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Article
Chitosan-iron oxide coated graphene oxide nanocomposite
hydrogel: A robust and soft antimicrobial bio-film.
Achyut Konwar, Sanjeeb Kalita, Jibon Kotoky, and Devasish Chowdhury
ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07510 • Publication Date (Web): 20 Jul 2016
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Page 1 of 12 ACS Applied Materials & Interfaces

1
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5
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7 Chitosan-iron oxide coated graphene oxide nanocomposite
8
9
hydrogel: A robust and soft antimicrobial bio-film
10
11
Achyut Konwara, Sanjeeb Kalitab, Jibon Kotokyb and Devasish Chowdhury*a
12 a
Material Nanochemistry Laboratory, Physical Sciences Division, Institute of Advanced Study in Science and Technology,
13
Paschim Boragaon, Garchuk, Guwahati, 781035, India. E-mail: devasish@iasst.gov.in; Fax: +91 361 2279909; Tel: +91 361
14
2912073
15 b
16 Drug Discovery Laboratory, Life Sciences Division, Institute of Advanced Study in Science and Technology, Paschim
17 Boragaon, Garchuk, Guwahati, 781035, India.
18
19
Keywords: iron oxide coated graphene oxide, iron oxide nanoparticles, chitosan hydrogel, antimicrobial, nanocomposite,
20
magnetic film
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22
23
24 ABSTRACT: We report a robust biofilm with antimicrobial properties fabricated from chitosan-iron oxide coated graphene oxide
25 nano-composite hydrogel. For the first time, the co-precipitation method was used for the successful synthesis of iron oxide coated
graphene oxide (GIO) nanomaterial. Following this, the films were fabricated by gel casting technique aided by the self-healing
26
ability of chitosan hydrogel network system. Both the nanomaterial and the nanocomposite films were characterized with the tech-
27 niques, viz., Scanning Electron Microscope, FT-IR spectroscopy, X-Ray Diffraction, Vibrating Sample Magnetometer etc. Thermo-
28 dynamic stability and mechanical property measurements reported a significant improvement in its thermal and mechanical proper-
29 ties. Moreover, the stress-strain profile indicated the tough nature of the nanocomposite hydrogel films. These improvements, there-
30 fore, indicated an effective interaction and good compatibility of the GIO nanomaterial with the chitosan hydrogel matrix. In addi-
31 tion, it was also possible to fabricate films with tunable surface properties like hydrophobicity simply by varying the loading per-
32 centage of GIO nanomaterial in the hydrogel matrix. Fascinatingly, the chitosan-iron oxide coated graphene oxide nanocomposite
33 hydrogel films displayed a significant antimicrobial activity against both gram-positive as well as gram-negative bacterial strain, for
34 instance, Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus and Escherichia coli and also with opportunistic der-
35 matophyte Candida albicans. Antimicrobial activities were tested on the films by agar diffusion assay and antimicrobial test based
36 on direct contact. A comparison of antimicrobial activity with that of individual chitosan-graphene oxide and chitosan-iron oxide
37 nanocomposite films demonstrated a higher antimicrobial activity of chitosan-GIO nanocomposite hydrogel films compared to that
38 of the former two types of films in both the methods. In-vitro hemolysis potentiality test and MTT assay of the nanocomposite films
39 indicated a non-cytotoxic nature of the films which conveyed a possibility of potential application of such soft and tough films in
40 biomedical and as well as in food industry.
41
42
43 INTRODUCTION mon method for preparation of such hydrogel network struc-
44 Development of biomaterials plays a significant role in ture of chitosan involves its crosslinking with glycerol in an
45 the area of health and hygiene. Formation of nanocomposite is acidic medium. Kiuchi et al. reported the preparation of chi-
46 one of the ways through which improvement in the desired tosan hydrogel film cross-linked with Diepoxy Poly(ethylene
47 properties of such biopolymeric materials can be achieved glycol) with improved mechanical and swelling behavior
48 with the possibility to impart new interesting properties. Chi- compared to the chitosan hydrogel film cross-linked with
49 tosan is one of the most important raw materials for biopoly- poly(ethylene glycol).5 Recently our group has reported the
50 meric advance material. It is the most amazing natural poly- fabrication of chitosan hydrogel film cross-linked with glycer-
51 mer with wide applications in different fields like biomedical, ol and improved its mechanical, thermal as well as optical
food industry, textile and paper industry. The NH2 functional properties by incorporation of carbon dots nanoparticle syn-
52
group present in the C-2 position of the D-Glucosamine re- thesized from Assam tea. There are also reports of using dif-
53
peating unit makes the polymer unique in its chemical behav- ferent nanomaterials like silver, gold, carbon nanotube etc. for
54 the preparation of chitosan hydrogel nanocomposites.3
55 ior. Chitosan, the cationic (1-4)-2-amino-2-deoxy-<beta>-D-
glucan is industrially obtained from partly acetylated chitin1,2. An extensive use of biopolymer in the biomedical appli-
56
It is the only pseudo-natural polymer which can be applied as cation and food industry has led the researchers to work on
57
solution, film, fiber and also in hydrogel form. The hydrogels incorporating antimicrobial property to the polymer itself and
58 consist of three-dimensional network structures of polymer its composite form. In doing so, two types of approaches are
59 which are scientifically proven to entrap a large amount of mainly used. One is the chemical approach which includes
60 water without losing the network structure3,4. The most com- mainly the chemical modification of its functional groups with
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 ACS Applied Materials & Interfaces
antibacterial agents like quaternary ammonium or phosphoni-
1 um salts. This is of course a tedious process and might lose the
2 cross-linking ability of the polymer 6,7,8. Another approach is
3 the preparation of chitosan-based composites in the form of
4 gels or films. Till date, generally the silver nanoparticle has
5 been used as the antimicrobial agent with chitosan9, 10, 11, 12.
6 There are few reports on development of antimicrobial chi-
7 tosan nanocomposite film with clay, layered silicates, metal
8 oxides and graphene oxides, etc..13,14,15 In recent years, im-
9 mense attention has been drawn by the graphene-based nano-
materials because of their great potential in applications such
10
as electrochemical devices, energy storage, catalysis, adsorp-
11 tion of enzyme, cell imaging, drug delivery as well as biosen-
12 sors and many more. Particularly, graphene oxide (GO) chem-
13 ically exfoliated from oxidized graphite is considered as a
14 promising material for biological applications owing to its
15 excellent aqueous processability, amphiphilicity, surface func-
16 tionalization ability, surface enhanced Raman scattering
17 (SERS) property and fluorescence quenching ability. GO is
18 dispersible in an aqueous medium and is experimentally found
19 to exhibit antimicrobial property. Here, it is worth mentioning
20 that GO is proven to be non-cytotoxic at low concentration
21 although it has some adverse effect at high concentration. The-
se antibacterial GO and other graphite based materials act
22
23 through physical damages upon direct contact to bacterial
membranes by sharp edges of graphene sheets16,17,18,19. Metal
24
oxides are another important group of nanomaterials, which
25
acts as antimicrobial agents. Copper oxide, iron oxide, nickel
26 oxide, zinc oxide etc. are found to possess the antimicrobial
27 activity. Among these metal oxides, iron oxide is one of the
28 most widely studied antimicrobial nanomaterials which acts
29 via physical damage of the cell membrane20,21,22, 23 .
30
In this work, we endeavoured to synthesize antimicrobi-
31 al bio-film from chitosan-iron oxide coated graphene oxide
32
nanocomposite hydrogel. This was successfully achieved by
33 first preparing iron oxide coated graphene oxide (GIO) nano-
34 materials by a modified co-precipitation method generally
35 used for the synthesis of iron oxide nanoparticles. There are
36 already few reports in the literature of synthesizing iron oxide
37 coated GO where the iron oxides were attached on the surface
38 of the GO by refluxing the system at high temperature or with
39 other tedious processes24,25,26. Excitingly, through our process,
40 we could successfully decorate the surface of GO with iron
41 oxide nanoparticles in one-step at room temperature. The as-
42 prepared GIO nanomaterial showed quite well dispersibility in
aqueous media compared to that of iron oxide alone, thereby
43
helping in an efficient and easy dispersion of the nanomaterial
44
in the polymeric matrix. In the second step, GIO nanomaterial
45 was successfully incorporated within the chitosan hydrogel
46
network system aided by secondary interactions like hydrogen
47 bonding as well as the electrostatic interaction between the
48
nanomaterial and the matrix phase. The hydrogel films were
49 fabricated by gel casting technique for the purpose. Interest-
50 ingly, chitosan-based hydrogels were found to exhibit a self-
51 healing property, the ability by which two different hydrogel
52 systems, when kept in contact with each other, can stick to-
53 gether and become one. Mainly, the strong electrostatic and
54 hydrogen bonding interaction abilities of chitosan is suspected
55 to act for this self-healing phenomena27,28,29. Here, we effec-
56 tively utilized this self-healing ability of the chitosan hydrogel
57 to prepare such films. The nanocomposite films were found to
show a broad spectrum antimicrobial activity with significant
58
inhibitory effect when tested against both gram-positive and
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gram-negative bacterial strains, responsible for infection in
wounds and contamination of foodstuffs. Most importantly,
the films also exhibited antimicrobial activity against Methi-
cillin-resistant Staphylococcus aureus (MRSA). This bacterial
strain is considered to be one of the most resistant ones and a
major portion of medicinal chemist and biotechnologists are
engaged for the development of antibiotics to combat the
MRSA resistance30. These fabricated films are also found to be
non-cytotoxic in nature which is the prerequisite for biomedi-
cal application of materials. Moreover, the nanocomposite
hydrogel film acquired a noteworthy improvement in thermal
and mechanical properties compared to that of the bare chi-
tosan hydrogel film. The speciality of these hydrogel films lies
in their softness because of the three-dimensional hydrogel
network structures. However, the loading of GIO nanomaterial
made the films robust by improving its mechanical properties
without hampering its softness much. Therefore, such soft but
robust antimicrobial films may find high potential application
in the biomedical field.
EXPERIMENTAL SECTION
Oxidation and exfoliation of graphite nanopowder: The
commercially available graphite nanopowder was oxidized
and exfoliated to graphene oxide following the method already
reported by our group31. In this simple and single step method,
0.01 g graphite nanopowder was dispersed in 3:1 acid mixture
of concentrated H2SO4 (98 wt%) and HNO3 (16 M). This mix-
ture was then sonicated in a bath sonicator for 24 h followed
by dilution. The graphene oxides formed were collected by
centrifugation and washed with Milli-Q water for several
times to remove all the acids. Then, these graphene oxide na-
noplatelets were again dispersed in the required amount of
Milli-Q water and sonicated with the help of an ultra-probe
sonicator.
Preparation of iron oxide coated graphene oxide (GIO)
nanomaterial: The magnetic iron oxide coated graphene ox-
ides were prepared by in-situ co-precipitation of magnetic iron
oxide nanoparticles on the surface of graphene oxides. Here,
we applied the basic principle of co-precipitation technique for
iron oxide synthesis32,33,34 with necessary modifications. For
this 5 mL solution of Fe3+ and Fe2+ ions in the ratio 2:1 (2.5
mL 0.2 M Fe3+ + 2.5 mL 0.1 M Fe2+) was mixed with 5 mL of
graphene oxide dispersion (of concentration 1mg/mL) fol-
lowed by mechanical stirring for 2 hrs. Then, the mixture was
added at a time in a 50 mL of 1 M aqueous NH4OH. An inert
atmosphere was provided to the system by a continuous sup-
ply of nitrogen and the whole system was vigorously stirred at
1200 rpm with a mechanical stirrer for 30 minutes. The black
precipitate obtained at the end was collected using a strong
magnet and washed several times with Milli-Q water. After
which, the product was dispersed in the required amount of
Milli-Q water.
Fabrication of chitosan hydrogel film: Chitosan hydrogel
films were fabricated by the technique already reported by our
group3. Chitosan hydrogel was first prepared by dissolving 0.1
g of chitosan in a 10 mL mixture of 0.1 M acetic acid and
glycerol in the ratio of 3:2 followed by neutralization with 5 N
NaOH. The resulting hydrogel obtained was washed thorough-
ly with Milli-Q water to remove the unreacted monomers. The
Page 3 of 12 ACS Applied Materials & Interfaces
gel was then cast on glass slides and allowed to dry at 60 oC in
1 a hot air oven. The films were peeled out from the glass slides
2 just by wetting with water and again kept for drying at room
3 temperature in a vacuum oven.
4 persions containing tested bacteria and C.albicans at various
5 Fabrication of chitosan-GIO nanocomposite hydrogel
6 films: Chitosan-GIO nanocomposite hydrogel films were fab-
7 ricated following the similar gel casting technique used for the
8 blank chitosan hydrogel films. For the preparation of the chi-
9 tosan-GIO nanocomposite hydrogel, required amount of chi-
10 tosan solution (in a mixture of 0.1 M acetic acid and glycerol
11 in the ratio of 3:2) and GIO dispersion (in water) was mixed
12 together with the help of a mechanical stirrer at 1000 rpm for 2
13 h. The nanocomposite hydrogel obtained after neutralization
with 5 N NaOH was washed thoroughly and cast on glass
14
slides. After drying at 60 oC, the films were peeled out and
15
16 kept in a vacuum oven for further drying.
17
18 CHARACTERIZATION
19 The nanomaterials and the nanocomposite films were
20 characterized by Fourier transform infrared spectrometer (Ni-
21
colet 6700 FT-IR) to investigate different types of interactions.
22 Scanning electron microscope (SEM) images were collected
23 on a ‘Carl Zeiss ∑igma VP’ instrument. Powder X-ray diffrac-
24 tion (XRD) spectra were collected on a ‘Bruker D8 Advance
25 diffractometer’. Magnetic properties of the nanomaterial and
26 the nanocomposite film were studied with a ‘Lake Shore
27 Model 7410’ vibrating sample magnetometer (VSM) instru-
28 ment. To analyze the thermostability of the films, the thermo-
29 gravimetric analysis (TGA) thermograms for the hydrogel
films were recorded in ‘Perkin Elmer 4000’ and the analysis
30
was performed in the range of 35-800 oC at a heating rate of
31
10 oC/min and with the nitrogen flow rate of 20 mL/min.
32
The films were cut into small pieces and dried overnight in a
33
vacuum oven before TGA was performed. To study the me-
34
chanical properties of the films we used a universal testing
35
machine (‘Tinius Olsen 5ST’ model) with a load cell of 2.5
36
KN capacity. The speed of the testing was maintained at 5
37 mm/min. The static contact angles of the hydrogel films were
38 measured using the Contact Angle Analyzer (KRüSS DSA 30,
39 Germany).
40 Antimicrobial test: The anti-microbial activities of the as-
41 fabricated hydrogel films were evaluated against Methicillin-
42 resistant Staphylococcus aureus (ATCC 25923), Staphylococ-
43 cus aureus (MTCC 3160), Escherichia coli (MTCC 40) and
44 Candida albicans (MTCC 183) using standard agar diffusion
45 test35 and also the one direct contact test method36. In the agar
46 diffusion test, the bacterial and Candida cultures were grown
47 on nutrient agar and Sabouraud Chloramphenicol (SC) agar
48 plates, respectively and maintained in slants at 4°C. The bacte-
rial inoculums were prepared in lysogeny broth medium and
49
kept in an incubator at 37 °C. With the help of a sterile cotton
50 swab, 1 mL (1.0×107 colony forming units [CFUs]) of 24-
51
hour-old bacterial and Candida culture (prepared in SC broth)
52 strains suspension were uniformly spread over the solidified
53 nutrient agar and SC agar plates respectively. The samples
54 were carefully placed on the plates using a sterilized forceps.
55 The plates were then incubated for a specific time and temper-
56 ature [37 °C for 24 hrs (bacteria); 28 °C for 72 hrs (Candida)],
57 and the antagonistic activities of the samples were quantita-
58 tively evaluated by measuring the zone of inhibition around
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the films37. The tests were conducted in triplicate and the aver-
age was taken with standard error.
In the direct contact test method, the fabricated films
were sterilized in 75 v/v% ethanol aqueous solution and dis-
concentrations of 107 CFU/mL, 106 CFU/mL and 105 CFU/mL
was introduced onto the sample to a density of 60 µL/cm2.
After 24 h of incubation at 37 °C bacterial dispersion and 72
hrs at 28 °C for C. albicans, the dissociated microorganism
dispersions were collected and inoculated into the nutrient
agar and SC agar culture medium respectively. After the incu-
bation period, the culture plates were photographed to have an
evidence of the microbial colony forming units. The potential
problem of water evaporation was addressed by filling up the
remaining empty wells with deionized water. Further, a con-
tainer filled with deionized water was put in the incubator. The
tests were conducted in triplicate to ensure the validity of re-
sults.
Toxicity level determination: To check the toxicity level, we
performed two standard tests, viz. hemolysis assay and MTT
assay.
Hemolysis assay: In-vitro hemolytic potential of the hydrogel
films were performed according to the standardized method38.
5 mL of venous blood were collected from a healthy volunteer
and stabilized with sodium citrate (3.8%). The blood was
centrifuged (8 minutes at 2000 rpm) and the pellet was washed
three times with sterile isotonic phosphate buffer solution
(PBS) (pH 7.2±0.2) by centrifugation at 1,500 rpm for 5
minutes. CH-GIO films with three different concentrations
(0.05, 0.1 and 0.2) were separately incubated for 1 hour at
37°C in siliconized tubes, each containing 10 mL of blood
(diluted with PBS in 1:9 ratio). The mixtures were incubated
for 30 minutes at 37°C and centrifuged at 1,500 rpm for 10
minutes. The free hemoglobin in the supernatants was meas-
ured by a UV-Vis spectrophotometer at 540 nm. PBS and dis-
tilled water were used as minimal and maximal hemolytic
controls. Each experiment was performed in triplicate at each
concentration. The level of percentage hemolysis by the tested
samples was calculated according to the following formula:
(Abssample – Abs –ve control) x 100
Hemolysis (%) = x100
Abs + ve control – Abs –ve control
MTT assay: To evaluate the cytotoxicity level of CH-GIO
films towards mammalian cells, MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide) dye conversion assay
(Mossman) was performed against mouse L929 fibroblastic
cell line (obtained from NCCS, Pune). L929 cells at a density
of 1×104 per well were cultured in a 100 µL volume of cell
culture medium (Dulbecco’s Modified Eagle’s Medium
[DMEM]) supplemented with 10% fetal bovine serum in a 96-
well cell culture plate. After 24 hours, cultured cells were
treated with CH-GIO1, CH-GIO2 and CH-GIO3 separately (1
cm2) in DMEM without serum and incubated further for 24, 48
and 72 hours. Wells with complete medium, MTT reagent and
solubilizing buffer; without cells were used as blanks. Cells
without treatment were kept as a negative control; whereas
cells treated with 1% triton X 100 were considered as positive
control. This was followed by removal of the media and treat-
ment with MTT dye at a final concentration of 0.5 mg/mL and
further incubated for 4 hours. Finally, 100 µL of DMSO was
added to each well to dissolve the blue formazan precipitate,
 ACS Applied Materials & Interfaces Page 4 of 12

and absorbance was measured at 570 nm using a microplate These magnetic nanoparticle-decorated graphene oxide nano-
1 reader (Bio-Rad Model 680; Bio-Rad Laboratories Inc., Her- material systems could be separated with a strong magnet as
2 cules, CA, USA). The cell viability was expressed as a per- shown in the photograph given in the scheme (Figure-1 (B,
3 centage of the control by the following equation: C)). The coating of iron oxide nanoparticle onto graphene
4 Viability (%) = Nt/Nc ×100 oxide was further confirmed by scanning electron microscope
5 images as shown in figure 2. The SEM images clearly show
Here, Nt is the absorbance of the cells treated with samples,
6 the deposition of iron oxide nanoparticles exclusively on the
Nc is the absorbance of the untreated cells (n=3; where n is the
7 surface of the GO nanosheets.
number of independent experiments)39.
8 We used FTIR spectroscopy to study the interaction be-
9 tween the GO and magnetic iron oxide nanoparticle (IO) (Fig-
10 ure-3 (a)). The FTIR spectra of GO shows a peak at 3125 cm-1
11 which is due to the O-H stretching of the COOH group. The
12 peak at 1700 cm-1 is due to C=O stretching. The intense peak
13 occurring at 1391 cm-1 is due to O−H deformations of the
C−OH groups and peak at 1103 cm-1 represents C-O-C stretch-
14
ing. In GIO, the interaction of the O-H group with the iron
15
oxide nanoparticle leads to the broadening of the O-H stretch-
16 ing peak at 3388 cm-1. The peak at 1633 cm-1 is due to the
17 carbonyl group interacting with the Fe atom of the iron oxide.
18 The consecutive peaks at around 1532 and 1451 cm-1 indicate
19 covalent interaction of the COO- group with the Fe atom on
20 the iron oxide surface. The peak for Fe-O stretch of iron oxide
21 arises at 578 cm-1 24,25,40.
22 Figure 3 (b) shows the XRD diffraction pattern of the
23 GO and GIO. In the XRD pattern of GO, the intense peak at
Figure 1: (A) Schematic presentation of synthesis of GIO nano-
24 material (B) GIO nanomaterial in aqueous medium attracted to- 2θ = 7.4 is the characteristic of GO. XRD spectra of GIO
25 wards magnet (C) aqueous dispersion of GIO nanomaterial in the clearly show the XRD pattern for iron oxide. In the co-
26 absence of magnetic field. precipitation technique both types of iron oxides, magnetite
27 and maghemite were obtained with a different percentage.
28 Both of these oxides have inverse spinal structure. In the XRD
29 pattern of GIO, the intense peaks arising at 2θ values 30.37,
30 35.7, 43.4, 53.9, 57.9, 57.5 and 63.07 correspond to the iron
31 oxide nanoparticles attached on the surface of GO. Here, it
32 was observed that the peaks relative to the 2θ value in the
XRD spectra of GIO resembled more closely to the ma-
33
ghemite according to the JCPDS 00-013-0458 data. The peak
34 Figure 2: SEM images of the GO and GIO nanomaterial. at 2θ = 26.3 in the XRD spectra of GIO appears to increase in
35 intensity compared to that of the GO indicating the reduction
36 of some oxygen-containing functional groups of GO in the
RESULTS AND DISCUSSION
37 GIO composite nanomaterial 24,41. The (M - H) loop measure-
38 Antimicrobial bio-film of chitosan-iron oxide coated gra-
ments of the iron oxide coated nanomaterial (GIO) were taken
39 phene oxide nanocomposite hydrogel was prepared by a two-
at 300K and are shown in figure-3 (c). The hysteresis loop
step process. In the first process, iron oxide coated graphene
40 shows ferromagnetic behaviour34. The coercivity values were
oxide nanocomposite was prepared. Initially, graphene oxide
41 determined to be 22.040 KOe and saturation magnetization
was prepared by oxidation and exfoliation of graphite na-
42 0.98945 emu.
nopowder as depicted in figure 1A. Iron oxide coated gra-
43 phene oxide nanomaterial (GIO) was formed in-situ by co-
44 precipitation of Fe3+ and Fe2+ ions (2:1 ratio) as iron oxide
45 nanoparticles on the surface of graphene oxide nanosheets.
46
47
48
49
50
51
52
53
54
55
56
57
58
59
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Figure 3: (a) FT-IR and (b) XRD spectra of the GO and GIO nanomaterial (c) Magnetization–hysteresis (M–H) loops of GIO nano-
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CH, CH-GIO1, CH-GIO2 and CH-GIO3 for films containing

1 0, 0.05, 0.1 and 0.2 % GIO nanomaterial respectively. SEM
2 images of the chitosan hydrogel films (Figure-5) show packing
3 of the macrostructure hydrogel network systems in the films.
4 As seen from the SEM images surface roughness is the unique
5 morphology of this type of films prepared by gel casting tech-
6 nique. The chitosan-GIO nanocomposite films has more com-
7 pact packing of hydrogel networks macrostructures as evident
8 from the SEM images. The images are also showing the ho-
9 mogeneous distribution of nanomaterials in the polymeric
hydrogel matrix.
10
11 FTIR spectroscopy provides us information regarding
12 the structure and different types of interaction in the hydrogel
13 matrix and with nanomaterials (Figure-6 (a)). In the FTIR
Figure 4: Scheme of formation of the chitosan-GIO hydrogel spectrum of chitosan hydrogel film, the peak at 3393 cm-1 and
14 nanocomposite film. 1652 cm-1 represents the vibration for O-H stretching and C-N
15
respectively. Intense peak at 1043 cm-1 is due to the ether link-
16 age present in the polymer. In the nanocomposite hydrogel
17 film, the increase in the intensity of the peak at 1592 cm-1 in-
18 dicates the increase in C=C stretching due to the increased
19 graphitic structure in the system. The presence of iron oxide
20 nanoparticle in the nanocomposite system is indicated by the
21 peak at 568 cm-1 3, 32.
22
23
Thermogravimetric analysis of the films: Thermogravimet-
24 ric analysis of all the chitosan hydrogel films was carried out
25 Figure 5: SEM images of chitosan hydrogel and chitosan hydro-
gel nanocomposite films. in order to decipher the role of the nanomaterial (GIO) in
26 thermostability of the films. TGA thermograms (Figure-6 (b))
27 The second step to prepare antimicrobial bio-film from of the films show two-stage degradation patterns for all the
28 chitosan-iron oxide coated graphene oxide nanocomposite films. For the chitosan hydrogel film (CH), the major fraction
29 hydrogel was carried out using chitosan-iron oxide nanocom- of the sample starts to degrade at 180 oC which is mainly at-
30 posite hydrogel matrix system prepared by homogeneous dis- tributed to the degradation of the side chains of chitosan and
31 persion of GIO nanomaterials in the aqueous polymeric solu- glycerol structure. For the nanocomposite film CH-GIO1, this
32 tion before allowing crosslinking by neutralization with first stage degradation temperature increases to 267 oC and for
NaOH. CH-GIO3 it increases as high as 274 oC. This tremendous im-
33
34 In this particular hydrogel system, crosslinking occurred provement in thermostability of the hydrogel nanocomposite
35 by electrostatic interaction between the NH3+ group of chi- films can be explained by the existence of strong interaction of
36 tosan and the –OH group of glycerol on neutralization along
with other types of secondary interactions like hydrogen bond-
37
ing3,4. Glycerol plays an important role in the fabrication of
38
such robust chitosan hydrogel nanocomposite films. With the
39 help of three hydroxyl groups glycerol helps to join the poly-
40 meric chains of chitosan by electrostatic as well as hydrogen
41 bonding interactions and thus acts like a chain extender. In the
42 absence of glycerol, upon neutralization of a mildly acidic
43 solution of chitosan does not produce any hydrogel network
44 system. Chitosan becomes positively charged in the acidic
45 medium due to the protonation of the NH2 groups present in
46 the polymer to produce NH3+ ions. Again neutralization of the
47 aqueous polymeric solution with NaOH in the presence of
48 glycerol leads to the crosslinking as depicted in the scheme
49 (Figure-4), and hence, forms the stable hydrogel network sys-
50 tem. The continuous robust hydrogel films were fabricated
51 with the successful application of self-healing ability of the
chitosan hydrogel macro systems in contact with each other.
52
This self-healing ability arises as a result of the strong hydro-
53
gen bonding interaction among the hydrogel network mac-
54 rosystems27, 28, 29.
55
We have fabricated chitosan-GIO nanocomposites hy-
56
drogel films with varying percentage of GIO viz. 0.05, 0.1 and Figure 6: (a) FT-IR spectra (b) TGA thermogram (c) Mechanical
57
0.2 with respect to the total amount of chitosan plus glycerol. property and (d) contact angle values of the chitosan hydrogel and
58 For the convenience of reporting, we have coded the films as chitosan hydrogel nanocomposite films.
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the nanomaterial with the polymeric matrix decreasing the Swelling study of the hydrogel films: A systematic swelling
1 mobility of the polymeric side chains. The presence of graphit- study was also undertaken for the hydrogel films. For this
2 ic structure within the matrix system also acts as a thermal study, films of 1 cm2 size were dried in a vacuum oven for 2
3 barrier leading to its improvement in thermal stability. The days. The swelling ratio was determined according to the fol-
4 second stage degradation starts above 450 oC because of the lowing formula:
5 degradation of aromatic moieties. The percent weight loss at Swelling ratio (%) = [(W s - W d)/ W d] x 100%
6 this stage is higher in the case of the nanocomposite films due
Where Ws and Wd are the weights of the swollen and dried
7 to the presence of higher amount of aromatic structures com-
samples, respectively.
8 pared to that of blank hydrogel films3,34.
It was observed that, when kept in water for 24 h, CH film
9 sample showed a swelling ratio (SR) of 45%. For the chitosan-
10 Mechanical properties of the hydrogel films: Loading of GIO nanocomposite hydrogel films, the SR values were 75, 69
11 GIO nanomaterial into the chitosan hydrogel matrix system and 57% for CH-GIO1, CH-GIO2 and CH-GIO3 film samples
12 led to an improvement in the mechanical strength with possi- respectively. The increase in SR values in case of nanocompo-
13 ble tuning by varying the amount of nanomaterials (Figure- site films is attributed by the increase in polar groups in the
14 6(c)). The stress-strain profile indicates tough nature of the interior of the hydrogel matrix system. But, with an increase in
15 nanocomposite films. Measurement of mechanical properties GIO nanomaterial concentration, the crosslinking density also
16 shows that CH film possesses a tensile strength (TS) 17±2 increased which predominates the increase of polar group ef-
17 MPa along with a maximum elongation at break 10±1 % and fect on SR of the hydrogel films. Hence, among the nanocom-
modulus 0.8±0.1 MPa. After loading of 0.05 % GIO nano- posite hydrogel films, the SR value decreases continuously
18
material, the TS of the hydrogel nanocomposite films was with higher nanomaterial loading3,34.
19
increased abruptly to 29±2 MPa along with improved modulus
20 value to 2.3±0.3 MPa. This improvement could be attributed
21 by the strong secondary interactions like hydrogen bonding as
22 well as polar-polar interactions between different functional
23 groups present in the matrix and the reinforcing nanomaterial
24 phase. These interactions led to the formation of a compact
25 structure as depicted by the SEM images. Above the optimum
26 amount, the tensile strength and modulus value of the nano-
27 composite films seemed to deteriorate on increasing the na-
28 nomaterial loading. The values of mechanical properties of the
29 hydrogel films are summarized in the SI (table-S1, in the SI).
30 An elongation at break was also measured for all the samples.
31 It was observed that % elongation tend to decrease after incor-
32 poration of the nanomaterial. This decrease is due to the stiff-
ening effect of the nanomaterial as a result of the increase in
33
the cross-linking density of the nanocomposite system3,34,42.
34
35
36 Contact angle analysis: The contact angle of water drops on
37 the surface of the hydrogel films was measured to study its
38 surface property. A comparative bar diagram plot of contact
39 angle values of the hydrogel films are given in figure 6 (d).
40 The contact angle value of CH film was found to be 62.1 ± 6
degree indicating that the surface of the films is hydrophilic in
41
nature. Contact angle value depends mainly on the presence of
42 the polar or nonpolar substance on the surface of the substrate
43 and also upon its smoothness. For a hydrophilic substrate,
44 surface smoothness facilitates the increase in contact angle
45 value and vice versa. After incorporation of 0.05% GIO, this
46 CA value increases to 85.7 ± 5 degree from 62.1±6 degree in
47 the case of bare chitosan hydrogel film. This increase in CA
48 value intending a transformation towards hydrophobicity is
49 mainly attributed due to the increase in cross-linking density
50 and compactness (as evidenced from SEM images) of the
51 three-dimensional hydrogel network system leading to the
52 surface smoothness of the films. But, with an increase in the Figure 7: (a) Photographic image of the antimicrobial activity of
53 percentage of GIO nanomaterials, the surface functionality the chitosan hydrogel nanocomposite films against MRSA (b, c)
54 increased along with the polarity because of the presence of SEM images of the dead bacterial cell after treatment (d) Com-
iron oxide nanoparticles attached to it. Thus, this increase in parative antimicrobial activity of the chitosan hydrogel (CH),
55
polarity on the surface of the hydrogel films pre-dominated chitosan-grapheme oxide (CH-GO), chitosan-iron oxide (CH-IO)
56
other factors responsible for higher CA values, thereby de- and chitosan-iron oxide coated grapheme oxide (CH-GIO) hydro-
57 creasing CA values at higher GIO concentration3,43. gel nanocomposite films against different micro-organisms.
58
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Figure 8: Typical photographs of re-cultivated microorganism colonies for Methicillin-resistant Staphylococcus aureus (MRSA), Staphy-
30 lococcus aureus (S.aureus), Escherichia coli (E.coli), and Candida albicans (C. albicans) on agar culture plates, with the seeded concen-
31 trations of bacteria onto graphene films being 105 CFU/mL.
32
33
34 Antimicrobial activity and mechanism of action: CH-GIO clear zone (Figure-7 (b, c)). SEM images clearly showed the
35 films exhibited significant antibacterial activity against patho- rupture of the bacterial membrane resulting the release of in-
36 genic bacteria, viz., Methicillin-resistant Staphylococcus aure- tracellular materials. The formation of this clear zone sur-
37 us, Staphylococcus aureus, Escherichia coli and opportunistic rounding the film sample indicated the release of some loosely
38 dermal pathogen Candida albicans (Figure-7(a) and Figure- bound nanomaterial from the surface of the hydrogel matrix.
39 S2, in the SI). We carried out the detail antimicrobial activity The sharp nano-sheet on direct contact with the microbial cell
evaluation of the hydrogel films by two different methods. resulted in membrane stress and ensuing superoxide anion
40
One of them is the conventional agar diffusion method35. But independent oxidation, which in turn led to the oxidation of
41 as our product is a solid film, so realising its potential applica- proteins, lipids and nucleic acid and ultimately resulted in
42 bility we endeavoured to perform an additional test method36 membrane damage and cell death47,48,16,17. The antimicrobial
43 which is based on the direct contact between the film sample activity of iron oxide nanoparticle could be attributed to the
44 and the microorganisms under test. The antimicrobial activity penetration of the small particles into the membranes of mi-
45 test by agar diffusion method revealed that though, CH films crobial cells, leading to oxidative stress and disruption of the
46 didn’t show a considerable zone of inhibition, it inhibited the cell membrane. Further, it is earlier reported that Fe2+ reacts
47 bacterial biofilm formation on the film surface. When tested with oxygen to create hydrogen peroxide49,20,21 which conse-
48 bacteria and Candida albicans came in contact with the CH quently reacts with ferrous ion via the Fenton reaction and
49 film, the ionic interaction between some residual positively produces hydroxyl radicals, which are known to damage the
50 charged amino groups in chitosan and negatively charged bac- macromolecular structure of the microbial cells50,51. We also
51 terial surface molecules such as Lipopolysaccharide occurred compared the antimicrobial effectiveness of the individually
52 resulting in alterations in membrane permeability of bacteria fabricated chitosan-graphene oxide (CH-GO) and chitosan-
53 and eventually microbial cell died44,45,46. On the other hand, the iron oxide (CH-IO) hydrogel nanocomposite films with simi-
GIO containing hydrogel nanocomposite films (CH-GIO2) lar concentrations of nanomaterials to that of the chitosan-
54
produced a considerable zone of inhibition as evidenced by the GIO2 hydrogel nanocomposite film used for the test. From
55
photographic images (Figure-7(a) and Figure-S2, in the SI). To figure 7(d), we can see that the bacterial inhibition activity of
56 study the mode of action of this antimicrobial activity, we the films is different for different bacterial strains and Candia
57 performed the FESEM analysis of the bacterial colony collect- albicans. But, irrespective of the type of microbial pathogens
58 ed from the edge of the tested, the chitosan-GIO hydrogel nanocomposite films
59
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 ACS Applied Materials & Interfaces
showed higher antibacterial inhibition compared to that of the
1 individually prepared chitosan-graphene oxide and chitosan-
2 iron oxide hydrogel nanocomposite films to that of CH-GIO2
3 nanocomposite hydrogel film. This improvement could be
4 explained by the cause of more effective rupturing of bacterial
5 cell wall through combined effect of the individual nano-
6 materials together in the composite GIO nanomaterial.
7 The test method based on direct contact with the sample
8 and microorganisms under the test established the antimicrobi-
9 al activity of such films and their effectiveness more clearly.
10 Figure 8 represents the typical photographs of re-cultivated
11 (105 CFU/mL) MRSA, E. coli, S. aureus and C. albicans colo-
12 nies after treatment against six types of films (viz. CH, CH-
13 GIO1, CH-GIO2, CH-GIO3, CH-GO and CH-IO). Photo-
graphs of such colonies of microorganisms with higher con-
14
centrations (106 and 107 CFU/mL) are given in figure S3 (a, b)
15 in the SI). The test results clearly depicted the absence of any
16
distinct growth or minimal growth of microorganism colonies
17 on treating with CH-GIO films, while in the case of CH films
18
excessive growth was observed. Moreover, the growth of col-
19 onies seemed to decrease with increase in GIO concentration.
20 And, in the case of CH-GIO3 film, the number of distinct col-
21 onies almost disappeared for most of the microorganism or
22 their concentration used during the test. This phenomenon
23 occurred because of the obvious reason of the presence of
24 higher amount of GIO nanomaterial on the surface of the
25 nanocomposite films at higher concentration of GIO. On the
26 other hand, CH-GO and CH-IO films having the similar con-
27 centration of nanomaterial with CH-GIO2 although decreased
the growth of the microorganism after treatment, yet they were
28
seemed to be less effective compared to that of CH-GIO2 film.
29
The results obtained from both the tests seemed to support
30 each other. Most importantly, the fabricated films exhibited
31
anti-MRSA activity which could be beneficial for various bi-
32 omedical applications.
33
34
In vitro Cytotoxicity of CH-GIO films: We have also en-
35
36 deavoured to study the biocompatibility of the hydrogel nano-
composite films. The fabricated CH-GIO nanocomposite film
37
exhibited very low to mild hemolytic activity (% hemolysis)
38 towards the human erythrocytes. The tested films demonstrat-
39 ed concentration dependent hemolysis of human RBCs (Red
40 blood cells) (Figure S4, in the SI). The main component of the
41 film, i.e. chitosan is regarded as a biocompatible polymeric
42 material, whereas the other component iron oxide has been
43 claimed to be bio-safe as they are bio-inert and Fe is essential
44 for living organisms. The surface chemistry, physical structure
45 and surface degradation are the critical aspects which influ-
46 ence the reaction of blood with film surfaces52,53. The extent of
47 hemolytic activity of the films depends on exfoliation and the
48 particle size of the GO. It is also reported that coating GO with
49 chitosan eliminates the hemolytic potential54. In our case, the
50 GO nanosheets were coated with iron oxide and embedded in
the chitosan films, which in turn resulted in the reduction in
51
GO’s toxicity towards RBCs. The permeable limit of hemoly-
52
sis for blood containing biomaterial was reported to be 5 % 55,
53 whereas the nanocomposite films exhibited a maximum of
54 0.37 % hemolysis. The results of our study indicated that the
55 prepared film material does not possess hemolytic components
56 in its surface.
57 To further explore the cytotoxicity of CH-GIO films;
58 MTT assay was performed. The MTT results for CH-GIO1,
59
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CH-GIO2 and CH-GIO3 showed dose-dependent effects on
the mouse L929 fibroblastic cells (Figure-S5 in the SI). Even
after 24, 48 and 72 hours of post-treatment with CH-GIO1,
CH-GIO2 and CH-GIO3 films, L929 fibroblastic cells showed
a much higher viability (80%-93%) compared to negative con-
trol; whereas estimated cell viability for positive control was
only 1.5%-3%. Notably, no absorbance was recorded for the
wells kept as blank. Prior to any in vivo application of the
nanocomposite films, high dose tolerance exhibited by a
mammalian cell line is very important.
CONCLUSIONS
A robust and soft chitosan-iron oxide coated graphene
oxide nanocomposite films (CH-GIO) were fabricated by
proper utilization of different types of secondary interactions
like electrostatic and hydrogen bonding as well as self-healing
ability of a chitosan hydrogel network system. The iron oxide
coated graphene oxide (GIO) nanomaterial used here was
chemically synthesized in-situ by co-precipitation of iron ox-
ide nanoparticles on the graphene oxide surface. Both the GIO
nanomaterial and the CH-GIO nanocomposite hydrogel films
were characterized by different spectroscopic and other tech-
niques which revealed the effective interaction and good com-
patibility between the iron oxide and graphene oxide in GIO
nanomaterial as well as GIO nanomaterial and chitosan hydro-
gel matrix in CH-GIO nanocomposite hydrogel system. The
property evaluation of hydrogel films reported a tremendous
improvement in thermostability. First phase degradation tem-
perature of the chitosan-GIO nanocomposite films increased
up to a maximum of 93 oC after incorporation of 0.2% GIO
nanomaterial compared to that of bare chitosan hydrogel film.
The tensile strength of the nanocomposite films was also im-
proved about 12 MPa and Young’s modulus up to 1.4 GPa. It
was also possible to improve and tune the hydrophobicity of
the nanocomposite films. In addition, the fabricated nanocom-
posite hydrogel film exhibited antimicrobial activity against
many harmful microorganisms as well as against MRSA,
which commonly infects wounds and food products. Antimi-
crobial activities were tested on the films by agar diffusion
assay and antimicrobial test based on direct contact. Both the
antimicrobial test demonstrates the effectiveness of nanocom-
posite films against gram-positive as well as gram-negative
bacterial strain. So this kind of biofilm may have high poten-
tial application in biomedical as well as in the food industry.
Even the hemolysis test indicating the non-cytotoxic nature of
the films proved its suitability in such applications.
ASSOCIATED CONTENT
Supporting information.
Mechanical property data of the chitosan hydrogel and chitosan
hydrogel nanocomposite films, Magnetization–hysteresis (M–H)
loops of CH-GIO hydrogel nanocomposite film, antimicrobial
activity datas of the hydrogel films, hemolytic activity and MTT
assay results of the nanocomposite hydrogel films.
Author Contributions
The manuscript was written through contributions of all authors.
Page 9 of 12 ACS Applied Materials & Interfaces

ACKNOWLEDGEMENT 15. Jiang, Y.; Gong, J. L.; Zeng, G. M.; Ou, X. M.; Chang, Y. N.;
1 Deng, C. H.; Zhang, J.; Liu, H. Y.; Huang, S. Y. Magnetic
2 DC thanks SERB, New Delhi, Grant No. SB/S1/PC-69/2012 and Chitosan–Graphene Oxide Composite for Anti-Microbial
3 BRNS, Mumbai, Grant No. 34/14/20/2014-BRNS for funding. JK and Dye Removal Applications. Int. J. Biol. Macromol.
4 thanks DBT, Grant No. BCIL/NER-BPMC/2015. We also thank 2016, 82, 702–710.
CIF, IIT Guwahati for helping with magnetic measurement. A.K 16. Liu, S.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.;
5
wants to thank IASST for fellowship. Jiang, R.; Kong, J.; Chen, Y. Antibacterial Activity of Graph-
6 ite, Graphite Oxide, Graphene Oxide, and Reduced Graphene
7 oxide: Membrane and Oxidative Stress. ACS Nano. 2011, 5,
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