Design and application of (Fe3O4)-GOTfOH based AgNPs doped starch/PEG-poly (acrylic acid) nanocomposite as the magnetic nanocatalyst and the wound dress

Article abstract of DOI:10.1016/j.molstruc.2020.128142

As a novel, recyclable nanocatalyst, (Fe₃O₄)-GO_TfOH based Ag nanoparticles doped Starch/PEG-poly (acrylic acid) nanocomposite (Fe₃O₄?GO_TfOH/Ag/St-PEG-AcA) was applied for one-pot synthesis

Full text of DOI:10.1016/j.molstruc.2020.128142

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Design and application of (Fe O )-GO
3 4 TfOH
based AgNPs doped starch/PEG-poly
(acrylic acid) nanocomposite as the magnetic nanocatalyst and the wound dress
Shayan Forouzandehdel, Maryam Meskini, Mina Rezghi Rami
PII:
S0022-2860(20)30467-1
DOI:
https://doi.org/10.1016/j.molstruc.2020.128142
MOLSTR 128142
Reference:
To appear in: Journal of Molecular Structure
Received Date: 9 February 2020
Revised Date: 15 March 2020
Accepted Date: 25 March 2020
Please cite this article as: S. Forouzandehdel, M. Meskini, M.R. Rami, Design and application of
(Fe O )-GO
3 4 TfOH
based AgNPs doped starch/PEG-poly (acrylic acid) nanocomposite as the magnetic
nanocatalyst and the wound dress, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
j.molstruc.2020.128142.
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Design and application of (Fe3O4)-GOTfOH based AgNPs doped Starch/PEG-
poly (acrylic acid) nanocomposite as the magnetic nanocatalyst and the wound
dress
Shayan Forouzandehdela, Maryam Meskinib, Mina Rezghi Ramic*
a Department of Chemistry, Sharif University of Technology, P. O. Box 11365-9516, Tehran,
Iran
b Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
c Department of Chemistry, K. N. Toosi University of Technology, P. O. Box 15875-4416,
Tehran, Iran.
*rezghi@siptc.ir
Credit author statements:
Mina Rezghi Rami: Methodology, Conceptualization, Writing - Original Draft, Supervision,
Project administration; Shayan forouzandehdel: Validation, Formal analysis, Resources,
Funding acquisition; Maryam Meskini: Data Curation, Writing - Review & Editing,
Visualization

Design and application of (Fe3O4)-GOTfOH based AgNPs doped Starch/PEG-poly
(acrylic acid) nanocomposite as the magnetic nanocatalyst and the wound dress
Shayan Forouzandehdela, Maryam Meskinib, Mina Rezghi Ramic*
a Department of Chemistry, Sharif University of Technology, P. O. Box 11365-9516, Tehran, Iran
b Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
c Department of Chemistry, K. N. Toosi University of Technology, P. O. Box 15875-4416, Tehran,
Iran.
*rezghi@siptc.ir
Abstract: As a novel, recyclable nanocatalyst, (Fe3O4)-GOTfOH based Ag nanoparticles doped
Starch/PEG-poly (acrylic acid) nanocomposite (Fe3O4@GOTfOH/Ag/St-PEG-AcA) was applied for
one-pot synthesis of 2,4,6-triarylpyridine derivatives under water solvent conditions. The prepared
nanocomposite was also evaluated in terms of biocompatibility for wound healing.
Fe3O4@GOTfOH/Ag/St-PEG-AcA could be easily removed from the mixture of the reaction by an
external magnet and recycled without a considerable decrease of activity even after 10 runs. The
new nanocatalyst offered better efficiencies than other commercially available sulfonic acid
catalysts. In terms of the bioactivity of nanocatalyst, good antimicrobial efficiency was confirmed
on E. coli bacteria. Besides, histology of repaired wounds in Fe3O4@GOTfOH/Ag/St-PEG-AcA for a
healed group of mice showed better fibroblast distribution and more compact collagen fiber
organization compared to wounds in the control group.
Keywords: Magnetic nanocatalyst; Hydrogels; Wound healing; 2,4,6-triarylpyridines.
Introduction
On account of the most important and obvious advantages of heterogeneous catalysts such as easily
separation and reusability for the production of fine chemicals[1] and increasing environmental
concerns about liquid mineral acids[2], application of solid acids and development of efficient and
recoverable heterogeneous catalysts has long been a goal in catalysis research[3].
It is renowned that graphene as a two-dimensional and one-atom-thick sheet of sp2 bonded carbon
atoms that are densely packed in a honeycomb crystal lattice and graphene oxide has been widely
used in many different fields such as sensors[4], catalysis[5], adsorption[6], and energy storage[7].
Nano sheets of graphene oxide with high surface areas are covered by hydroxyl, epoxy, and

carboxylic groups which offer high water solubility for many applications. Graphene oxides
contain a range of reactive oxygen functional groups, which offers it a good candidate for use in the
mentioned applications through chemical functionalization [8]. Recently, GO-based poly(lactic
acid) (PLA) stereo complex crystals (SCs) were designed by Xu et al. [9] to show elucidation of
acceptable thermal and barrier properties, which may influence further extension of other material
combinations. In the other study, Xu et al. [10] revealed that decrease the planar size of graphene
oxide quantum dots (GOQDs), and the intercalation of functional groups containing oxygen,
diminished the filler aggregation and improved the interfacial contacts with the host polymer. Their
findings afforded intangible understandings of the significance of the dimensionality and surface
chemistry of GO in the promising field of bionanocomposites.
Nanocomposite (NC) gels are considered due to their improved properties, for instance, the
extraordinary mechanical property of organic-inorganic hybrid networks of nanocomposite
gels[11]. Nano dispersions of metal nanoparticles are used to prepare nanocomposites due to their
special properties, such as the large surface area to volume ratio and multiple applications[12].
Incorporation of silver nanoparticles (AgNPs) into the hydrogels has developed both mechanical
properties and chemical stability of hydrogels, as well as increasing the adsorption capacity[13].
Besides, due to the antimicrobial properties of AgNPs, biocompatible polymers containing AgNPs
have proven to be promising candidates for the biomedical area due to their superior antibacterial
properties[14].
From a medical point of view, using NC for wound healing offers the possibility to absorb the
exudate and reducing water evaporation to keep dehydration away. Preparing these conditions is
considered an ideal environment for the wound healing process[15]. AgNPs and hydrogel-based
biomaterials are non-cytotoxic and safe for patients in wound care management[16]. The unique
intrinsic features of these materials promote wound healing and effectively control the growth
of microorganisms at the wound site. This strategy plays an important role in the treatment of both
acute and chronic wounds[17]. Recently, the development of AgNPs impregnated chitosan-poly
ethylene glycol (PEG) hydrogel to accelerate wound healing in diabetic patients. The results
illustrated a good porosity, high degree of swelling, and water vapor transition rate (WVTR) for
AgNPs impregnated hydrogel as well as advanced antimicrobial and antioxidant properties in-
vitro and enhanced wound healing capability in-vivo in diabetic rabbits[18]. Besides, stimuli-
responsive chitosan (CS) and poly (N-vinyl-2-pyrrolidone) (PVP) have attained hydrogel properties
in the presence of 74% neutralized polyacrylic acid (PAA). The biocompatibility of prepared
hydrogel made them pertinent to drug delivery, and their release profile is examined

spectrophotometrically using silver sulfadiazine showed 91.2% of drug release in a consistent and
controlled manner[19].
Over the past few years, the N-heterocyclic compounds, such as pyridines, are very helpful material
for the development of molecules of pharmaceutical or biological interest[20]. Furthermore,
conjugated molecules that have a pyridine core as the key unit, such as 2,4,6-triarylpyridines have
also attracted much attention in previous years due to providing the impetus for further studies in
utilizing this scaffold in new therapeutic drug classes[21, 22].
In our continuing efforts toward effective synthetic methodologies [23, 24], new environmental
catalysts improvement, we aimed to formulate a new type of magnetically separable nanocomposite
(Fe3O4)-GOTfOH based AgNPs doped Starch/PEG-poly (acrylic acid) nanocomposite
(Fe3O4@GOTfOH/Ag/St-PEG-AcA) and its application for the synthesis of pyridine derivatives as
well as a biocompatible nanocomposite as medication for wound healing. To the best of our
knowledge, there has no such reported on the synthesis of a novel magnetic graphene-based
bionanocomposite consist of starch, polyethylene glycol, acrylic acid, and AgNPs for simultaneous
investigation of antimicrobial activities, promoting wound healing, and organic synthesis.
Experimental
Material
Acrylic acid (AcA; MW =72.06 g mol-1, d= 1.06 g/cm3) and ammonium persulfate (APS) were
purchased from Fluka and used after vacuum distillation. N, N-methylene bis acrylamide (MBA)
were purchased from Merck and used as received. Nanosilver liquid (20 nm, 99 %) was purchased
from Mehregan Chemistry company. Soluble starch (Mw = 342.30), polyethylene glycol (PEG
4000, average mol wt 1,450 and d=1.0919 g/mL at 60 °C) and all other chemicals were purchased
from Sigma-Aldrich. Escherichia coli (ATCC-25922) strain was kindly donated by the
microbiology laboratory of Islamic Azad University, Tehran, Iran.
Preparation of Fe3O4@GOTfOH
The GO dispersion was prepared using the Hummers method[25]. Then, the resulted GO is filtered
and washed several times with ionized water. By coprecipitation and dispersion of 300 mg ferrous
(Fe2+) and 800mg ferric (Fe3+) ions (50 ml) in the suspension of 40 mg GO and 40 ml H2O under
ultrasonication, Fe3O4@GO is achieved. Then, 2g trifluoromethanesulfonic acid was added to a
suspension of Fe3O4@GO (1g) in 5ml CH2CH2, while mechanically stirred at 40ºC. The mixture

was filtered after 1 h, and the residue was washed with CH2CH2 and heated at 50 oC for 24 h under
vacuum to obtained triflic acid-functionalized magnetic GO (Figure 1).
Figure 1: Synthesis of (Fe3O4)-GO-TfOH
Synthesis of Fe3O4@GOTfOH/Ag/St-PEG-AcA
Briefly, starch (0.5 g) was solved in warm water at 80 °C. After 15 min, MBA (0.05 g) was
dissolved in water (5 mL) and added to the aqueous solution with continuous mechanical stirring
(300 rpm) until a homogeneous viscous mixture was obtained. The reaction temperature was
controlled at 50 °C under an argon atmosphere, then a certain amount of 70 % neutralized AcA (6
mL), PEG (0.24 g) was added to the reaction mixture. At the next step, a definite amount of APS
solution (0.08 g) was added into the mixture and was stirred for 10 min until the hydrogel was
produced. After adding APS, the as-prepared AgNPs colloidal solutions (100-400ppm) and
Fe3O4@GOTfOH (0.5 g) were mixed with the above homogeneous solution and allowed to stir for 10
min. When the reaction was completed, the product was washed thoroughly with ethanol (100 mL)

to remove the unreacted materials (Figure 2). After filtering and drying of the samples, the
viscosity of synthesized Fe3O4@GOTfOH/Ag/St-PEG-AcA was determined using HVU
481viscometer (Herzog Contracting Corp-Germany Model) about 2.45 cP at 20 °C in order to
investigate the size distribution of AgNPs.
\
Figure 2: Synthesis of Fe3O4@GOTfOH/Ag/St-PEG-AcA
General procedure for the synthesis of 2,4,6-triarylpyridines:
A mixture of an aryl aldehyde (1 mmol), acetophenone (2 mmol), ammonium acetate (1.3 mmol),
Fe3O4@GOTfOH/Ag/St-PEG-AcA and water (2 mL) was stirred at room temperature for the
indicated times (Scheme 1). After completion of the reaction, the precipitated solid after
magnetically-separation of the catalyst was collected by filtration under suction, washed with cold
water, and then recrystallized from n-hexane to afford pure products 1-13 (Table 2) in high yields.

Scheme 1: Synthesis of 2,4,6-triarylpyridines
Characterizations
Melting points were recorded on a Buchi B-540 apparatus. IR spectra were recorded on an ABB
1
Bomem Model FTLA200-100 instrument. H and 13CNMR spectra were measured on a Bruker
DRX-300 spectrometer, at 300 and 75MHz, using TMS as an internal standard. Chemical shifts (δ)
were reported relative to TMS, and coupling constants (J) were reported in hertz (Hz). X-ray
powder diffraction (XRD) was carried out on a Philips X’Pertdiffractometer with CoKα radiation.
The morphology characteristics of synthesized samples were evaluated using field emission
scanning electron microscopy (SEM, Hitachi S-4800). The thermogravimetric analysis (TGA) was
measured under a nitrogen atmosphere with a TG/DTA7300 from room temperature to 700°C with
a heating rate of 10°C/min. The crystal phases of the samples were analyzed using X-ray
diffractometer (XRD, M21X, MAC Science Ltd., Japan) with Cu-Kα radiation. UV-vis diffuse
reflectance spectra (DRS) were recorded on a UV-vis spectrophotometer (Cary 300, USA) with an
integrating sphere. The chemistry of the surface of nanocatalyst was also determined using X-ray
photoelectron spectroscopy (XPS, K-Alpha 1063, Thermo Fisher Scientific, England).
Swelling measurements
A 100 mesh nylon screen, i.e., tea bag containing Fe3O4@GOTfOH/Ag/St-PEG-AcA (0.2 ± 0.01 g)
with average particle sizes between 40–60 mesh (250–350 mm) was immersed in distilled water or
0.1 M of NaCl solution (100 mL) and allowed to soak for 5 h at room temperature. The teabag was
suspended for 10 min to the excess fluid to drain. The equilibrated swelling (ES) was determined in
duplicate using the following Eq. 1[26]:
Eq. 1
The accuracy of the measurements was ±3%.

Swelling kinetics
In order to measure the absorbency rate of the prepared material, different particle sizes of samples
(0.2 ± 0.01 g) were prepared, immersing in 100 mL distilled water using teabags. At pre-
determined time intervals, the equilibrium swelling capacity of the materials was evaluated using
the above procedure[27].
Antibacterial measurements
The potential of prepared nanocomposite in the field of antibacterial properties was examined using
the agar diffusion method against a gram-negative E. coli. as a model bacterium. In this method, the
bacteria cultures were grown onto nutrient agar plates after sterilized by autoclaving for 1.5 hours
at 100 °C. Then, nanocomposite hydrogel (0.1 g) was suspended in distilled water after sonication
and loaded onto the above nutrient agar plates. Incubation of the inoculated plates was done at 38
ºC for 38 h, then, the diameters (mm) of the inhibition zones were determined in three directions,
and the average was calculated.
In vivo study
Fe3O4@GOTfOH/Ag/St-PEG-AcA was tested in vivo for its ability to promote the wound healing
process. Ten young C57BL/6NHsd (Charles River) male mice, aged 20–22 months, were selected
and separated into two treatment groups of control (5 mice) and Fe3O4@GOTfOH/Ag/St-PEG-AcA
healed (5 mice). They were kept under standard laboratory conditions in terms of food and water ad
libitum. These procedures were approved by the local Ethics Committee for Animal Welfare[28].
The backs of the mice were firmly shaved and subsequently pinched (with the diameter of 12 mm)
between two ceramic disc magnets with a thickness of 5 mm, a weight of 2 g, and strength of 3,740
G. Three ischemia-reperfusion (IR) injury cycles were performed. A single cycle consisted of
dorsal skin magnet pinch for 12 h followed by a rest period of 12 h. Ulcer formation was diagnosed
by an appearance of a disrupted epidermis. The mice were then anesthetized using an
intraperitoneal injection of Avertine (tribromoethanol, 250 mg/kg, Sigma, Germany). On the
control group, ulcers were wetted with sterile saline and then bandaged with cotton gauze while in
Fe3O4@GOTfOH/Ag/St-PEG-AcA healed group, ulcers were treated with scaffolds secured in place
using 5-0 nylon suture. After 1, 5, 15, and 20 days, the wound size was measured by using a
Vernier caliper and a subsequent calculation of wound area (mm2) using length and width
measurements. On day 21 of the experiment when the wound had completely healed, the skin
samples were taken for histological study.

Results and Discussion
Figure 3 showed the FTIR spectra of prepared Fe3O4@GOTfOH/Ag/St-PEG-AcA compared with
Fe3O4@GO-TfOH and starch. The FT-IR band of resulted catalyst in the region of 1168 and 680
cm-1 is attributed to the stretching vibrations of the (S=O) and (F-O), respectively. The strong-broad
peak appeared at about 3340 cm-1 is due to the stretching of (O-H) groups in the acidic portion of
CF3SO3H. The broadband of Fe3O4@GOTfOH/Ag/St-PEG-AcA at 3000-3600 cm-1 was related to the
stretching vibration of OH groups on both starch and PEG polymers. The skeletal mode vibration
of the glycoside linkage is illustrated at 900-950 cm-1. The peak around 2962 cm-1 and 1472 cm-
1are related to the CH stretching and bending mode. Meanwhile, in this graph, two other
characteristics peaks of carboxylate ion were visible[29]. The first peak is at about 1403 cm−1,
assigned to the symmetric stretching mode of the carboxylate ion, while the second peak is at 1580
cm−1 due to the asymmetric stretching mode of COO− groups[30]. These results suggested that the
grafted acrylic acid monomers were a presence in two forms of non-ionized (COOH) and ionized
(COO−) onto the polymer backbone[31].
Figure 3: The FT-IR spectra of Fe3O4@GOTfOH/Ag/St-PEG-AcA compared to starch and
Fe3O4@GO-TfOH

Figure 4 shows the UV-vis spectroscopy spectra of Fe3O4@GOTfOH/Ag/St-PEG-AcA compared
with Fe3O4@GO-TfOH and AgNPs. As can be seen, Fe3O4@GO-TfOH absorbed below 400 nm in
the UV-vis spectrum. However, Ag nanoparticles appeared a single significant peak with a
maximum wavelength at about 400 nm, which assigned to the typical surface Plasmon resonance
(SPR) of spherical silver nanoparticles. However, in the spectrum of the Ag nanoparticles stabilized
by nanocomposite revealed a sharp absorption peak with a slightly shifting in SPR peak value in
the range of 400-415 nm with the appearance of a secondary absorbance peak below 400 nm[32].
Based on the above results and the absence of aggregation of Ag nanoparticles in the
nanocomposite hydrogel matrix, it is concluded that very stable nanoparticles of Ag were formed
and dispersed.
Figure 4: The UV spectra of Fe3O4@GOTfOH/Ag/St-PEG-AcA compared to AgNPs and
Fe3O4@GO-TfOH
SEM, TEM, EDX, and AFM techniques have been used to evaluate the incorporation of AgNPs
into the nanocomposite matrix, the morphology, and the topography of prepared material. In
Figure 5a and b, it was clearly indicated that the embedded nanoparticles of Fe3O4@GO-TfOH
were presented in the range of 83-99 nm with uniform-rough morphology. At the same time,
Fe3O4@GOTfOH/Ag/St-PEG-AcA had porosity structure. Investigating the interphase
characterization, the Ag/St-PEG-AcA hydrogel network placed primarily alongside the surface of
Fe3O4@GO-TfOH, Fe3O4@GOTfOH/Ag/St-PEG-AcA showed a mixed type of growth behavior.
This is due to the changing interaction of the hydrogel layer with catalyst surface caused by the
difference in surface morphology, texture, composition, and surface area to volume ratio of GO
sheets and hydrogel networks [33]. Close inspection of SEM images (Figure 5a and b) exhibits that
agglomeration of Fe3O4@GO-TfOH and Ag/St-PEG-AcA with the fine state of dispersion.

In Figure 5c and d, the presence of elements was illustrated in Fe3O4@GOTfOH/Ag/St-PEG-AcA.
Figure 5e showed the elemental composition of nanocatalysts; it was calculated that
Fe3O4@GOTfOH/Ag/St-PEG-AcA contained about 65.69 % of carbon, 16.86 % of oxygen, 9.68 %
of nitrogen, 4.00 % of sulfur, 2.02 % of sodium, 1.00% iron and 0.42 % of silver. The results
confirmed the deposition of Ag nanoparticles in the nanocomposite hydrogel matrix.
The morphology, shape, and distribution of AgNPs size were determined by analyzing the TEM
images of the nanocomposite hydrogel. The images of TEM were shown in Figure 6. It confirmed
a highly uniform distribution of the spherical AgNPs with the average of size particles ranges
between 20-30 nm inside the nanocomposite hydrogel.

Figure 5: The SEM images, EDX spectra, and the element percentages of Fe3O4@GO-TfOH (a, c
and d), Fe3O4@GOTfOH/Ag/St-PEG-AcA (b, d, and d), respectively.

Figure 6: TEM images of Fe3O4@GOTfOH/Ag/St-PEG-AcA
In Figure 7, AFM images were depicted the surface morphology of the nanocatalysts. Comparing
the 2D and 3D images in Figure 7 a-d, the surface morphology for Fe3O4@GO-TfOH was shown
to be a smooth while for Fe3O4@GOTfOH/Ag/St-PEG-AcA nanocomposite was a rough surface with
clustered features. The histogram in Figure 7e plotted the longitudinal section diagram of
nanocomposite hydrogel indicated the homogeneous size distribution of Ag nanoparticles with an
average size of 45-50 nm.

Figure 7: AFM 2D and 3D images of Fe3O4@GOTfOH/Ag/St-PEG-AcA (a, b), and Fe3O4@GO-
TfOH (c, d), respectively and the longitudinal section diagram of Fe3O4@GOTfOH/Ag/St-PEG-AcA
(e)
Analyzing the structure of Fe3O4@GO-TfOH and Fe3O4@GOTfOH/Ag/St-PEG-AcA using X-ray
diffraction was illustrated in Figure 8. As shown in the XRD patterns, Fe3O4@GO-TfOH showed a
sharp diffraction peak at 34.20°, which originated from the diffraction GO on its (002) layer planes.
In general, synthetic nanocatalyst showed diffraction peaks indexed to planes of cubic Fe3O4 units
including (220), (311), (400), (422) and (511), appearing at 30.11°, 34.20°, 43.80°, 56.03° and
60.99◦, respectively. In contrast, Fe3O4@GOTfOH/Ag/St-PEG-AcA exhibited relative sharp peaks at
2θ = 57.1°, 67.2° and 102.34° which were assigned to planes of the silver structure and 2θ =
31.01°, 33.19°, 46.20° and 55.90° which were related to planes of GO and Fe3O4 nanoparticles. In
addition, the slight difference between XRD patterns of the nanocomposite and AgNPs can be
concluded that the AgNPs into nanocomposite matrix were well crystallized. Furthermore, no peaks

related to the impurities were observed in XRD patterns. The average particle size of the trapped
AgNPs into the Fe3O4@GOTfOH/Ag/St-PEG-AcA Based on the Debye-Scherrer equation was found
about 45 nm with the good agreement with the results of AFM and TEM analysis [34].
Figure 8: XRD patterns of Ag, Fe3O4@GO-TfOH and Fe3O4@GOTfOH/Ag/St-PEG-AcA
Figure 9 represented the TGA/DTG analysis of Fe3O4@GO-TfOH and Fe3O4@GOTfOH/Ag/St-
PEG-AcA. Two steps of decomposition were seen for Fe3O4@GO-TfOH. The first weight loss at
150-250 °C was due to the evaporation of adsorbed water, and the second rapid weight loss was
due to the thermal degradation of the organic content of Fe3O4@GO-TfOH structure in the range
400-500 °C (Figure 9a). Similar decomposition steps were also seen for Fe3O4@GOTfOH/Ag/St-
PEG-AcA (Figure 9b). Also, the total weight loss of Fe3O4@GOTfOH/Ag/St-PEG-AcA and
Fe3O4@GO-TfOH was about 66 % and 81% at 800 °C, respectively. According to these results and
values of nanocomposite compared to Fe3O4@GO-TfOH, such as 195.91 °C, which was higher
compared to that of the hydrogel, the higher thermal stability of Fe3O4@GOTfOH/Ag/St-PEG-AcA
was concluded. The presence of silver nanoparticles might be the reason for this high thermal
stability in the nanocomposite matrix[35].

Figure 9: TGA/DTG analysis of Fe3O4@GO-TfOH (a) and Fe3O4@GOTfOH/Ag/St-PEG-AcA (b)
Swelling characteristics of prepared Fe3O4@GOTfOH/Ag/St-PEG-AcA nanocomposite hydrogel are
shown in Figure 10a-e with 0.1, 0.3, 0.5 and 0.6 mol L-1 APS concentration, 0.002, 0.004 and
0.006 mol L-1 MBA concentration, 0.5 and 1.2 mol L-1 AcA concentration, 0.75 and 1.5 wt% PEG
concentration, 100, 200, 400, 600 and 800 ppm AgNPs. All of these swelling experiments were
carried out at a pH of 7 in water. Swelling ratios of samples synthesized with optimized reaction
parameters are also shown in Figure 10f at pH 6.5, 7, and 8.5. In Figure 10a, it is observed that
increasing the concentration of APS from 0.1 to 0.3 mol L-1 leads to higher swelling ratios, which
may be attributed to the decrease of hydrogel molecular weight due to more chain ends and
imperfection of gel network. In the present work, optimum APS concentration was found to be 0.3

mol L-1. Similar trends for the swelling ratio of the hydrogels are also observed for increasing
MBA, AcA, and PEG concentrations (Figure 10b-d). This may be ascribed to lower crosslinking,
higher reactivity, and increased hydrophilicity of the hydrogels, respectively. From Figure 10e, it is
obvious that increasing the concentration of AgNPs from 100 to 400 ppm, leads to higher swelling
ratios which may be related to the capability of AgNPs embedded in the hydrogel to increase the
pores and free spaces within the structure of the network of the prepared hydrogel and as a
consequence adsorbs more water. Thus, the water absorption properties of the hydrogel improve.
However, further increase of silver concentration leads to decreased swelling properties due to
filling up the void spaces of the network chains and chelation with some -OH and -C=O groups of
Fe3O4@GOTfOH/Ag/St-PEG-AcA while neutralizing the repulsions in the networks. Based on the
swelling kinetics curves, Fe3O4@GOTfOH/Ag/St-PEG-AcA present higher and faster swelling ratios
compared to the corresponding pure hydrogel. This may be attributed to the presence of AgNPs
within the surface charges of the gel network and the presence of different sizes of AgNPs in an
efflux of water to balance the build-up of ion osmotic pressure.
From Figure 10f, it is observed that at higher pH swelling ratio increases, which may be due to
ionization of hydrophilic -COOH groups of the hydrogels. The swelling data of the hydrogels
including ESR, the initial rate of swelling (ri), rate constant (krs) and statistical parameters such as
R2, χ2 , F of the kinetic fittings confirmed close fitting of the swelling data to second-order rate
equation due to regression coefficient R2 (0.97-0.99) of these fittings are close to unity. At the same
time, chi-square χ2 (0.007-0.2) of these fittings are very low and F values are very high (900-4400)
and also calculated ES ratio of pure hydrogel (11.9) and Fe3O4@GOTfOH/Ag/St-PEG-AcA (13.1)
was also very close to their experimental ES ratios as shown in Figure 10f.

Figure 10: Swelling characteristics of prepared Fe3O4@GOTfOH/Ag/St-PEG-AcA nanocomposite
hydrogel under the effect of a) crosslinker (MBA), b) initiator concentration (APS), c) AcA
monomer concentration, d) PEG monomer concentration, e) AgNPs solution concentration and f)
pH on swelling ration data
The antimicrobial effect (in vitro) of prepared materials was investigated by comparing the
diameter of the growth inhibition zones of E. coli. As can be seen from Figure 11, both

Fe3O4@GOTfOH/Ag/St-PEG-AcA and Fe3O4@GO-TfOH showed the antibacterial effect on the
bacteria, E. coli, after 24 h of incubation at 37 °C. So that, paper discs soaked for Fe3O4@GO-
TfOH with two different concentrations exhibited inhibition zone values of 3.83 mm (for 1.5 mg L-
1) and 5.70 mm (for 3 mg L-1) against E. coli. Similarly, according to Fig. 14,
Fe3O4@GOTfOH/Ag/St-PEG-AcA exhibited inhibition zone values of 10.56 mm (for 1.5 mg L-1),
and 13.32 mm (for 3 mg L-1) against E. coli with the same concentrations. In this study, the results
obtained that the Fe3O4@GOTfOH/Ag/St-PEG-AcA showed further potential antibacterial activity
than the Fe3O4@GO-TfOH under similar test conditions. This could be related to the presence of
AgNPs and the presence of a more porous structure of nanocomposite hydrogel, which during the
swelling process, released the silver nanoparticles efficiently into the media and led to its
interaction with the lipid layer of the bacterial cell membrane[36]. Since gram-negative E. coli has
a thin lipid layer of the cell wall may lead to a facilitate penetration of released silver nanoparticles
into the bacterial cell membrane[37]. The released AgNPs can then destroy with the lipid layer of
the cell membrane.
Figure 11: Antimicrobial effect (in vitro) of Fe3O4@GOTfOH/Ag/St-PEG-AcA and Fe3O4@GO-
TfOH
The in vivo model of ulceration was performed as described in the experimental section. Ulceration
was induced by magnet pressure. In treated animals, the epidermis showed visible signs of
ulceration at least 3 days after the last cycle of magnet treatment. A significant reduction of the area
of lesion was observed following treatment of animals with Fe3O4@GOTfOH/Ag/St-PEG-AcA H at

5 and 15 days when compared with the control group (Figure 12a). It is interesting to observe that
the Fe3O4@GOTfOH/Ag/St-PEG-AcA treatment significantly increased the wound closure at 15
days compared with the treatment at 5 days. Furthermore, the results also showed the signs that the
wounds treated and covered with the samples of Fe3O4@GOTfOH/Ag/St-PEG-AcA healed very
faster than wounds treated with the control group (Figure 12b). The expressively expedited wound
healing can also be perceived in the Fe3O4@GOTfOH/Ag/St-PEG-AcA-treated wounds at day 15.
Figure 12: The in vivo model of ulceration
Figure 13 shows the skin histology on day 21. The Fe3O4@GOTfOH/Ag/St-PEG-AcA healed group
showed complete healing, as indicated by the uniformly packed parallel collagen fibers. These
suggest that the beginning of the macromolecular organization of the granulation tissue. For control
wounds, the tissue contained large numbers of fibroblasts, which were irregularly arranged, and

there was a less compact arrangement of collagen fibers compared to Fe3O4@GOTfOH/Ag/St-PEG-
AcA healed wounds. The dermis of mice treated with Fe3O4@GOTfOH/Ag/St-PEG-AcA showed
similar fibroblast and fibrillar arrangement of the control wounds.
Figure 13: The skin histology on day 21
With the increasing demand in modern organic synthesis for more pure compounds, we decided to
deliberate the efficiency of Fe3O4@GOTfOH/Ag/St-PEG-AcA as a novel magnetic catalyst in the
synthesis of 2,4,6-triarylpyridines in water. The reaction between acetophenone, 4-
chlorobenzaldehyde, and ammonium acetate in water solvent system was selected as a model
(Scheme 2). Increasing the reaction temperature and amount of the catalyst up to room temperature
and 0.03 g, respectively, increased the yield of products, whereas the further increase in both
temperature and amount of catalyst was met not to affect the formation of the products. On the
other hand, very low catalytic activity is observed for varied organic solvents such as EtOH,
MeOH, MeCN, and CHCl3, under reflux condition and in the presence of 0.03 g of
Fe3O4@GOTfOH/Ag/St-PEG-AcA (Table 1).

Scheme 2: The model reaction between acetophenone, 4-chlorobenzaldehyde, and ammonium
acetate in water in the presence of Fe3O4@GOTfOH/Ag/St-PEG-AcA
Table 1: Optimizing the condition of the model reaction
Entry
1
2
3
4
5
6
7
Catalyst (g)
-
Solvent
-
Temperature (oC)
Time (min)
Yielda (%)
120
r.t.
r.t.
r.t.
r.t.
300
60
10
6
6
6
60
60
60
60
Trace
50
88
94
94
94
60
66
68
0.01
0.01
0.03
0.05
0.03
0.03
0.03
0.03
0.03
-
H2O
H2O
H2O
H2O
80
EtOH
MeOH
MeCN
CHCl3
reflux
reflux
reflux
reflux
8
9
10
70
aIsolated yields
All the reaction with substituted benzaldehydes preceded very cleanly at optimized reaction
conditions, and no undesirable side-reaction were observed, although the yields were dependent on
the substituent. To check the versatility of this method, we had also studied the various hetero
arylaldehydes summarized in Table 2.
For demonstrating the matchless catalytic behavior of Fe3O4@GOTfOH/Ag/St-PEG-AcA and
considering the differences between its activity with other catalysts, the comparison of the model
reaction system was performed in Table 3 between Fe3O4@GOTfOH/Ag/St-PEG-AcA and other
reported catalysts[38-44]. It was determined that Fe3O4@GOTfOH/Ag/St-PEG-AcA is the most
effective catalyst for this purpose due to the high surface area and very strong acidity of -SO3H
groups.

Table 2: The reaction time (min) and the yield (%) of 2,4,6-triarylpyridines
Time
(min)
6
Yield*
(%)
97
Entry
Product
Ar
1
2
4a
4b
4-Cl-Ph
Ph
9
98
87
83
78
77
87
90
93
86
92
97
94
3
4c
4d
4e
4f
4-Br-Ph
6
4
4-F-Ph
6
5
4-Me-Ph
11
12
10
5
6
4-OMe-Ph
4-CN-Ph
7
4g
4h
4i
8
4-NO2-Ph
3-NO2-Ph
3,4-diCl-Ph
4-OH-Ph
9
7
10
11
12
13
4j
5
4k
4l
10
5
naphthalen-1-yl
thiophen-2-y
4m
5
Table 3: Comparison of model reaction system between Fe3O4@GOTfOH/Ag/St-PEG-AcA and
other reported catalysts
Temperature (oC)
Time (min)
Yield*
(%)
95 [38]
Entry
Catalyst
1
2
PEG-DAILsa, 10% mol
Activated Fuller’s earth
45
90
110
110
90 [39]
80 [40]
97 [41]
93 [42]
94 [43]
80 [44]
This work
20
480
50
60
60
6
120
150
80
3
4
5
6
7
8
Fe3O4@TiO2@O2PO2(CH2)2NHSO3H
-
HNTf2b, 1% mol
LPSFc, 0.01 g
60
130
r.t.
ChVO, 5 mg
Fe3O4@GOTfOH/Ag/St-PEG-AcA
a poly(ethylene glycol)–linked dicationic acidic ionic liquids
b triflimide
c Fe3O4\SiO2\propyltriethoxysilane\L-proline
d chitosan supported vanadium-oxo

The conceivable mechanism is shown in Figure 14. On the high surface area of magnetic
nanocatalyst, the reactants are easily absorbed. Because of intrinsic Brönsted acidity of -SO3H and
Lewis acidity of the Fe3+, the carbonyl moiety of acetophenone is activated.
Figure 14: Proposed mechanism for the synthesis of 2,4,6-triarylpyridines in the presence of
Fe3O4@GOTfOH/Ag/St-PEG-AcA
Anyway, this catalyst can be removed in the presence of an external magnet and be recovered in
subsequent runs with the low observation of a significant decrease in activity after 10 runs (Figure
15).

Figure 15: Recovery of catalyst in ten runs
Conclusions
In summary, our new idea that has been done in this project is leading to prepare
Fe3O4@GOTfOH/Ag/St-PEG-AcA as a magnetically recoverable nanocatalyst for one-pot synthesis
of 2,4,6-triarylpyridine derivatives under water solvent by functionalizing (Fe3O4)-GO with
Trifluoromethanesulfonic acid and combining with hydrogel network of Ag/St-PEG-AcA. This
catalyst with better activities to other commercially available sulfonic acid catalysts has made the
reaction condition distinctly superior over to many different protocols reported earlier. The
prepared nanocatalyst exhibits excellent antimicrobial efficiency toward E. coli bacteria. Besides,
histology of repaired wounds in Fe3O4@GOTfOH/Ag/St-PEG-AcA healed a group of mice showed
better fibroblast distribution and more compact collagen fiber organization compared to wounds in
the control group.
Acknowledgment
We gratefully thank the Department of chemistry, Institute of Science and Research (SIPTC),
Tehran, Iran (www.siptc.ir) for applying the research facilities.
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