Al‑Rajhi et al.

Applied Biological Chemistry (2024) 67:75

https://doi.org/10.1186/s13765-024-00926-2

ARTICLE Open Access

The green approach of chitosan/

Fe2O3/ZnO‑nanocomposite synthesis
with an evaluation of its biological activities
Aisha M. H. Al‑Rajhi1, Tarek M. Abdelghany2* , Mohammed S. Almuhayawi3,4, Mohammed H. Alruhaili3,5,
Soad K. Al Jaouni6 and Samy Selim7

Abstract
Biopolymers embedded with nanoparticles of metal oxides (MOs) demonstrate a wide range of bio-functions. Chi‑
tosan-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 func‑
tions. 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 Staphylococ-
cus 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 concentra‑
tions 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.
Keywords Coprecipitation, Chitosan, Nanocomposite, Bimetallic, Antimicrobial, Antioxidant, Wound healing

*Correspondence:
Tarek M. Abdelghany
[email protected]
Full list of author information is available at the end of the article

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Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 2 of 13

Introduction hematite (α-Fe2O3), magnetite ­(Fe3O4), and maghemite

The development of biological applications has been (γ-Fe2O3). These are extensively applied in medicinal
greatly influenced recently by green nanotechnology. [17]. Because of its special qualities, iron oxide is desir-
Researchers’ interest in metallic oxide nanoparticles able NPs for use in biological applications. In particu-
coated with bio-organic polymers has grown recently lar, ­Fe2O3 NPs can increase the therapeutic agents’
because of their numerous uses in the pharmacologi- permeability and stability through tissues, resulting
cal and biomedical sectors [1–3]. The surface of metal- in a prolonged circulation time [18]. As a result, using
lic oxide nanoparticles (NPs) can be changed using ­Fe2O3-based nanocarriers offers a successful treat-
organic polymers to improve their chemical, biological, ment with a lower dosage requirement for medication.
and physical characteristics [4–6]. Chitosan has gained According to Raisi et al. [19], incorporation of F ­ e 2O 3
a lot of attention among the different polymers because NPs with carboxymethyl chitosan possess good proper-
of its special qualities, which include low levels of toxic- ties of wound healing. Zinc oxide’s ability to modify the
ity, biocompatibility, biodegradability, and antibacterial iron oxide NPs surface is gaining more and more inter-
capabilities [7–9]. Chitosan surface modification of metal- est in biomedical studies. Essential metal oxide NPs,
lic oxide NPs improves their adaptability and biological iron oxide NPs have a variety of therapeutic uses [20].
characteristics [10]. According to several investigators, Several investigators studied the interaction among
the integration of chitosan–inorganic nanocomposites nanocomposites of metallic oxides and biological activ-
enhances its antibacterial properties, weak mechanical ities. For instance, Gamboa-Solana et al. [21] reported
strength, adsorption, and characteristics of drug-delivery the antibacterial activity of zinc NPs but their activ-
[11]. Because of its numerous uses in medical science, ity enhanced via doped with chitosan. Also, in recent
catalytic processes, and breakdown, different metal- study, anti-diabetic, antibacterial, and wound healing
lic oxide NPs and their surface covering of chitosan on properties of zinc oxide NPs and chitosan/zinc oxide
them have drawn a lot of interest [12]. A new generation NPs were investigated; a promising result was recorded
of biopolymer nanocomposites is produced by combining using NPs and chitosan/zinc oxide NPs compared to
metallic oxide NPs with chitosan in nanocomposites. Chi- zinc oxide NPs Gracias et al. [22]. The excellent anti-
tosan ZnO NPs demonstrated inhibitor potential versus bacterial activity of metals NPs loaded with chitosan
Escherichia coli [13]. Numerous investigations indicated as mentioned by Yue et al. [23] was attributed to vari-
that chitosan improve the antimicrobial activities of NPs ous features of the surface of bacteria and chitosan i.e.,
through interaction among negatively charged and posi- the positive charge of chitosan and negative charge on
tively charged of plasma membrane of bacteria and amino the bacterial cell surface increased attraction between
groups of glucosamine, respectively [14]. In the current them. Because addition of chitosan to zinc oxide NPs
decade, Chitosan-ZnO nanocomposite attracted abun- to creating nanocomposite recently [24], the wound
dant importance for their potential utilize as UV protec- closure and antioxidant were enhanced not compared
tor and biological activity [15]. only to zinc oxide NPs without chitosan or standard
In medicine, magnetic materials are used for a vari- drug. Also, the previous results of Bharathi et al. [25]
ety of purposes, such as tissue repair, hyperthermia, cell indicated that chitosan loaded with iron oxide NPs as
separation, and continuous drug delivery to specific tar- nanocomposite possess higher antimicrobial and anti-
geted organs or cells. The phenomenon known as super oxidant potential than that obtained by unloaded iron
para-magnetism is exhibited by magnetic NPs, which oxide NPs.
lose their magnetism when exposed to a magnetic field, Co-precipitation is the greatest ubiquitous process
thereby posing a risk of particle aggregation. The mag- for fabrication of iron oxide NPs. Moreover, it is more
netic particles synthesized from magnetic transition met- convenient and facile as compared with other creation
als such as iron, zinc, cobalt and nickel oxidized readily, methods. Co-precipitation is a proficient approach to
whereas iron oxides, such as magnetite, are more stable create the bimetallic NPs with chitosan coating [17]. In
against oxidation. Particles of nanomagnetite exhibit our study, preliminary experiments were performed on
robust ferrimagnetic properties and reduced susceptibil- Zinc NPs and Iron NPs individually including antimi-
ity to oxidation. Due to the presence of iron ions, mag- crobial, antioxidant, and wound healing activities but
netite particles belong to a class of materials that are both giving unpromising, therefor we decided to develop the
biologically compatible and non-toxic, this has attracted these NPs. Aim of the current paper was done to syn-
a lot of attention to them [16]. thesis of chitosan/Fe2O3/ZnO-nanocomposite via the
Iron oxide NPs represent one of the promising con- coprecipitation method with studying the biological
stituent with different types and derivatives comprising activities of the synthesized nanocomposite.
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 3 of 13

Materials and methods created. After 18/72 h of bacterial/fungal growth on nutri-

Preparation of chitosan/Fe2O3/ZnO‑nanocomposite ent/potato dextrose broth at 37/30 °C, pure cultures of
First, a real solution of 0.5% chitosan was prepared by bacteria/fungi were swapped out uniformly across each
mixing it well with 0.3% acetic acid, which was then fil- plate using sterile cotton swabs. Utilising inverted micro
tered to obtain a well-homogeneous solution. Secondly, tips (6 mm), wells were created in agar layer. Each well
4 mM ferric nitrate and 4 mM zinc nitrate solutions were was filled with dispersed nanocomposite, and Gentamy-
added to the solution of chitosan, and then this mix- cin/ketoconazoledrugs (positive control), and Dimethyl
ture was heated under magnetic stirring at 80 ℃ for 2 h. sulfoxide (DMSO) (negative control). The diameter of
Drops of 2 M NaOH were gradually added during this inhibition zone was used to measure the antimicrobial
procedure to vary the pH value of the solution until a activities of nanocomposite in the seeded plates after they
discernible color shift took place, resulting in the devel- were incubated for 24/96 h at 37/30 °C [26].
opment of a brown precipitate. A centrifuge was used to
separate the nanocomposite precipitate, and it ran for 5 Estimation of the minimum bactericidal concentration
min at a speed of 3000 rpm. To get rid of contaminants, (MBC) and minimum inhibitory concentration (MIC)
the precipitate was then repeatedly washed with distilled The microdilution strategy was utilized, per the CLSI, to
­H2O. The moist powder was then dried by leaving it in an decide the MIC. To begin with, Müeller-Hinton broth
oven overnight at 90 °C. The final chitosan/Fe2O3/ZnO- was included to each well on the microdilution plates.
nanocomposite was produced by annealing the dry pow- While later, nanocomposite was added at different con-
der for three hours at 200 °C in an oven. This procedure centrations. The suspensions of bacteria were balanced
made it easier for the intended chitosan/Fe2O3/ZnO- to 0.5 on the McFarland scale, then diluted, and added
nanocomposite structure to develop and stabilise. a last concentration of 2 × ­105 CFU/mL in wells. Next,
the plates were incubated (37 °C for 24 h). Employing a
Characterization of chitosan/Fe2O3/ZnO‑nanocomposite spectrophotometric examination at 620 nm, the stand-
X-ray diffraction (XRD) was achieved by Shimadzu XRD- ard medication’s least inhibitory concentration (MIC)
6000. The Cu Kα radiation was set as 40 kV, 20 mA. The that might hinder microbial development was found.
rate of scan was 1o/min starting at 10° to 80° (2θ). The Based on the MIC information, MBC was made. Ten
functional groups that were responsible for stabilization µL of aliquot was taken from each well that appeared no
of chitosan/Fe2O3/ZnO-nanocomposite were detected by signs of bacterial development. The aliquot was at that
Fourier transform infrared spectroscope (JASCO, FT-IR point aseptically excluded and put on Müeller-Hinton
6100). Using TEM- JEOL 1010, the NPs’ dimensions and agar. At optimum growth temperature (37 °C), the plates
shape were evaluated. The NPs mixture was added to the were incubated for 24 h. Following this incubation, dur-
carbon/copper TEM grid until complete adsorbed took ing which no microorganisms grew, MBC had the lowest
place and examination. SEM examination was performed concentration [27].
to examine the surface form, boundary region size, and
dispersion of the chitosan/Fe2O3/ZnO nanocompos- Assessment of composite against biofilm formation
ite. The elemental makeup, utmost purity and spatial of studied bacteria
distribution of the chitosan/Fe2O3/ZnO nanocompos- Using a microtiter plate test, anti-biofilm activity of
ite elements were studied using an EDX-BRUKER from nanocomposite was performed in plates (96-well polysty-
Germany. rene flat-bottom) that were supplemented with sub-lethal
doses of nanocomposite (75, 50, and 25% of MBC) and
Antibacterial activity of nanocomposite fresh broth of trypticase soy yeast (TSY) (300 μL/well).
Employing the agar-well diffusion procedure, the anti- A bacterial inoculation of 1 ­ 06 CFU/mL was applied to
bacterial activity of nanocomposite was assessed against the plates followed by incubation (48 h at 37 ℃). After
tested bacteria and fungi. The examined tested microor- that, the broth was separated, and sterile distilled water
ganisms included Bacillus subtilis (ATCC 6633), Escheri- (SDW) was used to remove any remaining free-floating
chia coli (ATCC 8739), Staphylococcus aureus (ATCC bacterial cells. Following a 30-min air drying period, the
6538), and Pseudomonas aeruginosa (ATCC 90274) which plates were stained for 17 min at 25°Cemploying 0.1%
provided from was obtained from Ain Shams University solution of crystal violet (CV) dissolved in SDW. After
Hospital, Cairo, Egypt as well as Aspergillus niger, and removing the extra CV, the plates underwent three SDW
Candida albicans which provided from center of mycol- washes. To solubilize the linked dye to bacterial cells, 250
ogy at Assiut University, Egypt. By dispersing nanocom- μL of 95% ethanol was added to each well. Via a micro-
posite in sterile water (2 mg/mL), stock solution was plate reader, the absorbance (Ab) was recorded at 570 nm
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 4 of 13

after the incubation period of 15 min. The subsequent nanocomposite necessary to scavenge (50%) of the free
equation was utilized to compute the inhibition of bacte- radicals. Ascorbic acid was used as standard in DPPH and
rial film formation (IBF): ABTS assays with the same concentration of tested sample.
Treated Ab − Blank Ab
%IBF = 1 − ×100 Wound healing of nanocomposite via scratch assay
Control Ab − Blank Ab As previously mentioned [31], the scratch wound-healing
Blank indicated the Ab of the media without any com- assess was conducted by minor altering as follow: Five hun-
posite treatment; the Ab of bacteria from the treatment dred HFB4 cells (Cell line of normal human skin which
by composite was represented by treatment with nano- were purchased from VACSERA, Egypt). were sowed in
composite. On the other hand, the control group showed 6-well cell plates, then permitted to grow as a monolayer
the level of bacteria absorption when not treated with until they reached 70–80% confluence. A sterile pipette tip
nanocomposites [28]. 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
Antioxidant potential of nanocomposite via DPPH radical process of scratching, the growth medium was taken out,
scavenging assay then the wells were cleaned twice employing PBS solution.
The antioxidant capacity of the nanocomposite was Each well was filled with fresh medium including 5% V/V
assessed spectrophotometrically at OD517 nm using the heat-inactivated FBS and composite, and the cells were
1,1-diphenylpicrylhydrazyl (DPPH) analysis, with ascor- grown for a full day. Using a fluorescence invert micro-
bic acid serving as a standard. The nanocomposite was scope fitted with a digital camera, imaginings were taken
distributed in methanol at varying doses (10 to 100 μg/ from the fields next the scratching (t0) and immediately
mL), with ascorbic acid serving as the standard drug after 24 h (Nikon Eclipse TE200: Nikon, Tokyo, Japan).
[29]. The next equation was used to decide each sample’s Scratched cells that were not treated served as the control.
capacity to scavenge DPPH free radicals:

Control OD − Sample OD
Percentage of DPPH activity (% scavenging) = × 100
Control OD

Scavenging activity of ABTS radical The wound closure percentage was estimated via the next
With minor adjustments, the 2,2-azino-bis(3-ethylb- equation:
enzothiazoline-6-sulphonic acid) (ABTS) radical scav-
Wound area at t0 − Wound area at t
enging potential of nanocomposite was calculated [30]. Wound closure (%) = × 100
Wound area at t0
ABTS was dissolve in water for concentration of 7 mM.
ABTS stock solution was reacted with 2.45 mM potas-
sium persulfate (final concentration), and letting the Effect of nanocomposite on viability of normal cells
mixture withstand for 15 h in the dark at 25 ℃ before Nanocomposite at different doses was examined against
employing, ABTS radical cation (ABTS. +) was created. viability of normal WI-38 cells (Normal human lung fibro-
­H2O was added to the solution of ABTS. + until the Ab at blast cell (American Type Culture Collection, Rockville,
734 nm was reach to 0.70. Three mL of the ABTS radical MD) employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
and 0.07 mL of the nanocomposite made up the reaction nyltetrazolium bromide (MTT) approach. The treated cells
mixture. The Ab was measured in a spectrophotometer at (2 × ­105 cells) by nanocomposite were incubated under
734 nm following a 6-min incubation period. ­CO2/37 °C/48 h. Once the incubation period ended, the
Via the following equation, the antioxidant potential propagated cells were treated by MTT solution (10 μL of 5
was calculated mg/mL), and then followed continue period of incubation
at above mentioned conditions up to 4 h. The developed
Ab control − Ab sample
%Inhibition = × 100 color from formazone crystals was liquefied employing 100
Ab control μL of DMSO [7]. Finally, at 570 nm, the wavelength was
Abcontrol = Ab of negative control at the moment of measured to determine percentage of cell viability (CV) %
nanocomposite solution preparation. While A­ bsample = Ab through the subsequent equation:
of reaction mixture after 6 min. The quantities of I­C50
Wavelength of treated cells
were estimated via graph which signifies the dose of CV (%) = × 100
Wavelength of untreayted cells
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 5 of 13

Statistical calculations are represented by the band at 3462.37 ­cm−1, the primary
The programs of Minitab version 19 and Microsoft Excel amide’s C–O stretching is 1639.56 ­cm−1, the secondary
version 365 were performed for statistical calculations at amide group’s N–H bending is 1553.73 ­cm−1, and the
level the 0.05 of probability. The analysis of variance, one- C–N axial shift (amine group) is represented by the band
way ANOVA, and post hoc Tukey’s test were employed at 1412.92 ­cm−1 [34]. The band containing C–O–C glyco-
to investigate quantitative results with a parametric dis- sidic linkages at 1111.05 ­cm−1 and 1021.35 ­cm−1 [35]. The
tribution. Standard deviation (SD) was calculated from previous bands refer to chitosan in the composite. The
three replicates of results. compound’s different functional categories and metallic
oxide bonds were observed employing the FTIR spec-
Results and discussion trum. A prominent vibration range in the FTIR spectra,
Characterization of nanocomposite spanning 400 ­cm−1 to 500 ­cm−1, is attributed to the dis-
Nanotechnology structures have attracted interest as tinctive extending phase of the ZnO bond [30]. The Fe–O
a rapidly developing class of substances with a diverse group is represented by a number of peaks in the 600–900
variety of applications [32]. When an organic synthesis cm–1 area, including those at 622.07 c­ m−1, 799.53 c­ m−1,
operation is carried out, it is required to establish either and 868 c­ m−1 [36, 37].
the composition or structure of the final result, which TEM is the most efficient method for figuring out the
can be performed using a range of methodologies rang- size and morphological configuration of a created nano-
ing from structural elucidation to verifying the pure state structure. The produced chitosan/Fe2O3/ZnO nano-
of the product under consideration. The XRD pattern for composite was spherical and had diameters between 20
chitosan/Fe2O3/ZnO nanocomposite (Fig. 1) indicates and 95 nm, according to the TEM picture (Fig. 3A). The
the existence of chitosan at 17.8° and 22.6°. ­Fe2O3 was spherical particles have a thin coating of chitosan encir-
identified by a XRD distribution with peak intensities at cling the composite’s core. Figure 3B displays the area
2θ ~ 26.8°, 36.01°, 40.27°, 53.29°, and 61.3°. The XRD pat- selective diffraction of electrons (SAED) pattern of chi-
tern of ZnO exhibits many peaks, including 31.9, 34.09, tosan/Fe2O3/ZnO nanocomposite, which shows good
36.01, 47.26, 57.61, 66.64, and 71.62. This implies the cre- crisp rings and validates the crystalline structure of the
ation of the chitosan/Fe2O3/ZnO nanocomposite and its Au nanoscale. It’s important to observe that the circular
high purity [33]. shape of the chitosan/Fe2O3/ZnO nanocomposite made
FT-IR analysis was employed to recognize the func- with chitosan is consistent with previous green synthesis
tional groups of the chitosan/Fe2O3/ZnO nanocom- techniques reported in the scientific literature [35].
posite spectra, and the resulting distinctive peaks were SEM, mapping, and EDS were employed to note and
shown in Fig. 2. The stretching vibration of –OH bond analyse the quantity of element as well as arrangement

Fig. 1 X-Ray diffraction pattern of chitosan/Fe2O3/ZnO nanocomposite

Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 6 of 13

Fig. 2 FTIR spectra of chitosan/Fe2O3/ZnO nanocomposite

analysis, mapping of elements images were created and

are shown in Fig. 4(iii-vi).

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 nanoparti-
cles’ low reactivity, oxidation, and agglomeration can
be reduced by coating with another metallic oxide NPs,
Fig. 3 TEM image (A) and SAED pattern (B) of chitosan/Fe2O3/ZnO
like zinc oxide NPs [40]. Applying a surface coating to
nanocomposite
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
on the composite face in order to compare the crystal- efficacy. The findings in Fig. 5 and Table 1 reflected
line and morphological characteristics of chitosan/ that the tested microorganisms were more sensitive to
Fe2O3/ZnO nanocomposite Fig. 4 presents the findings. nanocomposite than standard drug. For instance, the
The sample’s SEM picture, shown in Fig. 4 (i), revealed inhibition zones were 25, 28 and 27 mm in case of S.
its uneven shape. The goal of adding polymer is to con- aureus, B. subtilis and E. coli, respectively as a result
trol the composite’s surface shape and improve the uni- of exposure to nanocomposite. Nanocomposite didn’t
formity of the loaded active materials on support [35, showed any effect on the filamentous fungus A. niger
38]. This is demonstrated by the mapping results of but the unicellular fungus C. albicans was affected,
Fig. 4, which demonstrate the rather uniform loading of where the inhibition zone was 27 mm. Different signifi-
­Fe2O3/ZnO on the chitosan surfaces. It is also evident cant was appeared between treatment by nanocompos-
from Fig. 4, which represent the DES results for the ite and antibiotic used (Control) with all tested bacteria
sample from Fig. 4 (ii), that F
­ e2O3/ZnO was effectively while there’s no different significant in case C. albicans.
loaded onto the chitosan. The mapping of elements Low MIC value (15.62 µg/mL) of nanocomposite was
images and spectrum of EDS verify the well-distrib- attributed to C. albicans, B. subtilis and E. coli, while
uted presence of C (green), O (blue), Zn (red), and Fe high MIC value (62.50 µg/mL) was attributed to P.
(yellow), in the chitosan/Fe2O3/ZnO nanocomposite. aeruginosa. On the other hand, MBC of tested nano-
Because chitosan tape was used to bind the NPs, carbon composite was ranged from 15.62 to 125 µg/mL ver-
is present. Furthermore, so as to obtain a more accurate sus tested bacteria. MBC/MIC or MFC/MIC index
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 7 of 13

Fig. 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)

values were less than 4, these values were attributed to Surendra et al. [44] exhibited excellent inhibition of P.
cidal characteristics of nanocomposite. According to aeruginosa and E. coli growth. In the present investiga-
Bhushan et al. [42], Fe/Zn oxide nanocomposite was tion, because the small size of the created nanocom-
efficient and possess bactericidal properties against posite permeates their entry into the bacterial cells
several bacterial species belongs to Gram-positive and leading to injuries, disruption of respiration and ulti-
Gram-negative. Previously, the antimicrobial potential mately cause bacterial cell death. According to other
of Fe/Zn oxide nanocomposites dependent on [Zn]/ investigation, ­Fe3O4 NPs and ZnO NPs compared
[Fe] ratio, moreover, S. aureus was more affected than with its nanocomposite were experimented against S.
E. coli [43]. The biocreated Z
­ nFe2O4 NPs according to aureus where the zones of inhibition were 10, 15, and
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 8 of 13

Fig. 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

Table 1 Antimicrobial potential of nanocomposite, besides MIC, MBC, MFC and Index of MBC or MFC/MIC
Utilized microorganisms Inhibition zones (mm) MIC MBC or MFC MBC or
(µg/mL) (µg/mL) MFC/
Nanocomposite *Control MIC
Index

S. aureus 25 ± 0.1a 23 ± 0.3b 31.25 ± 1.33 62.5 ± 1.2 2

a
B. subtilis 28 ± 0.2 26 ± 0.1b 15.62 ± 0.5 15.62 ± 0.5 1
P. aeruginosa 21 ± 0.2a 17 ± 0.1b 62.5 ± 0.66 125 ± 1.0 2
E. coli 27 ± 0.1a 22 ± 0.2b 15.62 ± 1.2 31.25 ± 0.5 2
C. albicans 27 ± 0.2a 26 ± 0.3ab 15.62 ± 0.33 15.62 ± 0.33 1
A. niger NA 21 ± 0.3 NA NA NA
*
Standard control (Gentamycin/Nystatin), MIC (Minimum inhibitory concentration), MBC (Minimum bactericidal concentration), MFC (Minimum fungicidal
concentration). Values of the same row with the different letter are significantly different; however same letters are not significantly different by post hoc Tukey’s test
at P < 0.05

16 mm, correspondingly and versus E. coli where the 0.5 and 0.2 mm, respectively using F ­ e2O3- NPs without
zones of inhibition were 14, 15, and 26 mm, corre- chitosan.
spondingly [45]. Bharathi et al. [46] mentioned that chi- The formation of biofilm in the lifestyle of microor-
tosan incorporated with Zn–O to form nanocomposite ganism represents a vital stage for resistance to several
reflected inhibitory potential against several bacteria. antibiotics, moreover it linked to approximately 80% of
E. coli, K. pneumoniae, S. aureus, and B. subtilis were microbial; infectious diseases [48]. From the obtained
more affected by chitosan-ZnO nanocomposite than data, destruction of bacterial biofilm was observed but
unloaded ZnO by chitosan with inhibition zone 25.5, with different percentage of inhibition. Nanocomposite
24.5, 22.5, and 21 mm, respectively [46]. According to at different doses (25, 50, and 75% MBC) reflected lower
Kavitha [47], ­Fe2O3-chitosan nanocomposite caused anti-biofilm activity against C. albicans (66.80, 74.38,
inhibition growth of E.coli and S.aureus with 18, and and 88.60%) than other tested microorganisms includ-
12 mmof clear zone while very negligible clear zones ing B. subtilis (84.88, 92.21, and 96.99%), S. aureus 81.64,
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 9 of 13

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 bio-
film formation > 65% of some bacteria including Staphy-
lococcus epidermidis, E. coli, S. aureus, and P. aeruginosa,
moreover it reduce the established biofilm (up to 50%) at
recorded value of MIC.

Antioxidant activity of nanocomposite

Two techniques including DPPH and ABTS were per-
formed to estimate the antioxidant potential of nano-
composite. Ascorbic acid was used as positive controls
for DPPH and ABTS because ascorbic acid is strongest Fig. 7 DPPH Scavenging potential of nanocomposite and ascorbic
acid
antioxidant, moreover it contains OH groups, is attrac-
tive for its predictable high antioxidant capability. From
the obtained findings, the two methods documented the
antioxidant potential of nanocomposite with excellent Zn nanocomposite was 67.89 ± 0.31 µg/mL, while ascor-
­IC50 amount of 16.06 and 32.6 µg/mL of DPPH scav- bic acid possesses I­C50 value of 16.81 ± 0.10 µg/mL via
enging (Fig. 7) and ABTS scavenging (Fig. 8), respec- DPPH scavenging. Regarding antioxidant activity chi-
tively. Both DPPH scavenging % and ABTS scavenging tosan to nickel/iron NPs as a nanocomposite increased
% increased with increasing the dose of nanocomposite the % of scavenging from 35 to 42%, via DPPH assay
for obtaining high 89.1% and 95.8%, respectively 1000 [14]. This % of increase can be attributed to chitosan.
µg/mL. Our findings were matching with other report As mentioned in other study chitosan can join to ions
[50] indicating a dose-dependent manner of nanocom- of metal, like iron, copper, and zinc, preventing the
posite for DPPH scavenging %. The present experiment generation of harmful oxygen species besides declin-
was carry out in parallel to estimate the antioxidant ing oxidative injury [51]. The findings of our investiga-
activity of standard compound namely, ascorbic acid tion indicated that chitosan/Fe2O3/ZnO-nanocomposite
which reflected ­IC50 values of 2.08 and 10.55 µg/mL of can be applied as excellent antioxidant for accelerate the
DPPH scavenging and ABTS scavenging, respectively. wound heals as well as in the food field.
Elbrolesy et al. [50] mentioned that the I­ C50 value of Fe/

Fig. 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)
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 10 of 13

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%. Moreo-
ver, the values of RM (rate of migration) and area dif-
ference 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. Tri-
metallic CuO@AgO/ZnO nanocomposite remarkably
in the infected wound eradicated S. aureus and succes-
sively 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.
To examine the safety application of created nanocom-
posite, 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
Fig. 8 ABTS Scavenging potential of nanocomposite and ascorbic change in viability % of cells.
acid

Table 2 Wound Healing potential of nanocomposite using HFB4 cells

Treatment At 0 h At 28 h *RM um Wound Closure Area Difference %
% um
Area Width Area Width

Control cells 964.01 740,380.3 392.08 301,128.0 15.36a 76.75a 564,425.2a

HFB4 1006.02 772,645.0 246.01 188,939.8
952.05 725,467.2 124.06 94,537.1
888.01 680,252.2 202.09 154,808.7
1006.03 772,642.6 152.47 117,100.8
946.0 720,874.7 222.04 169,196.8
Mean
960.35 735,377.0 223.13 170,951.9
Treated cells 964.01 740,380.3 0.0 0.0 20.01b 100b 735,377.0b
1006.02 772,645.0 0.0 0.0
952.05 725,467.2 0.0 0.0
888.01 680,252.2 0.0 0.0
1006.03 772,642.6 0.0 0.0
946.0 720,874.7 0.0 0.0
Mean
960.35 735,377.0 0.0 0.0
*
RM, Rate of migration.Values of the same column with the different letter are significantly different; however same letters are not significantly different by post hoc
Tukey’s test at P < 0.05
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 11 of 13

Fig. 9 Scratch assay illustrate the effect of nanocomposite on the wound area at 0 and 48 h compared to untreated cells

Table 3 Normal cell (WI-38 cells) sensitivity to nanocomposite this nanocomposite might be employed to create effec-
Dose (µg/mL) Nanocomposite
tive wound heals without side effects. Hamouda et al. [53]
decide that Au/cellulose nanocomposites possess non-
Viability (%) Toxicity (%) toxic effect on normal lung fibroblasts because their ­IC50
0.0 100a 0.0 ± 0.0 was 182.75 ± 6.45 µg/µL. Also, from another study on chi-
31.25 100a 0.0 ± 0.0 tosan Mg0.5Co0.5Fe2O4- 5-fluorouracil nanocomposite,
62.5 97.86cde 2.14 ± 0.50 the authors mentioned this composite lacking to toxicity,
125 97.72cde 2.28 ± 0.25 where the normal human embryonic kidney not affected
250 96.91cde 3.09 ± 0.33 with recorded high quantity of ­IC50 (200 μg/mL) [54].
f
500 48.01 51.99 ± 0.2.66 Acknowledgements
1000 2.83g 97.17 ± 3.33 The authors would like to acknowledge Princess Nourah bint Abdulrahman
IC50 564.32 ± 1.46 µg/mL University Researchers Supporting Project number (PNURSP2024R217), Prin‑
cess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
Values of the same column with the different letter are significantly different;
however same letters are not significantly different by post hoc Tukey’s test at Author contributions
P < 0.05 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.
At the same time the recorded value of I­ C50 Funding
(564.32 ± 1.46 µg/mL) indicated that nanocomposite This research was funded by Princess Nourah bint Abdulrahman University
possess low toxicity on normal cells. The present result Researchers Supporting Project number (PNURSP2024R217), Princess Nourah
bint Abdulrahman University, Riyadh, Saudi Arabia.
certificated the biological application of this nanocom-
posite. Moreover these investigation showed that nano- Availability of data and materials
composite are nontoxic to normal cells, demonstrating All data that support the findings of this study are available within the article.
Al‑Rajhi et al. Applied Biological Chemistry (2024) 67:75 Page 12 of 13

Declarations multidrug-resistant Candida. Int J Biol Macromol 242(Pt 1):124709.

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