before employing, ABTS radical cation (ABTS. +) was created. H2O was added to the solution of ABTS. + until the Ab at 734 nm was reach to 0.70. Three mL of the ABTS radical and 0.07 mL of the nanocomposite made up the reaction mixture. The Ab was measured in a spectrophotometer at 734 nm following a 6-min incubation period.

Via the following equation, the antioxidant potential was calculated

$$\% \text{Inhibition }=\frac{\text{Ab control }-\text{ Ab sample}}{\text{Ab control }}\times 100$$

Abcontrol = Ab of negative control at the moment of nanocomposite solution preparation. While Absample = Ab of reaction mixture after 6 min. The quantities of IC50 were estimated via graph which signifies the dose of nanocomposite necessary to scavenge (50%) of the free radicals. Ascorbic acid was used as standard in DPPH and ABTS assays with the same concentration of tested sample.

Wound healing of nanocomposite via scratch assay

As previously mentioned [31], the scratch wound-healing assess was conducted by minor altering as follow: Five hundred HFB4 cells (Cell line of normal human skin which were purchased from VACSERA, Egypt). were sowed in 6-well cell plates, then permitted to grow as a monolayer until they reached 70–80% confluence. A sterile pipette tip of one milliliter was used to gently scrape the monolayer across the center of the well. To make a cross in each well, other scratch was made vertical to the first. Following the process of scratching, the growth medium was taken out, then the wells were cleaned twice employing PBS solution. Each well was filled with fresh medium including 5% V/V heat-inactivated FBS and composite, and the cells were grown for a full day. Using a fluorescence invert microscope fitted with a digital camera, imaginings were taken from the fields next the scratching (t0) and immediately after 24 h (Nikon Eclipse TE200: Nikon, Tokyo, Japan). Scratched cells that were not treated served as the control.

The wound closure percentage was estimated via the next equation:

$$\text{Wound closure }(\text{\%})=\frac{\text{Wound area at t}0 -\text{ Wound area at t}}{\text{Wound area at t}0}\times 100$$

Effect of nanocomposite on viability of normal cells

Nanocomposite at different doses was examined against viability of normal WI-38 cells (Normal human lung fibroblast cell (American Type Culture Collection, Rockville, MD) employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) approach. The treated cells (2 × 105 cells) by nanocomposite were incubated under CO2/37 °C/48 h. Once the incubation period ended, the propagated cells were treated by MTT solution (10 μL of 5 mg/mL), and then followed continue period of incubation at above mentioned conditions up to 4 h. The developed color from formazone crystals was liquefied employing 100 μL of DMSO [7]. Finally, at 570 nm, the wavelength was measured to determine percentage of cell viability (CV) % through the subsequent equation:

$$\text{CV }\left(\text{\%}\right) =\frac{\text{Wavelength of treated cells}}{\text{Wavelength of untreayted cells }}\times 100$$

Statistical calculations

The programs of Minitab version 19 and Microsoft Excel version 365 were performed for statistical calculations at level the 0.05 of probability. The analysis of variance, one-way ANOVA, and post hoc Tukey's test were employed to investigate quantitative results with a parametric distribution. Standard deviation (SD) was calculated from three replicates of results.

Results and discussion

Characterization of nanocomposite

Nanotechnology structures have attracted interest as a rapidly developing class of substances with a diverse variety of applications [32]. When an organic synthesis operation is carried out, it is required to establish either the composition or structure of the final result, which can be performed using a range of methodologies ranging from structural elucidation to verifying the pure state of the product under consideration. The XRD pattern for chitosan/Fe2O3/ZnO nanocomposite (Fig. 1) indicates the existence of chitosan at 17.8° and 22.6°. Fe2O3 was identified by a XRD distribution with peak intensities at 2θ ~ 26.8°, 36.01°, 40.27°, 53.29°, and 61.3°. The XRD pattern of ZnO exhibits many peaks, including 31.9, 34.09, 36.01, 47.26, 57.61, 66.64, and 71.62. This implies the creation of the chitosan/Fe2O3/ZnO nanocomposite and its high purity [33].

Fig. 1
figure 1

X-Ray diffraction pattern of chitosan/Fe2O3/ZnO nanocomposite

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FT-IR analysis was employed to recognize the functional groups of the chitosan/Fe2O3/ZnO nanocomposite spectra, and the resulting distinctive peaks were shown in Fig. 2. The stretching vibration of –OH bond are represented by the band at 3462.37 cm−1, the primary amide's C–O stretching is 1639.56 cm−1, the secondary amide group's N–H bending is 1553.73 cm−1, and the C–N axial shift (amine group) is represented by the band at 1412.92 cm−1 [34]. The band containing C–O–C glycosidic linkages at 1111.05 cm−1 and 1021.35 cm−1 [35]. The previous bands refer to chitosan in the composite. The compound's different functional categories and metallic oxide bonds were observed employing the FTIR spectrum. A prominent vibration range in the FTIR spectra, spanning 400 cm−1 to 500 cm−1, is attributed to the distinctive extending phase of the ZnO bond [30]. The Fe–O group is represented by a number of peaks in the 600–900 cm–1 area, including those at 622.07 cm−1, 799.53 cm−1, and 868 cm−1 [36, 37].

Fig. 2
figure 2

FTIR spectra of chitosan/Fe2O3/ZnO nanocomposite

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TEM is the most efficient method for figuring out the size and morphological configuration of a created nanostructure. The produced chitosan/Fe2O3/ZnO nanocomposite was spherical and had diameters between 20 and 95 nm, according to the TEM picture (Fig. 3A). The spherical particles have a thin coating of chitosan encircling the composite's core. Figure 3B displays the area selective diffraction of electrons (SAED) pattern of chitosan/Fe2O3/ZnO nanocomposite, which shows good crisp rings and validates the crystalline structure of the Au nanoscale. It's important to observe that the circular shape of the chitosan/Fe2O3/ZnO nanocomposite made with chitosan is consistent with previous green synthesis techniques reported in the scientific literature [35].

Fig. 3
figure 3

TEM image (A) and SAED pattern (B) of chitosan/Fe2O3/ZnO nanocomposite

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SEM, mapping, and EDS were employed to note and analyse the quantity of element as well as arrangement on the composite face in order to compare the crystalline and morphological characteristics of chitosan/Fe2O3/ZnO nanocomposite Fig. 4 presents the findings. The sample's SEM picture, shown in Fig. 4 (i), revealed its uneven shape. The goal of adding polymer is to control the composite's surface shape and improve the uniformity of the loaded active materials on support [35, 38]. This is demonstrated by the mapping results of Fig. 4, which demonstrate the rather uniform loading of Fe2O3/ZnO on the chitosan surfaces. It is also evident from Fig. 4, which represent the DES results for the sample from Fig. 4 (ii), that Fe2O3/ZnO was effectively loaded onto the chitosan. The mapping of elements images and spectrum of EDS verify the well-distributed presence of C (green), O (blue), Zn (red), and Fe (yellow), in the chitosan/Fe2O3/ZnO nanocomposite. Because chitosan tape was used to bind the NPs, carbon is present. Furthermore, so as to obtain a more accurate analysis, mapping of elements images were created and are shown in Fig. 4(iii-vi).

Fig. 4
figure 4

SEM (i), EDS (ii), of chitosan/Fe2O3/ZnO nanocomposite, Elemental mapping of C (iii), Elemental mapping of O (iv), Elemental mapping of Fe (v), Elemental mapping of Zn (vi)

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Antimicrobial activity of nanocomposite

The primary objective of antimicrobial activity is the development of NPs by change their surface, with no influence on normal cells [39]. Iron oxide nanoparticles' low reactivity, oxidation, and agglomeration can be reduced by coating with another metallic oxide NPs, like zinc oxide NPs [40]. Applying a surface coating to iron oxide NPs, according to Abbas and Krishnan [41], not only lessens their cytotoxicity but also improves their antimicrobial potential in terms of stability and efficacy. The findings in Fig. 5 and Table 1 reflected that the tested microorganisms were more sensitive to nanocomposite than standard drug. For instance, the inhibition zones were 25, 28 and 27 mm in case of S. aureus, B. subtilis and E. coli, respectively as a result of exposure to nanocomposite. Nanocomposite didn’t showed any effect on the filamentous fungus A. niger but the unicellular fungus C. albicans was affected, where the inhibition zone was 27 mm. Different significant was appeared between treatment by nanocomposite and antibiotic used (Control) with all tested bacteria while there's no different significant in case C. albicans. Low MIC value (15.62 µg/mL) of nanocomposite was attributed to C. albicans, B. subtilis and E. coli, while high MIC value (62.50 µg/mL) was attributed to P. aeruginosa. On the other hand, MBC of tested nanocomposite was ranged from 15.62 to 125 µg/mL versus tested bacteria. MBC/MIC or MFC/MIC index values were less than 4, these values were attributed to cidal characteristics of nanocomposite. According to Bhushan et al. [42], Fe/Zn oxide nanocomposite was efficient and possess bactericidal properties against several bacterial species belongs to Gram-positive and Gram-negative. Previously, the antimicrobial potential of Fe/Zn oxide nanocomposites dependent on [Zn]/[Fe] ratio, moreover, S. aureus was more affected than E. coli [43]. The biocreated ZnFe2O4 NPs according to Surendra et al. [44] exhibited excellent inhibition of P. aeruginosa and E. coli growth. In the present investigation, because the small size of the created nanocomposite permeates their entry into the bacterial cells leading to injuries, disruption of respiration and ultimately cause bacterial cell death. According to other investigation, Fe3O4 NPs and ZnO NPs compared with its nanocomposite were experimented against S. aureus where the zones of inhibition were 10, 15, and 16 mm, correspondingly and versus E. coli where the zones of inhibition were 14, 15, and 26 mm, correspondingly [45]. Bharathi et al. [46] mentioned that chitosan incorporated with Zn–O to form nanocomposite reflected inhibitory potential against several bacteria. E. coli, K. pneumoniae, S. aureus, and B. subtilis were more affected by chitosan-ZnO nanocomposite than unloaded ZnO by chitosan with inhibition zone 25.5, 24.5, 22.5, and 21 mm, respectively [46]. According to Kavitha [47], Fe2O3-chitosan nanocomposite caused inhibition growth of E.coli and S.aureus with 18, and 12 mmof clear zone while very negligible clear zones 0.5 and 0.2 mm, respectively using Fe2O3- NPs without chitosan.

Fig. 5
figure 5

Antimicrobial characteristics of nanocomposite (N), positive control (PC), and negative control (NC) against (1) P. aeruginosa, (2) E. coli, (3) S. aureus, (4) B. subtilis, (5) C. albicans, and (6) A. niger

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Table 1 Antimicrobial potential of nanocomposite, besides MIC, MBC, MFC and Index of MBC or MFC/MIC
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The formation of biofilm in the lifestyle of microorganism represents a vital stage for resistance to several antibiotics, moreover it linked to approximately 80% of microbial; infectious diseases [48]. From the obtained data, destruction of bacterial biofilm was observed but with different percentage of inhibition. Nanocomposite at different doses (25, 50, and 75% MBC) reflected lower anti-biofilm activity against C. albicans (66.80, 74.38, and 88.60%) than other tested microorganisms including B. subtilis (84.88, 92.21, and 96.99%), S. aureus 81.64, 90.52, and 94.64%), E. coli (85.0, 93.2, and 96.0%), and P. aurogenosa (90.11, 94.43, and 98.24%), respectively (Fig. 6). The outcomes of Sharma et al. [49] indicated that the chitosan-encapsulated ZnFe2O4 NPs suppress the biofilm formation > 65% of some bacteria including Staphylococcus epidermidis, E. coli, S. aureus, and P. aeruginosa, moreover it reduce the established biofilm (up to 50%) at recorded value of MIC.

Fig. 6
figure 6

A Inhibitory potential of nanocomposite against tested microorganisms, B Microtiter plate offered color shift as a sign of declined biofilm formation. Media + (0%), 25%, 50% and 75% of MBC of nanocomposite. B. subtilis (BS) S. aureus (SA), E. coli (EC), P.aeruginosa (PA), C. albicans (CA), and Control (C)

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Antioxidant activity of nanocomposite

Two techniques including DPPH and ABTS were performed to estimate the antioxidant potential of nanocomposite. Ascorbic acid was used as positive controls for DPPH and ABTS because ascorbic acid is strongest antioxidant, moreover it contains OH groups, is attractive for its predictable high antioxidant capability. From the obtained findings, the two methods documented the antioxidant potential of nanocomposite with excellent IC50 amount of 16.06 and 32.6 µg/mL of DPPH scavenging (Fig. 7) and ABTS scavenging (Fig. 8), respectively. Both DPPH scavenging % and ABTS scavenging % increased with increasing the dose of nanocomposite for obtaining high 89.1% and 95.8%, respectively 1000 µg/mL. Our findings were matching with other report [50] indicating a dose-dependent manner of nanocomposite for DPPH scavenging %. The present experiment was carry out in parallel to estimate the antioxidant activity of standard compound namely, ascorbic acid which reflected IC50 values of 2.08 and 10.55 µg/mL of DPPH scavenging and ABTS scavenging, respectively. Elbrolesy et al. [50] mentioned that the IC50 value of Fe/Zn nanocomposite was 67.89 ± 0.31 µg/mL, while ascorbic acid possesses IC50 value of 16.81 ± 0.10 µg/mL via DPPH scavenging. Regarding antioxidant activity chitosan to nickel/iron NPs as a nanocomposite increased the % of scavenging from 35 to 42%, via DPPH assay [14]. This % of increase can be attributed to chitosan. As mentioned in other study chitosan can join to ions of metal, like iron, copper, and zinc, preventing the generation of harmful oxygen species besides declining oxidative injury [51]. The findings of our investigation indicated that chitosan/Fe2O3/ZnO-nanocomposite can be applied as excellent antioxidant for accelerate the wound heals as well as in the food field.

Fig. 7
figure 7

DPPH Scavenging potential of nanocomposite and ascorbic acid

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Fig. 8
figure 8

ABTS Scavenging potential of nanocomposite and ascorbic acid

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Wound healing and cytotoxicity of nanocomposite

Wound healing experiment reflected the vital role of nanocomposite for accelerating the healing process (Table 2 and Fig. 9). After 48 h of treatment, the wound closer % becomes 100% compared to the untreated cells where the level of wound closer was 76.75%. Moreover, the values of RM (rate of migration) and area difference are other indications of the wound heals where increased from 15.36 (untreated) to 20.0 (treated) and from 564,425.2 (untreated) to 735,377.0% um (treated), respectively with different significant. Wound healing process may depend on the reducing the inflammation of tissue as well as minimize the oxidative stress. Trimetallic CuO@AgO/ZnO nanocomposite remarkably in the infected wound eradicated S. aureus and successively enhanced the wound healing [52]. Halarnekar et al. [24] demonstrated that nanocomposite of chitosan ZnO NPs exhibited positive effect on the process of wound heals with 95.67% while ZnO NPs showed 93% of wound closer, respectively.

Table 2 Wound Healing potential of nanocomposite using HFB4 cells
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Fig. 9
figure 9

Scratch assay illustrate the effect of nanocomposite on the wound area at 0 and 48 h compared to untreated cells

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To examine the safety application of created nanocomposite, its cytotoxicity against normal cells (WI-38 cells) was performed (Table 3). From the recorded results, however the viability of tested cells decreased with dose of nanocomposite increase but negligible cytotoxicity was viewed at dose up to 250 µg/mL. At 62.5, 125, and 250 µg/mL of nanocomposite, there is no significant change in viability % of cells.

Table 3 Normal cell (WI-38 cells) sensitivity to nanocomposite
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At the same time the recorded value of IC50 (564.32 ± 1.46 µg/mL) indicated that nanocomposite possess low toxicity on normal cells. The present result certificated the biological application of this nanocomposite. Moreover these investigation showed that nanocomposite are nontoxic to normal cells, demonstrating this nanocomposite might be employed to create effective wound heals without side effects. Hamouda et al. [53] decide that Au/cellulose nanocomposites possess nontoxic effect on normal lung fibroblasts because their IC50 was 182.75 ± 6.45 µg/µL. Also, from another study on chitosan Mg0.5Co0.5Fe2O4- 5-fluorouracil nanocomposite, the authors mentioned this composite lacking to toxicity, where the normal human embryonic kidney not affected with recorded high quantity of IC50 (200 μg/mL) [54].

Availability of data and materials

All data that support the findings of this study are available within the article.

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Acknowledgements

The authors would like to acknowledge Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

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Authors and Affiliations

  1. Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia

    Aisha M. H. Al-Rajhi

  2. Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, 11725, Egypt

    Tarek M. Abdelghany

  3. Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, 21589, Jeddah, Saudi Arabia

    Mohammed S. Almuhayawi & Mohammed H. Alruhaili

  4. Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine Application, Faculty of Medicine, King Abdulaziz University, 21589, Jeddah, Saudi Arabia

    Mohammed S. Almuhayawi

  5. Special Infectious Agents Unit, King Fahad Medical Research Center, King AbdulAziz University, Jeddah, Saudi Arabia

    Mohammed H. Alruhaili

  6. Department of Hematology/Oncology, Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine Application, Faculty of Medicine, King Abdulaziz University, 21589, Jeddah, Saudi Arabia

    Soad K. Al Jaouni

  7. Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, 72388, Sakaka, Saudi Arabia

    Samy Selim

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  4. Mohammed H. Alruhaili
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Contributions

A.M.H.A. and T.M.A. Conceptualization and methodology; M.S.A. and M.H.A.; investigation and formal analysis; S.K.A. and S.S. writing—review and editing. All authors agreed to the published version of the manuscript.

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Correspondence to Tarek M. Abdelghany.

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Al-Rajhi, A.M.H., Abdelghany, T.M., Almuhayawi, M.S. et al. The green approach of chitosan/Fe2O3/ZnO-nanocomposite synthesis with an evaluation of its biological activities. Appl Biol Chem 67, 75 (2024). https://doi.org/10.1186/s13765-024-00926-2

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