This article is licensed under CC-BY 4.

www.acsanm.org Article

Antiviral and Nontoxic Dermal Iron Oxide Nanoparticle/Biopolymer

Coatings for Cotton Fabric
Jamilly S. F. Constantino, Iran D. S. de Mesquita, João D. P. Moraes Segundo,
Raimundo N. F. Moreira Filho, Ana B. de Araújo, Marcia V. P. Ferreira, José J. A. de Almeida,
Gladyane S. da Silva, Francisco F. P. Souza, Marcos Vinicius Lorevice, Fábia K. Andrade,
Marisa M. Beppu, Kalyne Almeida Leal,* and Rodrigo Silveira Vieira*
Cite This: ACS Appl. Nano Mater. 2024, 7, 13991−14004 Read Online
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via UNIV ESTADUAL DE CAMPINAS on January 5, 2025 at 17:43:27 (UTC).

ACCESS Metrics & More Article Recommendations *

sı Supporting Information

ABSTRACT: In this study, hybrid coating systems comprising

biopolymers (chitosan, N-succinyl chitosan, or sodium alginate,
and sodium carboxymethylcellulose) and iron oxide nanoparticles
(IONPs) were synthesized, and their antiviral activity against the
coronavirus as well as their dermal toxicity in rats were evaluated.
The hybrid systems were applied as coating surfaces with virucidal
properties against the coronavirus. IONPs were synthesized by
using the coprecipitation method, with TEM images revealing their
crystalline structure and an average size of 5.6 nm. XRD analysis
confirmed the predominance of magnetite in the nanoparticles.
Zeta potential analysis assessed the suspension stability of the
biopolymer-based antiviral solutions at different IONP concen-
trations (1.4, 2.8, and 4.1 mM). The hybrid systems were designed for coating cotton fabric, and SEM, EDS, and FTIR characterized
the coated surfaces. Among the coatings, the N-succinyl chitosan-based (IONPs/NSC) coating showed the lowest iron ion release
after 24 h compared to other polymers. The IONPs/NSC hybrid coating achieved 99% antiviral activity within 5 min of contact, and
all coatings exhibited 99.9999% antiviral activity against coronavirus within 24 h, while being nontoxic to L929 fibroblast cells after
24 h of exposure. The acute dermal toxicity of the IONPs/NSC hybrid system was evaluated in accordance with OECD guidelines
402, demonstrating safety for topical use. For this, animals were treated with topical applications of increasing doses of IONPs/NSC
(1.5, 5, 14, and 40 mg/kg), benzalkonium chloride (750 mg/kg, toxic standard), and saline or white nanoparticle (WN, control
group or a polymeric solution without IONPs). Compared to the control group, no clinical or histological changes were observed for
the IONPs/NSC groups during the 14-day observation period. Conversely, benzalkonium chloride induced erythema, edema, and
histological alterations in rat skin. These coatings show promise for use on protective equipment, with the aim to mitigate the risk of
epidemics or pandemics.
KEYWORDS: iron oxide nanoparticles, N-succinyl chitosan, sodium alginate, coronavirus, protective equipment coating, dermal toxicity

1. INTRODUCTION From an epidemiological point of view, prevention should

COVID-19 is a complex, severe acute respiratory syndrome be the first strategy to diminish coronavirus spreading.9
However, airborne contamination by droplets spread by
caused by the SARS-CoV-2 virus.1 The rapid spread of the
exhalation is a challenge to controlling the transmissibility of
coronavirus and its variants still poses a threat worldwide.2
the disease.10 As such, the use of virucidal and/or micro-
Vaccines have brought us hope, resulting in a decline in the
biological materials, such as masks and filters, are excellent
risk of disease-related infections.3 However, the virus under- strategies to inactivate the coronavirus by contacting the
goes mutations,4 thereby affecting its properties and making it antimicrobial surface with viral droplets, thus preventing new
more aggressive and/or increasing the rate of transmissibility,
which may affect the efficiency of neutralizing antibodies.5 Received: February 15, 2024
These mutations usually change the spike glycoprotein,5 which Revised: May 15, 2024
comprises a highly preserved free fatty acid binding pocket Accepted: May 20, 2024
with unknown function and selection advantage,6 thus being Published: June 20, 2024
responsible for the entry of the coronavirus into cells7 and,
therefore, it is the target of almost all neutralizing antibodies.8
© 2024 The Authors. Published by
American Chemical Society https://doi.org/10.1021/acsanm.4c00951
13991 ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

waves of infection and ensuring the health of the sodium alginate to adhere to surfaces enhances its use for
population.11,12 chemical modifications in materials.32 Sodium carboxymethyl
Overall, nanoparticles are promising in the fight against cellulose is a high molecular weight anionic compound derived
SARS-CoV-2, its main variants and other pathogens.13 By from the reaction of cellulose with caustic alkali and acetic
definition, nanoparticles (1−100 nm) are atomic clusters with acid.33 Similar to sodium alginate, sodium carboxymethyl
crystalline or amorphous structures with unique physical and cellulose (CMC-Na) also contains numerous carboxyl and
chemical properties.14 Silver, iron, copper, and gold nano- hydroxyl groups in its macromolecular structure, rendering it
particle have been reported as presenting antiviral action.15 both mucoadhesive and a natural polymer. Thus, a hybrid
The major methods by which metallic nanoparticles inhibit coating utilizing N-succinyl chitosan and sodium alginate as
viruses are by (i) the disruption of viral capsid structures or primary fixative materials, along with CMC-Na as a blending
direct interaction with viral surfaces, proteins, and genetic monomer, can ensure enhanced adsorption.34 The incorpo-
material through the diffusion of nanoparticles and metallic ration of iron oxide nanoparticles (IONPs) as the main
ions, impairing viral integrity; (ii) production of metal ions and antiviral bioactive agent may provide greater durability and
reactive oxygen species (ROS) through chemical pathways that prolonged performance of the material under study.
adhere and then degrade viral components; and (iii) reactions IONPs have previously been employed as antiviral agents.
that break disulfide bonds within viral glycoproteins, For instance, they were impregnated in raw Citrus limetta peels,
denaturing them to reduce infectivity.16,17 The mutual where they significantly reduced the viral titer of CHIKV and
influence between the surface of the virus and the nano- viral RNA, showing more significant viral potential when
particles may occur due to the interaction between the compared to zinc.35 Researchers have developed similar
hydrophobic regions of the coating and the lipids on the virus’s technology, but using a multilayer coating of copper salt with
surface, promoting its denaturation.18 alginate on polypropylene masks.23 However, the use of metal-
Among metallic nanoparticles, iron oxide nanoparticles based nanoparticles remains a limiting factor when in direct
(IONPs) have been reported to have interesting features that contact with human skin due to dermal toxicity.36 According to
can be used as antiviral nanomaterials. For instance, IONPs are Kumar et al.,37 IONPs can bind to the virus and inhibit its
biocompatible,19 can cross biological membranes, and easily adsorption. Despite evidence proving the effectiveness of
conjugate with target ligands;20 in addition, the US Food and IONPs against viruses, no applications of these nanoparticles
Drug Administration (FDA, for short) has approved the use of in personal protective equipment have been found in the
iron nanoparticles in the treatment of anemia due to their literature. Motivated by these challenges, to our knowledge,
proven safety.21 Nevertheless, the air may easily oxidize this is the first communication to report the production of
IONPs, thus leading to loss of dispersibility and magnet- biodegradable hybrid systems (biopolymers and IONPs) as
ization.22 In this sense, using a polymeric matrix as a dispersion bioactive coatings against coronavirus with the potential to be
medium can be a favorable strategy to increase the stability and applied to cotton fabrics used in the production of hospital
adhesion of the metal to coat the target surface.23,24 personal protective equipment (e.g., masks) with dermal
Furthermore, the antimicrobial action of metallic nanoparticles toxicity tests in rats. Herein, the stability of hybrid systems
may be potentiated when combined with a polymeric matrix, was analyzed by zeta potential, and the particle size was
resulting in coatings capable of stabilizing the metal and observed by using transmission electron microscopy (TEM).
modulating its release. The surfaces coated with cotton fabrics were characterized by
Chitosan is a potential candidate for producing a coating scanning electron microscopy (SEM) and energy dispersive X-
containing iron nanoparticles since this biopolymer is ray spectroscopy (EDS). Atomic absorption spectrometry
biodegradable and biocompatible and possesses antibacterial, (AAS) was used to determine initial iron amount and the
antifungal, and antiviral properties. Notably, the antimicrobial amount of iron released by cotton fabrics after coating with
efficacy of chitosan varies based on several factors, including IONPs-chitosan (IONPs/Chi), IONPs-N-succinyl chitosan-
the type of microorganism, molecular weight, degree of sodium carboxymethyl cellulose (IONPs/NSC), or IONPs-
deacetylation, concentration, pH, chitosan source, and temper- sodium alginate-sodium carboxymethyl cellulose (IONPs/SA).
ature, as described by Li and Zhuang.25 Furthermore, The virucidal assay was performed by exposure to the MHV-3
contemporary research has explored the synergistic effects of Coronaviridae strain belonging to the beta coronavirus (β-
chitosan with other bioactives, such as essential oils26 and CoV) genus, the same genus as SARS-CoV-1 and SARS-CoV-
metallic nanoparticles,27 to enhance its antimicrobial prowess. 2, within 5 min and 24 h. In addition, the cytotoxicity of the
Additionally, chitosan exhibits good mechanical properties coatings for virucidal surfaces on L929 fibroblast cells was
suitable for both pharmaceutical and biomedical use.25,28 evaluated. During the development of personal protective
Furthermore, chitosan may be chemically modified to improve materials, it is essential to evaluate their safety. Therefore, this
its physicochemical and biological properties.29 For instance, study evaluated the acute preclinical dermal toxicity of the
inserting the succinyl group into the amino side (−NH2) of the IONPs/NSC in rats.
polymer chain is one way to obtain water-soluble chitosan,
thereby facilitating access to water and establishing a strong 2. EXPERIMENTAL METHODS
interaction between the iron nanoparticle and chitosan.30 2.1. Materials. Low molecular chitosan (77% degree of
Sodium alginate is another biopolymer with unique properties deacetylation, MW 78 kDa) was purchased from Sigma-
that are well suited for use with iron oxide nanoparticles. Its Aldrich (Brazil). Sodium alginate (medium viscosity, purity of
biocompatibility and ability to make hydrogen bonds, via its 99%) was purchased from Dinâmica (Brazil). Commercially
numerous active hydroxyl and carboxyl groups,31 make it a available methanol, ethanol, acetone, benzalkonium chloride,
mucoadhesive polymer. This property, along with its ability for and glacial acetic acid were purchased from Synth, São Paulo,
gelation and transdermal permeation enhancement, means that Brazil. Succinic anhydride (Sigma-Aldrich, Brazil) and sodium
it is widely used in the medical field. Furthermore, the ability of hydroxide (Vetec, Brazil) were acquired in analytic grade
13992 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

Scheme 1. Schematic Methodology for the Impregnation of a Hybrid Biopolymeric Coating Based on Iron Oxide
Nanoparticles (IONPs) and the Potential Chemical and Electrostatic Interactions Between the Polymers, the IONPs, and the
Cotton Surface (Created with BioRender.com)

a
(a) Synthesis of N-succinyl chitosan. (b) Formation of the hybrid system (IONPs/Biopolymers) and production of the hybrid surface/coating.
(c) Interaction of iron oxide nanoparticles with viral proteins or membranes upon contact with the virucidal surface, resulting in protein
denaturation and/or viral conformational dysfunction.

(>99% purity). Iron(II) chloride tetrahydrate (FeCl2·4H2O, 2.2. Synthesis of the Iron Oxide Nanoparticles
́
MW 198.81 g/mol) was obtained from Ê xodo Cientifica, and (IONPs). IONPs were synthesized by the coprecipitation
iron(III) chloride hexahydrate (FeCl3·6H2O, MW 270.30 g/ method, using the methodology described by Nehra et al.38
with modification. First, approximately 2.5 g of iron(II)
mol) was obtained from Neon. Sodium carboxymethylcellulose
chloride tetrahydrate and 6.5 g of iron(III) chloride
(high viscosity-Na Content 6.5−9.5%) was obtained from hexahydrate were dissolved in 200 mL of Milli-Q water
Neon. Ketamine hydrochloride and xylazine were obtained using a magnetic stirrer at a speed of 800 min−1 until a
from Vetanarcol, Konig S.A Laboratories, Argentina. homogeneous solution was formed. The solution was heated at
13993 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

65 °C in a water bath for 15 to 20 min, and then 28 mL of 25% allow for the drainage of excess solution, the fabric was
(w/v) sodium hydroxide (NaOH) was added. The chemical subsequently subjected to the drying process at 50 °C for 60
reaction ended when a dark color was observed, indicating the min.
formation of the IONPs. The solution was cooled to room 2.6. Morphological and Structural Characterization
temperature and filtered. The supernatant (solution) was of the IONPs by TEM, XRD, Particle Size, and Zeta
discarded, and the pellet was washed three times with Potential. The morphology and size distribution of IONPs
deionized water and alcohol. Finally, the IONPs were dried were acquired through transmission electron microscopy
overnight in a vacuum desiccator at room temperature. (TEM) analysis. Dried samples were first dispersed in water
2.3. Synthesis of N-Succinyl Chitosan (NSC). Chitosan (0.01 wt %) and submitted to sonicated (10 cycles 30 s on/30
was modified with N-succinyl based on the methodology s off) in 40% of amplitude and drop-casted onto carbon- and
described by Bashir et al.39 Briefly, 1.0 g of chitosan was added Formvar-coated copper grids. After deposition, grids were left
into 500 mL of acetic acid solution (5% v/v) and kept under to dry in room temperature for 30 min. The images were
agitation for 45 min at 60 °C until solubilization. Next, using a acquired with a JEOL JEM 2100 LabB6 operating at
round-bottomed flask in a glycerol bath, 50 mL of methanol accelerating voltage equal to 200 kV and equipped with a
was added to the system, followed by 3.5 g of solubilized TV (Gatan ES 500 W); CCD(Tvips-16MP). The size particle
succinic anhydride in 30 mL of acetone. The reaction lasted for distribution was determined by measurement of 100 randomly
48 h at 65 °C. Subsequently, to adjust the pH to 12, 1 M selected particles in diverse regions of three different TEM
NaOH was added, and the mixture was left to rest for 24 h at Images (see the Supporting Information).
room temperature (25 °C). After this period, approximately The crystallinity of the synthesized IONPs was examined by
1500 mL of ethanol was added to the mixture. The precipitate an X-ray diffraction technique. The average size of the
formed was separated by centrifugation (6000 rpm, 10 min) synthesized NPs was calculated using the Scherrer equation
and washed until neutral pH. Finally, the product was vacuum
filtered, dried for approximately 2 h at 70 °C, and stored at K·
D=
room temperature. The schematic representation of the · cos( ) (1)
synthesis is depicted in Scheme 1.
2.4. Preparation of Hybrid Coating Systems. An where K is a constant equal to 0.94, λ is the X-ray
amount of 0.26% (w/v) chitosan was mixed with 50 mL of a wavelength equal to 0.154 nm, β is the full width at half-
solution containing 2% (v/v) acetic acid to dissolve the maximum (fwhm) in radians, θ is the Bragg angle, and D is the
chitosan. The solution was homogenized in a mechanical size of the nanoparticles in nanometers (nm).
shaker for approximately 2 h. Next, concentrations of 1.4, 2.8, The zeta potential is a fundamental parameter that affects
and 4.1 mM iron oxide nanoparticles (Table S1) were added the stability of the metal in the solution as it measures the
to the solution and then sonicated in tip ultrasound (0.5 cycle, magnitude of repulsive or attractive electrostatic forces using
70% amplitude) for 20 min to disperse the nanoparticles. The the Zetasizer Ultra measuring instrument. For this, the hybrid
selection of these specific concentrations was guided by a system (containing chitosan, N-succinyl chitosan, or sodium
thorough analysis, considering some factors. Prioritizing safety, alginate and 0.10% CMC-Na) with a known concentration of
we assessed the nontoxicity of the iron nanoparticles through IONPs was added to an aqueous solution containing Milli-Q
dermal cytotoxicity assays, while recognizing the necessity for water and then diluted 1:15 to achieve a completely clear
comprehensive testing, particularly regarding direct skin solution. The pH of the dispersion for Z measurements was
application. We also aimed for uniformity, stability in the adjusted and monitored throughout the experiment using a pH
hybrid solution, fabric adhesion, and antiviral efficacy. meter (Digimed DM-22). The zeta potential for the hybrid
Following careful consideration, these concentrations emerged systems without the addition of CMC-Na was also analyzed to
as the most suitable for incorporation into the polymeric assess whether the polymer addition would affect the metal
matrix. For ultrasonic homogenization, the UP100H sonicator stability, with a total of 5 scans per sample. The hydrodynamic
model was utilized, operating at a power of 100 W and a diameters of the particles were also determined in the same
frequency of 30 kHz. The production of polymeric coatings experiment.
with modified chitosan (N-succinyl chitosan) and sodium 2.7. Fourier Transform Infrared Spectroscopy Anal-
alginate was obtained following the same methodology ysis�FTIR. In addition, to evaluate the chemical composition
described above but dissolved in Milli-Q water. Additionally, of the coatings, Fourier transform infrared spectroscopy
0.10% (w/v) sodium carboxymethyl cellulose (CMC-Na) was (FTIR) analysis was performed using a PerkinElmer IR
added to form a complex in the polymer solution with the spectrophotometer (model FT-IR/NIR FRONTIER) equip-
capability of forming films or coatings. Then, the solutions ped with a Pike Technologies ATR/MIRacle accessory
were stored for further characterization and applications for attached with a ZnSe crystal, with a resolution of 4 cm−1
coatings. and wavenumber ranging from 4000 to 650 cm−1. For FTIR
2.5. Application of the Coating on Cotton Fabric. Due analysis using KBr, iron oxide nanoparticles in powder form,
to the COVID-19 pandemic, protective masks have been N-succinyl chitosan, and chitosan were mixed and ground with
recommended as preventive measures against the COVID-19 potassium bromide (KBr), and subsequently analyzed over a
virus. In this context, cotton fabric, commonly used for the wavenumber range varying from 4000 to 400 cm−1.
production of masks and other hospital personal protective 2.8. Characterization of the Textile Coatings by SEM-
equipment, was selected to receive the virucidal coating. EDS. Scanning electron microscopy (SEM) coupled to energy
Cotton fabric pieces (2 × 2 cm2)) were immersed in 15 mL of dispersive X-ray spectroscopy (EDS) (Quanta 450-FEG,
each hybrid system for 20 min. The cotton fabric impregnated model Polaron SC500), operating with 5 kV voltage
with the solution was carefully extracted upon immersion using acceleration, was used to analyze the morphology of the
clean forceps. Following an approximate interval of 1−2 min to cotton fabric coated with the polymeric hybrid systems.
13994 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

2.9. Determination of Iron Release Kinetics by streptomycin and 10% FBS, and medium was changed every
Atomic Absorption Spectrometry (AAS). Release kinetics 3 days until cells reached 80% confluence. The cells were then
using the AAS methodology were performed using the detached with trypsin-EDTA, centrifuged at 200g for 6 min at
following procedure: coated fabric samples (6 × 6 cm2) were 25 °C, and resuspended in fresh DMEM. The cell density was
placed in Petri dishes in triplicate containing 20 mL of adjusted to 60,000 cells/mL, using the trypan blue exclusion
ultrapure water (18 MΩ cm) at 25 °C and shaken in a Dubnoff method (Sigma-Aldrich, São Paulo, Brazil) and counting in the
bath (Quimis, model: 0216M2). Aliquots (1.0 mL) were Neubauer chamber. Cells were seeded in 96-well plates
removed over 24 h (1, 5, 15, 30, 60, 120, 240, 480, and 1440 containing 100 μL/well of cell solution (6,000 cells/well)
min), and the percentage of iron oxide leached was quantified and incubated in an oven at 37 °C, 5% CO2, and 95%
by AAS, considering the cumulative amount of iron initially humidified atmosphere for 24 h. Afterward, the culture
incorporated. medium was removed and replaced by 100 μL of each test
To quantify total iron on the fabric-coated surface, samples sample and the cells were incubated for 24 and 48 h. Cells
(6 × 6 cm2) in triplicate were immersed in 10% HNO3 (v/v) exposed only to DMEM medium were used as negative control
for 24 h. The concentration of iron in each aliquot was (C−).
determined by flame AAS (AAS, AA 7000, 248.3 nm). It is To quantified the cytotoxicity, the resazurin colorimetric
important to note that the cumulative nature of the calculation method was used, a dark blue dye that, in the presence of
accounts for the gradual release of iron over time and the total viable cells, reduced by mitochondrial enzymes and acquires a
iron content on the fabric surface due to the addition of a pink color with high fluorescence emission.43 After 24 h, the
specific amount of water with nitric acid to the sample. extracts were discarded, and the wells were gently washed with
2.10. Virucidal Assay. The virucidal activity of the hybrid phosphate buffered saline (PBS) pH 7.4. Then, resazurin (25
systems after coating of the fabrics (strips 2 × 2 cm2) was μg L−1 252; 120 μL) solution in DMEM was added, before
determined in vitro by exposure to the MHV 3 Coronaviridae incubating the microplate for 4 h. Subsequently, 100 μL of the
strain, belonging to the genus β-coronavirus (β-CoV), the metabolized medium was transferred to a new microplate, and
same genus of SARS-CoV-1 and SARS-CoV-2, at room fluorescence was read in a microplate reader (λexcitation = 560
temperature. To perform viral inhibition and cytotoxicity nm and λemission = 590 nm). The percentage of cell viability was
assays, L929 fibroblast cells (NCTC clone 929) [L cell, L-929, calculated as follows:
derivative of Strain L] (ATCC CCL-1) were cultured in
Dulbecco’s Minimal Essential Medium (DMEM) containing Fsample
2% to 10% fetal bovine serum (FBS) in 5% CO2 at 37 °C. CV(%) = × 100
Fcontrol (4)
Coronavirus titration (Strain MHV-3) was performed by the
serial 50% infectious dose tissue culture method. Subsequently, where CV is the percentage of cell viability, Fsample is the
the treated virus solution was diluted in series (101 to 108) and fluorescence value of the sample, and Fcontrol is the fluorescence
transferred to the L929 fibroblast cell monolayer. The value of the negative control. Percentages of cell viability below
microplates with the products, viruses, and cellular systems 70% indicated cytotoxicity.
were incubated at 37 °C in an atmosphere with 5% CO2 for 48 2.12. Acute Dermal Toxicity Study. Female Wistar rats
h, and the cytopathic (CPE) and cytotoxicity effects were then (150−200 g) were kept under controlled temperature and
examined directly under an inverted microscope (quadrupli- humidity conditions in 12-h light/dark cycles, with free access
cate). For this, the amount of virus in each dilution is known. to food and water. The experimental protocol was approved by
Therefore, the observation of CPE in a diluted sample (by the Ethics Committee on Animal Use (CEUA) of the Federal
counting dead cells) indicates a sufficient amount of virus to University of Ceara (N° 7752300920). The acute dermal
promote such an effect. The infectivity rate was calculated toxicity study followed OECD guideline 402. Dorsal skin
based on the method of Reed and Muench,40 the log viral trichotomy was used to expose at least 10% of the area, and the
reduction (L) was calculated by eq 2, and the inhibition region was exposed to cotton with or without increasing doses
efficacy was expressed as percentage reduction (P), using eq 3, of iron oxide nanoparticles (IONPs/NSC; 1.5, 5, 14, and 40
iAy mg/kg), benzalkonium chloride (750 mg/kg), N-succinyl
Log reduction = log10jjj zzz chitosan solution with sodium carboxymethyl cellulose
kB{ (2)
(control group or white nanoparticles, WN), or saline. The
P = (1 10 Log reduction
)× 100[%] animals were monitored for 14 days, with daily observation of
(3)
clinical signs, including macroscopic skin evaluation, and
A is the number of viable viruses before treatment, and B is recording of body weight every 48 h. The evaluation of clinical
the number of viable viruses after treatment. signs was carried out according to a previous study (Table S2).
2.11. Cytotoxicity Assay. Sample preparation and the 2.12.1. Histological Analysis. The skin from the dorsal
cytotoxicity assay were carried out in accordance ISO 10993- region was removed (1 cm) from the animal and fixed in
1241 and ISO 10993-5,42 respectively. Samples were cut into buffered formalin (10%) on the 14th day after treatment. The
pieces of 1 cm2, sterilized by UV radiation (one cycle of 20 min tissue was processed, embedded in paraffin, cut into 4.0−5.0
for each side of the material), placed into 24-well plates with μm sections with a microtome (Lupetec, Brazil), and then
Dulbecco’s minimum essential medium (DMEM) (1 cm2/ stained with hematoxylin and eosin stain (Sigma-Aldrich,
mL), and incubated in an oven at 37 °C for 24 h. Brazil). The tissues were then examined by light microscopy
Subsequently, the supernatant of each well was collected (RML5Microscope, Askmania-Germany). Photomicrographs
(called extract) and used in the cytotoxicity assay. were taken at 400× magnification.
Fibroblast cells, acquired from the Rio de Janeiro Cell 2.12.2. Statistical Analysis. Statistical analyses were
Bank�BCRJ, were used for the cytoxicity assay. Cells were performed using GraphPad Prism 6.01 software (GraphPad
grown in DMEM supplemented with 1% penicillin and Software, Inc.). All results are expressed as means ± the
13995 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

Figure 1. TEM (a−d) micrographs and particle size distribution and (e) XRD pattern of the iron oxide NPs synthesized by coprecipitation. (a, b)
Overview of particle size and particle size distribution. (c, d) Zoomed area (it is not essentially the area indicated by the dot-dot square) and atomic
planes of the IONPs. (e) XRD pattern of IONPs.

standard error of the mean (SEM). The differences were stability. Z potential data serve as a valuable indicator of
assessed using nonparametric data, applying the Kruskal− stability, particularly when electrostatic repulsion or attraction
Wallis test followed by the Wilcoxon−Mann−Whitney post- dominates particle interactions. IONPs exhibit a magnetic
test. property that favors the formation of aggregation in
suspensions;49 thus, it was necessary to evaluate the stability
3. RESULTS AND DISCUSSION of the suspension through the analysis of the Z potential, which
is often used as a metric to measure the stability of particles in
3.1. Morphological and Structural Characterization the colloidal direction.50 In fact, it was imperative to assess
of the IONPs by TEM and XRD. The TEM images provided whether the addition of CMC-Na would affect the stability of
information regarding the morphology, aggregation, and size of the suspension. As a result, the IONPs/NSC hybrid system
the IONPs. Figures 1a and S1 summarize the morphological with the addition of CMC-Na exhibited a high negative Z
characteristics of IONPs that were successfully obtained from potential of −63.5 ± 1.85 mV (Table S3). Conversely, the
coprecipitation. The histogram (Figure 1b), determined by same system without the addition of CMC-Na showed a zeta
counting 100 randomly particles, indicated average particle size potential of −41.3 ± 0.89 mV. This intense negative charge on
around 5.6 nm, in accordance with recent IONPs synthesized particle surfaces leads to a high electrostatic repulsion between
via coprecipitation.38 Particles presented a crystal structure the nanoparticles, reducing the likelihood of aggregation. On
(Figure 1c) and have intrinsic tendency to aggregate due to the the other hand, in contrast to the previous system, the addition
dipole−dipole attraction (Figure 1a).44 of CMC-Na to the IONPs/SA hybrid system decreased the
The crystallinity of the synthesized IONPs was examined by stability of the system, resulting in a Z potential reduction from
the X-ray diffraction technique, and the XRD pattern is shown −69.43 ± 0.85 to −29.37 ± 1.25 mV. However, negative
in Figure 1e. The crystallinity of the synthesized IONPs was charges between the particles remained, indicating electrostatic
examined by the X-ray technique, and the XRD pattern is repulsion. CMC-Na is a negatively charged polymer, and its
shown in Figure 1e. The manufactured IONPs’ characteristic addition to the system may compete with the negative charges
peaks are in accordance with those of the literature.38,45 As of sodium alginate and, depending on the pH, with iron oxide
shown in Figure 1e, the peaks positioned at 2θ = 30.35°, nanoparticles. This can result in a net decrease in the overall
35.57°, 43.36°, 53.95°, 57.39°, 62.84°, and 74.35° are assigned negative charge of the system. The −COOH and −OH groups
to (220), (311), (400), (422), (511), (440), and (533) can establish hydrogen bonds with the −N�N− and −OH
crystalline planes of magnetite (Fe3O4), respectively. Likewise, groups and exhibit strong adsorption capacity under alkaline
diffraction peaks of maghemite (γ-Fe2O3) were observed at 2θ conditions.34 Negatively charged particles in a hybrid system
= 21.34, 26.42, and 59.24 and are attributed to (200), (211), can create electrostatic repulsion between the particles and the
and (520) crystal planes, respectively. These peaks were viral surface, potentially inhibiting virus adhesion and reducing
identified following the standard 2θ values for γ-Fe2O3, its ability to infect the cells. Additionally, the magnetic
reported by Yu and Kwak.46 This result depicts the spinal properties of the nanoparticles can also interact with the virus,
structure, crystalline nature, and predominance of magnetite of contributing to the virus inactivation mechanisms. According
the synthesized Fe3O4-NPs.47,48 The average size of the IONPs to Zachar,51 any nanoparticle that colloids with a highly
was estimated as 14.52 ± 4.30 nm. negative Z potential strength (>−20 mV) works effectively and
3.2. Zeta Potential. Before delving into the zeta potential causes the nanoparticles to bind selectively to the spike
analysis results, it is essential to acknowledge the significance of proteins of viruses, thus neutralizing their binding affinity to
electrostatic stabilization mechanisms in influencing colloidal the receptor. Conversely, the hybrid solution of chitosan
13996 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

(IONPs/Chi) exhibits a positive zeta potential of +67.6 ± 2.05

mV in a 2% acetic acid solution, attributed to the presence of
protonated −NH3+ residues in an acidic environment.52 The
electrostatic interaction between the positive charges of the
hybrid system and the surface of coronavirus could play a role
in the inactivation mechanism, given that viruses typically carry
a negative charge at neutral pH.52,53 Although the Z potential
indicated a high degree of repulsive or attractive charge, the
nanoparticles still exhibited a wide size range, as observed in
Table S3, with a high polydispersity index especially for the
IONPs/sodium alginate-CMC-Na (IONPs/SA) system. The
nanoparticles still exhibited a wide size range (0.4−8,635 nm).
This suggests that although the high repulsiveness/attractive-
ness visibly reduced nanoparticle aggregation, a wide size range
of IONPs was observed, indicating that other forces, such as
magnetic attraction, still dominate the interaction process. This
is also evident in the results obtained from electrophoretic
mobility, where the greater the mobility, the faster the
movement of these charged particles in an electric field.
3.3. Structural Characterization of IONPs and Fabric
by FTIR. The FTIR spectrum obtained for IONPs is shown in
Figure S2. The intense band observed at 575 cm−1 is attributed
to the deformation associated with metal−oxygen bonds (Fe−
O) in the magnetite crystal lattice, characteristic of almost all
iron structures.54 The bands at 3402 cm−1 are associated with
hydroxyl groups (−OH), which may be related to moisture
absorption.55
For the N-succinyl-modified chitosan spectrum (Figure 2a),
we observed the stretching vibration of the O−H hydroxyl
group, represented by the band at 3452 cm−1 and a slight C−H
axial elongation vibration at 2909 cm−1.56 The symmetrical
band at 1412 cm−1 corresponds to the formation of the
carboxylate derivative (−COO−) through the interaction
between the amino and carboxyl groups.39 N-succinylation
induced evident structural changes upon comparison of the
chitosan and N-succinyl chitosan spectra. For the chitosan
spectrum (Figure 2a), the slightly broader and more Figure 2. FTIR spectra of chitosan (Chi) and N-succinyl chitosan
pronounced band near 3380 cm−1 is formed by the (NSC) (a); and uncoated cotton surfaces and coated with hybrid
superposition of −OH and −NH2 stretching vibrations,57 solutions of IONPs/NSC, IONPs/Chitosan, and IONPs/SA (b).
which are characteristic of the chitosan polymer, along with a
secondary amide band at 1651 cm−1.39 These results are pores. These fabrics may be used in products such as curtains,
consistent with previous reports.56 hospital benches or sheets, lab coats, or even masks (after an
In Figure 2b, FTIR spectra for cotton fabrics coated with adequate porosity study).
hybrid systems containing 4.1 mM iron oxide nanoparticles are The EDS chemical analysis performed an area of 250 × 250
presented. The common band at 1036 cm−1, observed in all μm2 confirmed the presence of iron on the covered fabric
fabric spectra before and after the coatings, can be attributed to (Table S4 and Figure S3). The EDS plots indicated a greater
the stretching vibrations of C−O and C−C of the amount of iron oxide on fabric coated with the hybrid system
polysaccharide cellulose.58 Additionally, the strong band produced with a higher concentration of iron oxide nano-
observed at 3338 cm−1 is also characteristic of cotton fabric particles (4.1 mM). According to EDS maps, the iron content
(cellulose), attributed to the stretching vibration of the −OH represented 0.1−0.3% by weight for surfaces coated with a
group of water, becoming weaker and narrower in the other hybrid solution that contained a concentration of iron oxide
spectra.59 A decrease in the band intensity is also observed nanoparticles at concentrations of 1.4 and 2.8 mM. Fabrics
with the insertion of the hybrid coating into the fabric. coated with 4.1 mM IONPs showed a higher proportion of
3.4. Morphology and Chemical Composition of iron nanoparticles, as confirmed by EDS analysis, which
Virucidal Coatings. SEM images (Figure 3) indicate that showed 0.4 and 0.6% by weight of IONPs on the surfaces
the immersion of the cotton fabric in the virucidal hybrid coated with a hybrid solution of N-succinyl chitosan and
coating was effective, forming a film capable of completely chitosan, respectively. The EDS maps show that IONPs are
covering the surface of the cotton fabric. It is also possible to homogeneously distributed over the fibers, indicating that the
observe small particles (red color) in the EDS analysis (Figure technique may produce materials with a uniform surface
S3) and a homogeneous distribution of nanoparticles distribution.
throughout the surface. These particles are associated with 3.5. Determination of Iron Release Kinetics. The total
IONPs. Neither complete nor partial blocking of pores was amount of iron incorporated onto the surface of the cotton
observed that would reduce the diameter of the cotton fiber fabrics was determined by AAS, immersing the coated surface
13997 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

Figure 3. SEM images of (a) uncoated cotton surfaces (control) and

(b) surfaces coated with N-succinyl chitosan (NSC) at 1.4 mM
IONPs, (c) NSC/2.8 mM IONPs, (d) NSC/4.1 mM IONPs, (e)
sodium alginate (SA)/1.4 mM IONPs, (f) SA/2.8 mM IONPs, (g)
SA/4.1 mM IONPs, (h) chitosan/1.4 mM IONPs, (i) chitosan/2.8
mM IONPs, and (j) chitosan/4.1 mM IONPs.

in a 10% HNO3 (v/v) solution and quantifying the iron ions.

The results of the total amount of iron ions per area
impregnated in the fabric for the different hybrid systems
studied are described in Table S5. Figure 4. Release kinetics of different concentrations of IONPs (1.4,
Table S5 depicts the Fe retention results of the virucidal 2.8, and 4.1 mM) by hybrid system coating cotton surfaces (a) N-
coating on cotton fabric after immersion in an aqueous succinyl chitosan (NSC), (b) sodium alginate (SA), and (c) chitosan
medium for 24 h. All the hybrid solutions studied showed (Chi). The scatter columns represent the release behavior in
percentage (right axis) and quantifiable in mg/cm2 (left axis).
interesting results with iron retention in the fabric of between
41.34% ± 7.25 (for chitosan) and 68.49% ± 2.90 (for N-
succinyl chitosan). These results are encouraging and may be 3.6. Virucidal Test. Different biopolymeric coatings with
used to analyze and optimize these materials. IONPs at different concentrations, coating cotton fabrics
The kinetics of iron ion release (Figure 4) showed that commonly used in manufacturing PPE, were evaluated for their
coatings with different hybrid solutions showed a rapid release viral inactivation properties and cytotoxicity (cytopathic effect
of iron in an aqueous medium up to a time of approximately evaluation; see Table 1). The cotton-fabric control sample
480 min, which was released continuously during the 24 h (without coating) did not display virucidal activity, and
(1440 min) observed. The coating of the chitosan hybrid presented a low cytopathic effect, being nontoxic for L929
solution showed a greater release of iron ions, between 52.67 cells.
and 58.66% (see Figure 4a); on the other hand, the surface of In contrast, all fabrics coated with the hybrid solution
the fabric that was impregnated with the hybrid solution of N- showed a viral inactivation of 99.9999% for all samples studied,
succinyl chitosan showed greater resistance to the release of within 24 h. In addition, the N-succinyl chitosan with iron
iron, between 31.51 and 33.39% (see Figure 4b). For the nanoparticle hybrid surface (4.1 mM) was able to inactivate
sodium alginate hybrid solution, the release of iron ions during 99% of the virus within just 5 min of contact. These results are
the 24 h of immersion in water ranged from 47.12 to 50.49% encouraging and indicate that IONPs are virucidal against
(Figure 4c). The presence of the bioactive was also observed SARS-CoV-2, as the NPs interact with the spike glycoprotein
even after 24 h of fabric immersion in an aqueous medium. of the coronavirus, favoring a reaction with the viral lipid
These results are highly encouraging, as they demonstrate a structure, which may hinder or prevent the virus from entering
strong interaction of IONPs with the biopolymers and the host cell.6−8 In addition, the direct interaction of
excellent fabric adhesion even when immersed in water, not nanoparticles and metal ions with viral surfaces, such as
fully releasing iron ions. Iron release experiments demonstrate proteins and genetic material, may impair viral integrity,
the performance of the coating during viral inactivation and breaking and causing damage to the structure of the virus.16
indicate the potential release of the virucidal agent during Furthermore, releasing reactive oxygen species (ROS) may
fabric washing. degrade viral components through oxidation.60 Magnetite
13998 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

Table 1. In vitro Results of the Action of Hybrid Coatings Against Coronavirus (Strain MHV-3) and Cytotoxicity Test Using
the L929 Fibroblast Cell Linea
sample IONP concentration (mM) viral inactivation (%)* contact time In vitro toxicity to fibroblast cells (dilutions)
2
fabric (Cotton) 0 0% 24 h:1:10 (*) nontoxic
N-succinyl chitosan 1.4 99.9999% 24 h:1:102 nontoxic
sodium Alginate 99.9999% 24 h:1:102 nontoxic
chitosan 99.9999% 24 h:1:102 nontoxic
N-succinyl chitosan 2.8 99.9999% 24 h:1:102 nontoxic
sodium Alginate 99.9999% 24 h:1:102 nontoxic
chitosan 99.9999% 24 h:1:102 nontoxic
N-succinyl chitosan 4.1 99% 5 min:1:102 nontoxic
99% 60 min:1:102 nontoxic
99.9999% 24 h:1:102 nontoxic
sodium Alginate 99.9999% 24 h:1:102 nontoxic
chitosan 99.9999% 24 h:1:102 nontoxic
a
*Active dilution of the coronavirus.

nanoparticles coated with PEG and PVP could interact with sterilizing polymers.16 Scheme 1c shows a schematic
the virus and reduce the production of viral progeny, with a representation of the interaction of iron oxide nanoparticles
reduction of 3.5 and 2.5 log against the Influenza A/H1N1 with the virus protein or membrane, resulting in viral
virus for MDCK (Madin-Darby canine kidney) cells with a denaturation and/or conformational dysfunction.
concentration of 30 μg·mL−1 of nanoparticles.61 An aqueous solution of CMC-Na, the anionic surfactant
The nanometer dimension of IONPs allows them to interact used to stabilize the hybrid solution, was also investigated for
with different structures within living organisms.8,14 The extent viral inactivation and its toxicity to cells. The cytotoxicity test
of IONP release corroborates the AAS release kinetic profiles (maximum nontoxic dose or DMNT, for short) of the samples
presented herein. Furthermore, the in vitro cytotoxicity test for of CMC-Na in aqueous solution was tested in the 101 to 1010
the cells with the virucidal fabric demonstrated nontoxicity dilution in DMEM, in fibroblast cells, and without virus. The
within 24 h of contact. Due to their low cytotoxicity and virucidal test was performed with the contact time of the
approved in vivo use in iron replacement therapies,21 IONPs coronavirus (Strain MHV-3) with the CMC-Na solution in 5
have great potential for preventing and controlling infections in min. As a result, the biopolymer used for the dispersion of iron
healthcare settings. Therefore, their use in the manufacturing oxide nanoparticles did not show toxicity to cells; it also
formulations for antiviral coatings is suggested. In light of this, showed a 99.59 ± 0.27% reduction in the viral titer. Although
our results further indicate that the coatings stably release ions, the CMC-Na virucidal test was carried out in an aqueous
proven by low leaching, at a rate that produces virucidal solution, the result also implies the salt’s positive influence in
activity and is safe for human cells. the hybrid system and enhances the virucidal effect of the
Even though the studies on the mechanism of action by nanoparticles on the coating. Due to the presence of protons
which iron oxide nanoparticles act against the SARS-CoV-2 from sulfonic acid groups that become surface accessible after
virus are still inceptive, some insights have been reported. For exposure to an aqueous medium, anionic polymers can also
instance, Lin et al.16 studied some mechanisms that may inactivate the coronavirus.63
explain the interaction of metallic nanoparticles with the 3.7. Cytotoxicity Assay. The percentage of viability for
SARS-CoV-2 virus (e.g., reactive oxygen species (ROS) fibroblast ranged from 29.60 to 53.56% in 24 h, with no
generation, lipid peroxidation, and binding to viral surface statistical difference between the samples; and from 18.74 to
proteins to impair binding to host cells). The contact of iron 36.77% in 48 h, being higher for 4.1 mM IONPs/Chitosan in
oxide nanoparticles with the lipid envelope of the SARS-CoV-2 relation to the other samples (p < 0.05) (see Figure 5). All
membrane becomes vulnerable to lipid peroxidation, whereas samples compromised mitochondrial dehydrogenase activity,
ROS and nanoparticle binding may inhibit its integral thus showing a time-dependent cytotoxic effect. Cell viability
proteins.16 Oxidants may activate and inactivate transcription percentages were all below 70%, which is the minimum value
factors, membrane channels, and metabolic enzymes and to be considered as a noncytotoxic material, as established by
regulate calcium-dependent and phosphorylation signaling the International Organization for Standardization�ISO
pathways.62 Kumar et al.61 showed that iron oxide nano- 10993-5.42 Moreover, cells at 24 h after treatment presented
particles may interact directly with viral transcripts to inhibit morphological changes, including evidence of the presence of
the formation of new virions. Abo-Zeid et al.60 studied the intracellular vacuoles and cell disruption, when compared with
highly specific interaction of Fe2O3 and Fe3O4 with the S1- those of the control group (data not shown).
RBD of SARS-CoV-2. They found a lower binding free energy IONPs normally present concentration-dependent cytotox-
Fe3O4 (−10.66 kcal/mol) than Fe2O3 (−8.97 kcal/mol), icity, which can affect the cell size and morphology. For
thereby indicating greater stability of the Fe3O4/S1-RBD example, Sonmez et al.64 reported dose-dependent cytotoxicity
complex. They also illustrated the tendency of the iron oxide and genotoxicity of IONPs (iron oxide nanoparticles) on
nanoparticles to bind to the viral surface of receptors through human cell lines, and other authors have reported cytotoxicity
electrostatic interactions, blocking the ability of the virus to of IONPs based on impaired mitochondrial activity of
bind to receptors corresponding to target cells. Therefore, fibroblast cells, further showing intracellular accumulation of
ROS-generating nanomaterials (e.g., iron oxide nanoparticles) aggregates of nanoparticles in the cell membrane and
are suitable prophylactic agents for surface coatings and self- cytoplasm.65 The cytotoxicity mechanisms suggested for
13999 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

IONPs/NSC (N-succinyl chitosan-sodium carboxymethylcel-

lulose-iron oxide nanoparticles) over other studied systems for
preclinical assays. Let us highlight some reasons for this choice:
although all hybrid coatings showed antiviral activity of
99.9999% within 24 h of contact, the IONPs/NSC hybrid
system demonstrated 99% virucidal activity in just 5 min of
contact, exhibiting high efficiency in reducing viral load and
showing significant potential for antiviral applications. Another
point to note is the zeta potential: the IONPs/NSC system
showed as much stability as the IONPs/Chi system (chitosan-
iron oxide nanoparticles), despite opposite charges, slightly
decreasing this stability for the IONPs/SA system (sodium
alginate-sodium carboxymethylcellulose-iron oxide nanopar-
ticles) with the addition of CMC-Na. However, this did not
affect the good iron deposition of the IONPs/SA system in
leaching tissue, as observed in the leaching tests. Nevertheless,
the IONPs/NSC hybrid system continued to stand out as it
Figure 5. Cell viability profile of L929 cells exposed to different retained a greater amount of iron in solution in the tissue,
treatments. which is relevant for this research since, due to its magnetic
properties, among others, iron is the main bioactive for
IONPs consist of the impairment of the activities of key inactivation and should be released in a controlled manner. It
enzymes, increased permeability of the plasma membrane, and is also important to emphasize the absence of previous studies
oxidative damage to nucleic acids.66 It is important to highlight using this system (IONPs/NSC) for antiviral coatings, thus
that in vitro toxicity assays with monolayers cells are limited highlighting the originality and potential impact of this
and do not contemplate the diversity of biological reactions of research.
the in vivo environment, and the results must always be Therefore, topical administration of white nanoparticles or
interpreted with caution. Another point to be considered is the
hybrid solution without IONPs (WN) did not cause
form and/or site of application of the material. In the context
macroscopic changes in the skin of the animals, when
of this study, the IONPs would be applied as a covering for
PPE surfaces and would come into contact with the user’s skin compared to the saline group, and evaluated during 14 days
and not directly with skin cells, meaning a low risk of cytotoxic after treatment. Similar results were observed for the groups
reaction. The dermal toxicity risk was evaluated in an in vivo treated with IONPs/NSC (1.5−40 mg/kg), compared to the
model, as shown below. Due to the lower iron leaching on the WN group (Figure 6). On the other hand, the topical
surface and its high virucidal activity, an acute dermal toxicity administration of benzalkonium chloride�BC (750 mg/kg)
study was carried out for the IONPs/NSC hybrid system, in induced progressive alterations in the skin and in the degree of
accordance with OECD guideline 402. skin injury, expressed in scores (0−4). BC caused roughness,
3.8. Acute Dermal Toxicity Assay. Due to some altered natural color, and redness in the rat skin. On the 14th
limitations, such as the limited quantity of animals available day post-treatment with BC, the skin irritation progressed to
for the study’s development, we opted for the hybrid system exposed wounds (Figure 6). It is important to highlight that

Figure 6. Skin of rats before and after treatment with IONPs/NSC.

14000 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

trichotomy did not cause damage to the animals’ skin (data not nanoparticles, which can influence the formation of aggregates,
shown). such as particles of iron oxide, and the degree of the interaction
Figure S4 depicts the body weight progression of rats treated of the metallic nanoparticles with the biological system. Studies
with IONPs/NSC (1.5−40 mg/kg). Following treatment with show that proteins in the plasma adsorb to nanoparticles,
WN, animals gained body mass throughout the 14-day triggering, for example, the production of reactive oxygen
observation period, similar to observations for the saline species and cellular metabolism alterations.69 Additionally, the
group, which also gained body mass during the study period. route of administration, dose, and physical and chemical
Similar results were observed for the IONPs/NSC-treated characteristics of metallic nanoparticles influence their toxicity.
groups, where on the 14th day of evaluation, the average body A previous study in mice showed that IONPs (size: <50
mass of the group that was treated with the highest dose of nm), administered by inhalation, reached the animals’ brains
IONPs/NSC (40 mg/kg) was 209.3 ± 4.3 g, which was not without inducing any adverse effects.70 However, another
statistically different from that of the white nanoparticle (WN) study that evaluated the repeated dose toxicity of IONPs (size:
group (192.3 ± 10.9 g). The topical administration of BC to 155 nm), administered by oral route (30, 300, and 1000 mg/
the animals did not alter the body mass of the animals when
kg), reported on their toxic effects, which were possibly
compared to the saline group.
associated with oxidative stress.71 These results exemplify
Similarly to the saline group, the animals treated with WN
showed intact epidermis, well-defined skin layers, and the variables that can influence the toxicity of metallic particles,
absence of inflammatory cells (Figure 7a,b). This absence of such as the size and route of administration. However, the
influence of other variables cannot be ruled out, such as the
nature of the polymer, the preparation method, and the
stability of the system.72 In the present study, the IONPs did
not demonstrate dermal toxicity in female rats, which may be
associated, for example, with the administration route of the
IONPs. In addition, chitosan was employed as the polymer (β-
1,4-D-glucosamine) for the formulation of the IONPs under
study. Reports in the literature show that chitosan has
characteristics, such as biocompatibility and low toxicity, that
favor applications in the field of dermatology.25

4. CONCLUSION
Hybrid iron oxide nanoparticle coatings demonstrated
remarkable effectiveness against the MHV-3 Coronaviridae
strain. The IONPs exhibited strong interaction and homoge-
neous distribution on the cotton surface, indicative of the
efficacy of the immersion technique employed. Despite
inducing in vitro cytotoxicity against isolated fibroblasts, the
IONPs exhibited no signs of acute dermal toxicity in rats, as
Figure 7. Histology of the skin tissues of the rats treated, or not, with determined by clinical and histological evaluations. These
IONPs/NSC. (a) Skin tissue of saline group, (b) skin tissue of WN
group, (c, d, e, and f) skin tissue of IONPs/NSC (1.5, 5, 14, and 40
promising results obtained from our hybrid coatings inspire the
mg/kg, respectively), and (g) BC (750 mg/kg). Staining: Hematox- development of a novel and effective strategy for combating
ylin-eosin. Magnification 400×. The black arrow indicates the airborne viruses such as SARS-CoV-2 and other viral threats.
presence of parakeratosis. However, it is important to point out that the practical
application of these coatings will encounter significant
morphological changes in the animals’ skin was also observed challenges, including scalability and cost-related issues. In
in the groups treated with increasing doses of IONPs/NSC. future work, assessments of breathability and filtration
However, the BC group showed significant histological capability could be conducted to further enhance the utility
alterations, with the presence of parakeratosis and fibroblast of these coatings.
formation, indicating tissue replacement and loss of annexes
after injury, as well as the presence of red blood cells and
leukocytes (Figure 7g).
The dermal toxicity of BC, a toxic standard, has been
■ ASSOCIATED CONTENT
* Supporting Information
sı

classified as moderate to severe, and its effects are related to The Supporting Information is available free of charge at
the disruption of the lipid bilayers of the cell membrane.67 A https://pubs.acs.org/doi/10.1021/acsanm.4c00951.
positive aspect of employing BC in dermal toxicity studies is
that it allows confirmation that the methodology used in this Concentration of biopolymers for the hybrid systems
study is able to detect alterations in the skin of the investigated used in the coating, macroscopic evaluations used in the
animals; in other words, BC serves as an excellent standard for dermal toxicity test, TEM images of particle distribution
dermal toxicity studies. and FTIR spectra of iron oxide nanoparticles, EDS
Several factors have been associated with the toxicological analysis for the hybrid coatings, leaching and retention
profiles of metallic nanoparticles. These include the nature of of IONPs immersed in aqueous solution, and evaluating
the particulate metal, such as AgNP, CuNP, CuO NP, Fe2O3 the mass of rats treated with IONPs/NSC for 14 days
NP, ZnO NP, and TiO2 NP,68 the morphology of the metallic (PDF)

14001 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

■ AUTHOR INFORMATION
Corresponding Authors
Funding
Cearense Foundation to Support Scientific and Technological
Development (ROR identifier: P20-071-00034.01.00/20).
Kalyne Almeida Leal − Federal University of Ceará, Fortaleza
Notes
60455-760, Brazil; Department of Pharmacy, Fortaleza
60430-160, Brazil; Email: [email protected] The authors declare no competing financial interest.
Rodrigo Silveira Vieira − Federal University of Ceará,
Fortaleza 60455-760, Brazil; Department of Chemical
Engineering, Fortaleza 60455-760, Brazil; orcid.org/
■ ACKNOWLEDGMENTS
This research used facilities of the Brazilian Nanotechnology
0000-0003-4569-9655; Email: [email protected] National Laboratory (LNNano), part of the Brazilian Center
for Research in Energy and Materials (CNPEM), a private
Authors nonprofit organization under the supervision of the Brazilian
Jamilly S. F. Constantino − Federal University of Ceará, Ministry for Science, Technology, and Innovations (MCTI).
Fortaleza 60455-760, Brazil; Department of Chemical The TEM (Proposal Number 20240065) staff area acknowl-
Engineering, Fortaleza 60455-760, Brazil; orcid.org/ edged for their assistance during the experiments, and also to
0000-0002-1260-5459 the Department of Genetics, Evolution, Microbiology and
Iran D. S. de Mesquita − Federal University of Ceará, Immunology, University of Campinas for antiviral analyses.
Fortaleza 60455-760, Brazil; Department of Pharmacy,
Fortaleza 60430-160, Brazil
João D. P. Moraes Segundo − Federal University of Ceará,
■ REFERENCES
(1) McGonagle, D.; Bridgewood, C.; Meaney, J. F. M. A
Fortaleza 60455-760, Brazil; Department of Chemical Tricompartmental Model of Lung Oxygenation Disruption to Explain
Engineering, Fortaleza 60455-760, Brazil; Department of Pulmonary and Systemic Pathology in Severe COVID-19. Lancet
Materials Engineering and Bioprocesses, School of Chemical Respir. Med. 2021, 9 (6), 665−672.
Engineering, University of Campinas, Campinas 13083-852, (2) Qin, Z.; Sun, Y.; Zhang, J.; Zhou, L.; Chen, Y.; Huang, C.
Brazil Lessons from SARS-CoV-2 and Its Variants (Review). Mol. Med. Rep.
2022, 26 (2), 263.
Raimundo N. F. Moreira Filho − Federal University of Ceará, (3) Pinato, D. J.; Ferrante, D.; Aguilar-Company, J.; Bower, M.;
Fortaleza 60455-760, Brazil; Department of Chemistry, Salazar, R.; Mirallas, O.; Sureda, A.; Bertuzzi, A.; Brunet, J.;
Fortaleza 60455-760, Brazil Lambertini, M.; et al. Vaccination against SARS-CoV-2 Protects
Ana B. de Araújo − Federal University of Ceará, Fortaleza from Morbidity, Mortality and Sequelae from COVID-19 in Patients
60455-760, Brazil; Department of Pharmacy, Fortaleza with Cancer. Eur. J. Cancer 2022, 171, 64−74.
60430-160, Brazil (4) Ou, J.; Zhang, Y.; Wang, Y.; Zhang, Z.; Wei, H.; Yu, J.; Wang,
Marcia V. P. Ferreira − Federal University of Ceará, Fortaleza Q.; Wang, G.; Zhang, B.; Wang, C. ACE2-Targeting Antibody
60455-760, Brazil; Department of Pathology and Forensic Suppresses SARS-CoV-2 Omicron and Delta Variants. Signal
Medicine, Fortaleza 60430-160, Brazil Transduction Targeted Ther. 2022, 7 (1), 43.
José J. A. de Almeida − Federal University of Ceará, Fortaleza (5) Li, M.; Lou, F.; Fan, H. SARS-CoV-2 Variant Omicron:
Currently the Most Complete “Escapee” from Neutralization by
60455-760, Brazil; Department of Pharmacy, Fortaleza Antibodies and Vaccines. Signal Transduction Targeted Ther. 2022, 7
60430-160, Brazil (1), 28.
Gladyane S. da Silva − Federal University of Ceará, Fortaleza (6) Walls, A. C.; Park, Y.-J.; Tortorici, M. A.; Wall, A.; McGuire, A.
60455-760, Brazil; Department of Pharmacy, Fortaleza T.; Veesler, D. Structure, Function, and Antigenicity of the SARS-
60430-160, Brazil CoV-2 Spike Glycoprotein. Cell 2020, 181, 281−292.e6.
Francisco F. P. Souza − Federal University of Ceará, (7) Staufer, O.; Gupta, K.; Kohler, F.; Sigl, C.; Singh, G.; Vasileiou,
Fortaleza 60455-760, Brazil; Department of Chemical K.; Yagüe Relimpio, A.; Macher, M.; Fabritz, S.; Dietz, H.; Adam, E.
Engineering, Fortaleza 60455-760, Brazil A. C.; Schaffitzel, C.; Ruggieri, A.; Platzman, I.; Berger, L.; Spatz, J. P.
Marcos Vinicius Lorevice − Brazilian Nanotechnology Synthetic Virions Reveal Fatty Acid-Coupled Adaptive Immunoge-
National Laboratory (LNNano), Brazilian Center for nicity of SARS-CoV-2 Spike Glycoprotein. Nat. Commun. 2022, 13
(1), 868.
Research in Energy and Materials (CNPEM), Campinas (8) Cao, Y.; Wang, J.; Jian, F.; Xiao, T.; Song, W.; Yisimayi, A.;
13083-970, Brazil; orcid.org/0000-0001-5635-8608 Huang, W.; Li, Q.; Wang, P.; An, R.; Wang, J.; et al. Omicron Escapes
Fábia K. Andrade − Federal University of Ceará, Fortaleza the Majority of Existing SARS-CoV-2 Neutralizing Antibodies. Nature
60455-760, Brazil; Department of Chemical Engineering, 2021, 602, 657−663.
Fortaleza 60455-760, Brazil (9) Ghasemiyeh, P.; Mohammadi-Samani, S.; Firouzabadi, N.;
Marisa M. Beppu − Department of Materials Engineering and Dehshahri, A.; Vazin, A. A Focused Review on Technologies,
Bioprocesses, School of Chemical Engineering, University of Mechanisms, Safety, and Efficacy of Available COVID-19 Vaccines.
Campinas, Campinas 13083-852, Brazil; orcid.org/ Int. Immunopharmacol. 2021, 100, 108162.
0000-0001-6519-6970 (10) Corrêa, T. Q.; Blanco, K. C.; Vollet-Filho, J. D.; Morais, V. S.;
Trevelin, W. R.; Pratavieira, S.; Bagnato, V. S. Efficiency of an Air
Complete contact information is available at: Circulation Decontamination Device for Micro-Organisms Using
https://pubs.acs.org/10.1021/acsanm.4c00951 Ultraviolet Radiation. J. Hosp. Infect. 2021, 115, 32−43.
(11) Ruiz-Hitzky, E.; Darder, M.; Wicklein, B.; Ruiz-Garcia, C.;
Funding Martín-Sampedro, R.; Del Real, G.; Aranda, P. Nanotechnology
Responses to COVID-19. Adv. Healthcare Mater. 2020, 9 (19),
The Article Processing Charge for the publication of this 2000979.
research was funded by the Coordination for the Improvement (12) Weiss, C.; Carriere, M.; Fusco, L.; Capua, I.; Regla-Nava, J. A.;
Pasquali, M.; Scott, J. A.; Vitale, F.; Unal, M. A.; Mattevi, C.; et al.
of Higher Education Personnel - CAPES (ROR identifier: Toward Nanotechnology-Enabled Approaches against the COVID-19
00x0ma614). Pandemic. ACS Nano 2020, 14 (6), 6383−6406.

14002 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

(13) Tiwari, S.; Juneja, S.; Ghosal, A.; Bandara, N.; Khan, R.; Wallen, (30) Bashir, S.; Teo, Y. Y.; Ramesh, S.; Ramesh, K.; Khan, A. A. N-
S. L.; Ramakrishna, S.; Kaushik, A. Antibacterial and Antiviral High- Succinyl Chitosan Preparation, Characterization, Properties and
Performance Nanosystems to Mitigate New SARS-CoV-2 Variants of Biomedical Applications: A State of the Art Review. Rev. Chem.
Concern. Curr. Opin. Biomed. Eng. 2022, 21, 100363. Eng. 2015, 31 (6), 563−597.
(14) Singh, S.; Batish, Y.; Sharma, N.; Arora, S.; Kakkar, V.; Batra, S. (31) Shen, W.; Pei, P.; Zhang, C.; Li, J.; Han, X.; Liu, T.; Shi, X.; Su,
Unveiling the Nanotechnology-Based Drug Delivery Systems and Z.; Han, G.; Hu, L.; Yang, K. A Polymeric Hydrogel to Eliminate
Patent Literature for Diabetic Retinopathy. Biointerface Res. Appl. Programmed Death-Ligand 1 for Enhanced Tumor Radio-Immuno-
Chem. 2021, 12 (5), 6651−6667. therapy. ACS Nano 2023, 17 (23), 23998−24011.
(15) Chue-Gonçalves, M.; Pereira, G. N.; Faccin-Galhardi, L. C.; (32) Rostami, E. Recent Achievements in Sodium Alginate-Based
Kobayashi, R. K. T.; Nakazato, G. Metal Nanoparticles against Nanoparticles for Targeted Drug Delivery. Polym. Bull. 2022, 79 (9),
Viruses: Possibilities to Fight SARS-CoV-2. Nanomaterials 2021, 11 6885−6904.
(11), 3118. (33) Ren, H.; Gao, Z.; Wu, D.; Jiang, J.; Sun, Y.; Luo, C. Efficient
(16) Lin, N.; Verma, D.; Saini, N.; Arbi, R.; Munir, M.; Jovic, M.; Pb(II) Removal Using Sodium Alginate−Carboxymethyl Cellulose
Turak, A. Antiviral Nanoparticles for Sanitizing Surfaces: A Roadmap Gel Beads: Preparation, Characterization, and Adsorption Mecha-
to Self-Sterilizing against COVID-19. Nano Today 2021, 40, 101267. nism. Carbohydr. Polym. 2016, 137, 402−409.
(17) Zhang, S.; Go, E. P.; Ding, H.; Anang, S.; Kappes, J. C.; (34) Chen, C.; He, E.; Jia, W.; Xia, S.; Yu, L. Preparation of
Desaire, H.; Sodroski, J. G. Analysis of Glycosylation and Disulfide Magnetic Sodium Alginate/Sodium Carboxymethylcellulose Inter-
Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein. J. Virol. penetrating Network Gel Spheres and Use in Superefficient
2022, 96 (3), No. e01626−21. Adsorption of Direct Dyes in Water. Int. J. Biol. Macromol. 2023,
(18) Sportelli, M. C.; Izzi, M.; Kukushkina, E. A.; Hossain, S. I.; 253, 126985.
Picca, R. A.; Ditaranto, N.; Cioffi, N. Can Nanotechnology and (35) Choudhary, S.; Kumar, R.; Dalal, U.; Tomar, S.; Reddy, S. N.
Materials Science Help the Fight against SARS-CoV-2? Nanomaterials Green Synthesis of Nanometal Impregnated Biomass − Antiviral
2020, 10 (4), 802. Potential. Mater. Sci. Eng. 2020, 112, 110934.
(19) DeDiego, M. L.; Portilla, Y.; Daviu, N.; López-García, D.; (36) Liu, L.; Song, G.; Song, Z. Intrinsic Atopic Dermatitis and
Villamayor, L.; Mulens-Arias, V.; Ovejero, J. G.; Gallo-Cordova, Á .; Extrinsic Atopic Dermatitis: Similarities and Differences. Clin., Cosmet.
Veintemillas-Verdaguer, S.; Morales, M. P.; Barber, D. F. Iron Oxide Invest. Dermatol. 2022, 15, 2621−2628.
and Iron Oxyhydroxide Nanoparticles Impair SARS-CoV-2 Infection (37) Kumar, R.; Nayak, M.; Sahoo, G. C.; Pandey, K.; Sarkar, M. C.;
of Cultured Cells. J. Nanobiotechnol. 2022, 20 (1), 352. Ansari, Y.; Das, V. N. R.; Topno, R. K.; Bhawna; Madhukar, M.; et al.
(20) Fernández-Bertólez, N.; Costa, C.; Brandão, F.; Teixeira, J. P.; Iron Oxide Nanoparticles Based Antiviral Activity of H1N1 Influenza
Pásaro, E.; Valdiglesias, V.; Laffon, B. Toxicological Aspects of Iron A Virus. J. Infect. Chemother. 2019, 25 (5), 325−329.
Oxide Nanoparticles, In Nanotoxicology in Safety Assessment of (38) Nehra, P.; Chauhan, R.; Garg, N.; Verma, K. Antibacterial and
Nanomaterials. Advances in Experimental Medicine and Biology. Antifungal Activity of Chitosan Coated Iron Oxide Nanoparticles. Br.
Louro, H.; Silva, M. J., Eds. Springer International Publishing: J. Biomed. Sci. 2018, 75 (1), 13−18.
Cham, 2022; pp. 303350 (39) Bashir, S.; Teo, Y. Y.; Ramesh, S.; Ramesh, K. Synthesis,
(21) Bobo, D.; Robinson, K. J.; Islam, J.; Thurecht, K. J.; Corrie, S. Characterization, Properties of N-Succinyl Chitosan-g-Poly (Meth-
R. Nanoparticle-Based Medicines: A Review of FDA-Approved acrylic Acid) Hydrogels and in Vitro Release of Theophylline. Polymer
Materials and Clinical Trials to Date. Pharm. Res. 2016, 33 (10), 2016, 92, 36−49.
2373−2387. (40) Reed, L. J.; Muench, H. A Simple Method of Estimating Fifty
(22) Salehipour, M.; Rezaei, S.; Mosafer, J.; Pakdin-Parizi, Z.; per Cent Endpoints. Am. J. Epidemiol. 1938, 27 (3), 493−497.
Motaharian, A.; Mogharabi-Manzari, M. Recent Advances in Polymer- (41) International Organization for Standardization. Biological
Coated Iron Oxide Nanoparticles as Magnetic Resonance Imaging Evaluation of Medical Devices - Part 12: sample Preparation and
Contrast Agents. J. Nanopart. Res. 2021, 23 (2), 48. Reference Materials; International Organization for Standardization,
(23) Bataglioli, R. A.; Rocha Neto, J. B. M.; Calais, G. B.; Lopes, L. 2021.
M.; Tsukamoto, J.; Moraes, A. P.; Arns, C. W.; Beppu, M. M. Hybrid (42) International Organization for Standardization. ISO 10993−5,
Alginate−Copper Sulfate Textile Coating for Coronavirus Inactiva- Part 5: tests for in Vitro Cytotoxicity; International Organization for
tion. J. Am. Ceram. Soc. 2022, 105 (3), 1748−1752. Standardization, 2009.
(24) Das Jana, I.; Kumbhakar, P.; Banerjee, S.; Gowda, C. C.; Kedia, (43) Uzunoglu, S.; Karaca, B.; Atmaca, H.; Kisim, A.; Sezgin, C.;
N.; Kuila, S. K.; Banerjee, S.; Das, N. C.; Das, A. K.; Manna, I.; Karabulut, B.; Uslu, R. Comparison of XTT and Alamar Blue Assays
Gowda, C. C.; Mondal, A. Copper Nanoparticle−Graphene in the Assessment of the Viability of Various Human Cancer Cell
Composite-Based Transparent Surface Coating with Antiviral Activity Lines by AT-101 (−/− Gossypol). Toxicol. Mech. Methods 2010, 20
against Influenza Virus. ACS Appl. Nano Mater. 2021, 4 (1), 352−362. (8), 482−486.
(25) Li, J.; Zhuang, S. Antibacterial Activity of Chitosan and Its (44) Galvão, W. S.; Pinheiro, B. B.; Golçalves, L. R. B.; De Mattos,
Derivatives and Their Interaction Mechanism with Bacteria: Current M. C.; Fonseca, T. S.; Regis, T.; Zampieri, D.; Dos Santos, J. C. S.;
State and Perspectives. Eur. Polym. J. 2020, 138, 109984. Costa, L. S.; Correa, M. A.; et al. Novel Nanohybrid Biocatalyst:
(26) Hamedi, H.; Moradi, S.; Tonelli, A. E.; Hudson, S. M. Application in the Kinetic Resolution of Secondary Alcohols. J. Mater.
Preparation and Characterization of Chitosan−Alginate Polyelectro- Sci. 2018, 53 (20), 14121−14137.
lyte Complexes Loaded with Antibacterial Thyme Oil Nano- (45) Shahbazi, F.; Noghani, M.; Ahmadi, R. Effect of Synthesis
emulsions. Appl. Sci. 2019, 9 (18), 3933. Conditions on the Morphology, Composition and Magnetic Proper-
(27) Rajkumari, N. P.; Roy, P.; Siddika, S.; Adhikary, K.; Goswami, ties of the Iron Oxide Nanoparticles Prepared via Electric Discharge
P. Enhancing Anti-Inflammatory and Antibacterial Activity of Method. J. Magn. Magn. Mater. 2021, 536, 168090.
Curcumin by Nano Composing with Curcumin Reduced Copper (46) Yu, B. Y.; Kwak, S.-Y. Assembly of Magnetite Nanocrystals into
Nano for the Treatment of Bacterial Infection. Mater. Sci. Eng. 2023, Spherical Mesoporous Aggregates with a 3-D Wormhole-like Pore
292, 116416. Structure. J. Mater. Chem. 2010, 20 (38), 8320.
(28) Younes, I.; Rinaudo, M. Chitin and Chitosan Preparation from (47) Rahman, S. S. U.; Qureshi, M. T.; Sultana, K.; Rehman, W.;
Marine Sources. Structure, Properties and Applications. Mar. Drugs Khan, M. Y.; Asif, M. H.; Farooq, M.; Sultana, N. Single Step Growth
2015, 13 (3), 1133−1174. of Iron Oxide Nanoparticles and Their Use as Glucose Biosensor.
(29) Farinha, I.; Freitas, F. Chemically Modified Chitin, Chitosan, Results Phys. 2017, 7, 4451−4456.
and Chitinous Polymers as Biomaterials. In Handbook of Chitin and (48) Suryawanshi, P. L.; Sonawane, S. H.; Bhanvase, B. A.;
Chitosan; Elsevier, 2020; pp. 4369. Ashokkumar, M.; Pimplapure, M. S.; Gogate, P. R. Synthesis of

14003 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004
ACS Applied Nano Materials www.acsanm.org Article

Iron Oxide Nanoparticles in a Continuous Flow Spiral Microreactor Iron Oxide Nanoparticles: An in Vitro Biosafety Study. Arch. Biol. Sci.
and Corning® Advanced FlowTM Reactor. Green Process. Synth. 2018, 2016, 68 (1), 41−50.
7 (1), 1−11. (65) Hong, S. C.; Lee, J. H.; Lee, J.; Kim, H. Y.; Park, J. Y.; Cho;
(49) Furlan, D. M.; Morgado, D. L.; Oliveira, A. J. A. D.; Faceto, Â . Lee, J.; Han, D.C. Subtle Cytotoxicity and Genotoxicity Differences in
D.; Moraes, D. A. D.; Varanda, L. C.; Frollini, E. Sisal Cellulose and Superparamagnetic Iron Oxide Nanoparticles Coated with Various
Magnetite Nanoparticles: Formation and Properties of Magnetic Functional Groups. Int. J. Nanomed. 2011, 6, 3219−3231.
Hybrid Films. J. Mater. Res. Technol. 2019, 8 (2), 2170−2179. (66) Raghunath, A.; Perumal, E. Metal Oxide Nanoparticles as
(50) Pate, K.; Safier, P. Chemical Metrology Methods for CMP Antimicrobial Agents: A Promise for the Future. Int. J. Antimicrob.
Quality. In Advances in Chemical Mechanical Planarization (CMP); Agents 2017, 49 (2), 137−152.
Elsevier, 2016; pp. 299325. (67) Sekijima, H.; Oshima, T.; Ueji, Y.; Kuno, N.; Kondo, Y.;
(51) Zachar, O. Nanomedicine formulations for respiratory Nomura, S.; Asakura, T.; Sakai-Sugino, K.; Kawano, M.; Komada, H.;
infections by inhalation delivery: COVID-19 and beyond. Med. Kotani, H. Toxicologic Pathological Mechanism of Acute Lung Injury
Hypotheses. 2022, 159, 110753. Induced by Oral Administration of Benzalkonium Chloride in Mice.
(52) Ochi, D.; Freitas, E. D.; Kerwald, J.; d’Á vila, M. A.; Beppu, M. Toxicol. Res. 2023, 39 (3), 409−418.
M. Synthesis and Characterization of N-Phosphonium Chitosan and (68) Tortella, G.; Rubilar, O.; Fincheira, P.; Pieretti, J. C.; Duran, P.;
Its Virucidal Activity Evaluation against Coronavirus. Int. J. Biol. Lourenço, I. M.; Seabra, A. B. Bactericidal and Virucidal Activities of
Macromol. 2023, 246, 125665. Biogenic Metal-Based Nanoparticles: Advances and Perspectives.
(53) Calais, G. B.; Rocha Neto, J. B. M.; Bataglioli, R. A.; Chevalier, Antibiotics 2021, 10 (7), 783.
P.; Tsukamoto, J.; Arns, C. W.; Mantovani, D.; Beppu, M. M. (69) Liu, N.; Tang, M.; Ding, J. The Interaction between
Bioactive Textile Coatings for Improved Viral Protection: A Study of Nanoparticles-Protein Corona Complex and Cells and Its Toxic
Effect on Cells. Chemosphere 2020, 245, 125624.
Polypropylene Masks Coated with Copper Salt and Organic
(70) Kwon, J. -T.; Hwang, S. -K.; Jin, H.; Kim, D. -S; Minai-Tehrani,
Antimicrobial Agents. Appl. Surf. Sci. 2023, 638, 158112.
A.; Yoon, H. -J.; Choi, M.; Yoon, T. -J.; Han, D. -Y.; Kang, Y. -W.;
(54) Monteiro, R. R. C.; Neto, D. M. A.; Fechine, P. B. A.; Lopes, A.
Yoon, B. -I; Lee, J. -K.; Cho, M. -H. Body Distribution of Inhaled
A. S.; Gonçalves, L. R. B.; Dos Santos, J. C. S.; De Souza, M. C. M.;
Fluorescent Magnetic Nanoparticles in the Mice. J. Occup. Health
Fernandez-Lafuente, R. Ethyl Butyrate Synthesis Catalyzed by Lipases 2008, 50 (1), 1−6.
A and B from Candida Antarctica Immobilized onto Magnetic (71) Reddy, U. A.; Prabhakar, P. V.; Mahboob, M. Biomarkers of
Nanoparticles. Improvement of Biocatalysts’ Performance under Oxidative Stress for in Vivo Assessment of Toxicological Effects of
Ultrasonic Irradiation. Int. J. Mol. Sci. 2019, 20 (22), 5807. Iron Oxide Nanoparticles. Saudi J. Biol. Sci. 2017, 24 (6), 1172−1180.
(55) Sajjad, M.; Saad Almufarij, R.; Ali, Z.; Sajid, M.; Raza, N.; (72) Patil, U.; Adireddy, S.; Jaiswal, A.; Mandava, S.; Lee, B.;
Manzoor, S.; Hayat, M.; Abdelrahman, E. A. Magnetic Solid Phase Chrisey, D. In Vitro/In Vivo Toxicity Evaluation and Quantification
Extraction of Aminoglycosides Residue in Chicken Egg Samples of Iron Oxide Nanoparticles. Int. J. Mol. Sci. 2015, 16 (10), 24417−
Using Fe3O4-GO-Agarose-Chitosan Composite. Food Chem. 2024, 24450.
430, 137092.
(56) Qing, X.; He, G.; Liu, Z.; Yin, Y.; Cai, W.; Fan, L.; Fardim, P.
Preparation and Properties of Polyvinyl Alcohol/N−Succinyl
Chitosan/Lincomycin Composite Antibacterial Hydrogels for
Wound Dressing. Carbohydr. Polym. 2021, 261, 117875.
(57) Meng, J.; Cui, J.; Yu, S.; Jiang, H.; Zhong, C.; Hongshun, J.
Preparation of Aminated Chitosan Microspheres by One-Pot Method
and Their Adsorption Properties for Dye Wastewater. R Soc. Open Sci.
2019, 6 (5), 182226.
(58) Kaidi, S.; Belattmania, Z.; Bentiss, F.; Jama, C.; Reani, A.;
Sabour, B. Synthesis and Characterization of Silver Nanoparticles
Using Alginate from the Brown Seaweed Laminaria Ochroleuca:
Structural Features and Antibacterial Activity. Biointerface Res. Appl.
Chem. 2021, 12 (5), 6046−6057.
(59) Mohabatpour, F.; Yazdanpanah, Z.; Papagerakis, S.; Chen, X.;
Papagerakis, P. Self-Crosslinkable Oxidized Alginate-Carboxymethyl
Chitosan Hydrogels as an Injectable Cell Carrier for In Vitro Dental
Enamel Regeneration. J. Funct. Biomater. 2022, 13 (2), 71.
(60) Abo-Zeid, Y.; Ismail, N. S. M.; McLean, G. R.; Hamdy, N. M. A
Molecular Docking Study Repurposes FDA Approved Iron Oxide
Nanoparticles to Treat and Control COVID-19 Infection. Eur. J.
Pharm. Sci. 2020, 153, 105465.
(61) Kumar, S. R.; Paulpandi, M.; ManivelRaja, M.; Mangalaraj, D.;
Viswanathan, C.; Kannan, S.; Ponpandian, N. An in Vitro Analysis of
H1N1 Viral Inhibition Using Polymer Coated Superparamagnetic
Fe3O4 Nanoparticles. RSC Adv. 2014, 4 (26), 13409.
(62) Winterbourn, C. C.; Hampton, M. B. Thiol Chemistry and
Specificity in Redox Signaling. Free Radical Biol. Med. 2008, 45 (5),
549−561.
(63) Peddinti, B. S. T.; Downs, S. N.; Yan, J.; Smith, S. D.; Ghiladi,
R. A.; Mhetar, V.; Tocchetto, R.; Griffiths, A.; Scholle, F.; Spontak, R.
J. Rapid and Repetitive Inactivation of SARS-CoV-2 and Human
Coronavirus on Self-Disinfecting Anionic Polymers. Adv. Sci. 2021, 8
(11), 2003503.
(64) Sonmez, E.; Aydin, E.; Turkez, H.; Ö zbek, E.; Togar, B.; Meral,
K.; Ç etin, D.; Cacciatore, I.; Di, S. Cytotoxicity and Genotoxicity of

14004 https://doi.org/10.1021/acsanm.4c00951
ACS Appl. Nano Mater. 2024, 7, 13991−14004