Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539

https://doi.org/10.1007/s10904-018-0848-1

Effect of Biopolymer Blend Matrix on Structural, Optical and Biological

Properties of Chitosan–Agar Blend ZnO Nanocomposites
G. Magesh1 · G. Bhoopathi1 · N. Nithya1 · A. P. Arun2 · E. Ranjith Kumar3

Received: 19 December 2017 / Accepted: 7 April 2018 / Published online: 9 April 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
The present research is focused on the development of ecofriendly biopolymer blend based nanocomposites to enhance
the effect of cytotoxic activity. Novel eco-friendly synthesis of pure Chitosan–Agar blend and Chitosan–Agar/ZnO nano-
composites was successfully synthesized by in-situ chemical synthesis method. The influence of Chitosan–Agar (1:1 wt/
wt%) concentrations (0.1, 0.5, 1 and 3 g) was studied. The presence of ZnO nanoparticles in Chitosan–Agar polymer matrix
was confirmed by UV, FTIR, XRD, FESEM, EDAX and TEM. The crystallite size of the nanocomposites in the range of
12–17 nm is observed from XRD analysis. PL and UV reveal that Nanocomposites shows an blue shift by increase in the
blend concentrations. TEM analysis shows that 0.1 and 3 g of Chitosan–Agar/ZnO Nanocomposites are in spindle and spheri-
cal shape with polycrystalline nature. The prepared Nanocomposites shows the respectable Antibacterial activity against
Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Pseudomonas aureginosa and Klebsilla
pneumonia) bacteria. The potential toxicity of Chitosan–Agar/ZnO nanocomposites was studied for normal (L929) and
breast cancer cell line (MB231). The result of this investigation shows that the Chitosan–Agar/ZnO nanocomposites deliver
a dose dependent toxicity in normal and cancer cell line.

Keywords  Nanocomposites · Structural analysis · Morphological analysis · Antibacterial activity · Anticancer activity

1 Introduction biopolymers was increased considerably due to their excep-

Recently, bio polymer blending has been widely used for the
development of drug delivery systems and eco-friendly anti-
bacterial packaging materials as an alternative for synthetic
polymers [1]. Biopolymers are a versatile carrier due to their
enhanced biodegradability, nontoxic, biocompatibility, eco-
friendly property etc. [2]. Polysaccharide based bio polymers
are readily available, biocompatible, environmental friendly
and are also cheap. The performance of the polysaccharide
* G. Bhoopathi
bbijjju03@gmail.com
* E. Ranjith Kumar
ranjueaswar@gmail.com
1
Department of Physics, PSG College of Arts and Science,
Coimbatore 641 014, Tamilnadu, India
2
Department of Mechanical Engineering, Kumaraguru
College of Technology, Coimbatore 641035, Tamilnadu,
India
3
Department of Physics, Dr. N.G. P. Institute of Technology,
Coimbatore 643 048, India
tional property in biomedical application [3].
Chitosan is a cationic linear polysaccharide comprised of
β (1–4) 2-acetamido-2-deoxy-d-glucosamine [4]. Chitosan
has tremendous application in the biomedical field, owing
to its gel forming capability, solubility in dilute acidic acids,
biocompatibility, nontoxicity and chemical resistant proper-
ties [5]. The binding ability of chitosan with other polymer
which is attributed to the amino (–NH2) and hydroxyl (–OH)
functional groups [6]. To improve the biological properties
of Chitosan, several bio polymers are blended with chitosan
such as methyl cellulose [7], sodium alginate [8], gelatin [9],
collogen [10] and corn starch [11]. Agar is an unbranched
polysaccharide extracted from the marine algae that consists
of a mixture of agarose and agaropectin. It is one of the most
attractive material because of its low cost, good gel strength,
abundant in nature, non-toxicity, eco-friendly and recycla-
bility [12]. Saxena et al. studied that, Agar–Gelatin blend
had potential effectiveness of drug release.The characteris-
tics with a large array of functions and molecular diversity
structure of agar made it as a good material for biopoly-
mer in composites and blend [13]. Chitosan–Agar blend

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Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539 1529
has been already reported that, they have high miscibility
strength and good mechanical and chemical properties [14].
Hu et al., reported that, Chitosan–Agar blend has potential
application in food packaging material and targeted drug
delivery system [15]. The properties of biopolymer blend
(Chitosan–Agar) may lead it as biomaterial for the develop-
ment of new bio polyblend Nanocomposites.
Recently, polyblend nanocomposites have a great research
interest in the field of nanotechnology [16]. In-situ synthesis,
metal oxide nanoparticles were prepared from their corre-
sponding precursors in the presence of the polymer blend
matrix [17]. This successful method is used to prevent
agglomeration and improve the size distribution in a poly-
mer matrix which is attributed to the interfacial interaction
between organic and inorganic material [18]. It enhances
the biological properties because of their synergistic effect.
Among the metal oxide nanoparticles, ZnO nanoparticles
show a potential antibacterial activity against gram negative
and gram positive bacteria [19, 20] and cytotoxicity against
cancer cell line with lower toxicity towards the normal cell
line [21]. Alireza et al. observed that, PVA/PANI/ZnO nano-
composites exhibits a strong antibacterial activity against
gram negative and gram positive bacteria [20]. Shekhar et al.
suggested Chitosan–PVA hydrogel as a perfect applicant for
the effective in-situ synthesis of silver nanoparticles [22].
In this work, different concentration (0.1, 0.5, 1, 3 g) of
Chitosan–Agar blend ZnO nanocomposites were prepared
by in-situ chemical synthesis method. Here, Chitosan–Agar
blend was used to achieve controlled synthesis of ZnO nano-
particles. The Chitosan–Agar blend ZnO nanocomposites
were characterized by XRD, FESEM and EDX, HRTEM,
UV, FTIR, PL analysis and their biological properties (anti-
bacterial and anticancer activity) were investigated.
2 Materials and Methods
2.1 Chemicals
Chitosan with 85% degree of deacetylation and Agar was
purchased from Sigma Aldrich Co. Ltd. Acetic acid (Gla-
cial 100%, Pro analysis), sodium hydroxide and zinc acetate
dehydrate (Zn ­(CH3COO)2·2H2O) were purchased from
Merck. All chemicals were used without further purification
and freshly prepared solutions were used in all experiments.
The detailed sample preparation techniques are described
below.
2.2 Preparation of Chitosan and Agar Blend
The preparation of Chitosan–Agar Blend was carried at
(1:1 wt/wt%) ratio. Briefly, Chitosan was prepared by dis-
solving 0.5 g in 100 ml of acetic acid solution at room
temperature. The agar was prepared by preheated ultrapure
water followed by stirring at a room temperature of 90 °C
for 2 h until a clear solution obtained. The aqueous Agar
solution was added drop by drop to Chitosan solution,
under constant stirring until a homogeneous solution were
obtained. The homogeneous of Chitosan/Agar (1:1 wt/
wt%) solution was cast into a petri dish. The cast solution
was allowed to dry in oven at 60 °C for 24 h. The dried
film was carried and stored in a desiccator for futher char-
acterization. The blended films were prepared by ionically
cross-linked method is described by El-hefian et al. [14].
2.3 Preparation of Chitosan–Agar /ZnO
Nanocomposites
0.2M of zinc acetate was added into the equal quantity of
Chitosan–Agar blend under constant stirring for 2 h. The
required amount of NaOH solution was added dropwise to
the mixture with constant stirring until pH 12 is attained.
The transparent solution turned to milky white and the
reaction continued for 2 h. The white precipitate formed
was allowed to settle by keeping the mixture for 24  h
aging. The precipitate was separated by centrifugation
and then rinsed with distilled water for several times to
remove the excess of by-products. Finally, Chitosan–Agar/
ZnO Nanocomposites were dried at 60 °C for 24 h. A set
of four homogeneous samples named: (a) 0.1 g, (b) 0.5 g,
(c) 1 g, (d) 3 g was prepared by varying the concentration
of Chitosan and Agar blend at a fixed concentration of
ZnO and NaOH.
2.4 Material Characterization
The Chitosan–Agar /ZnO nanocomposites were character-
ized by XRD, FESEM with EDX, HRTEM, UV, FTIR and
PL. The phase purity of the samples was confirmed by X-ray
Diffraction (X-PERT-3, Philips PW3064/60) with CuKα1
(λ = 1.54060 Å) and CuKα2 (λ = 1.54443 Å) radiation oper-
ating at 30 mA and 45 kV. Data were collected over a 2Θ
range of 10°–90°. The structure morphology and composi-
tion of the samples were analyzed by (SIGMA HV-CARL
ZEISS) FESEM with Bruker Quantax-200-Z10 EDX detec-
tor and HRTEM images were taken in (JEOL–JEM-2010,
Japan) with an accelerating voltage of 200 kV using SAED
pattern were also performed. The optical properties of the
samples were characterized by UV spectrometer (JASCO-
V-770 Spectroscopy) at a wavelength range between 200 and
900 nm. PL spectrum was performed with FP-3800 spectro-
flurometer. FTIR spectroscopic analysis were recorded using
(Bruker Alpha FTIR spectrometer) at a wavenumber range
of 400–4000 cm−1.
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1530 Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539
2.5 Antibacterial Activity
The antibacterial activity of pure Chitosan–Agar blend and
various concentration of Chitosan–Agar/ZnO was performed
against Gram-positive (Bacillus subtilis and Staphylococcus
aureus) and Gram-negative (Pseudomonas aureginosa and
Klebsilla pneumonia) bacteria by disc diffusion method. The
microorganisms were inoculated in the fresh nutrient broth and
incubated to get required turbidity. The nutrient agar plate was
swabbed with the test bacterial culture and various concentra-
tions of sample discs (100 µg) (discs are soaked overnight in
nanoparticle suspension) was added into the well. After 24 h
incubation at 37 °C, the antibacterial activity was measured
based on the diameter of the zone of clearance.
2.6 Cytoxicity of the Samples
2.6.1 Cell Line and Cell Culturing
Breast cancer cell line (MB-231) and Normal Fibro Blast
(L929) cell line were obtained from National Centre for
Cell Science (NCCS), Pune and grown in Eagles Minimum
Essential Medium (EMEM) containing 10% Fetal Bovine
Serum (FBS). The cells were cultured in a humidified atmos-
phere with 5% ­CO2 at 37% in 96 well tissue plates.
2.6.2 MTT Assay
The Chitosan–Agar/ZnO Nanocomposites were evaluated
for cytotoxic activity of MB-231 (cancer cell) and L929
(Normal cell). The cells were plated in 96 well cell plates
(1 × 105) cells/well and left overnight at 37 °C. The medium
was then discarded and the cells were treated with differ-
ent concentrations of ZnO (25, 50, 75, 100 and 125 µg/ml).
Incubate the samples at 37° ± 1 °C for 18 h, then MTT was
added to all the wells and incubated for 4 h. After incuba-
tion, DMSO was added into the wells and the absorbance
read at 570 nm using a photometer. Cytotoxicity and Cell
viability of the samples were calculated using the formula
given below. The concentrations required for 50% Inhibition
of Viability ­(IC50) were determined graphically.
[ ]
Cytotoxicity = (Control − Treated)∕Control × 100
Cell Viability =(Treated∕Control) × 100
3 Result and Disccussion
3.1 Structural Evolution of Nanoparticles Through
X‑ray Diffraction Analysis
Figure 1 shows the structural properties of the pure Chi-
tosan–Agar blend and different concentrations (0.1, 0.5,
13
1 and 3 g) of Chitosan–Agar /ZnO nanocomposites were
studied by XRD analysis. The Chitosan–Agar blend shows
amorphous feature characterized by two halos around Bragg
angles 10.67° and 19.96° is shown in Fig. 1a. This property
is attributed by the formation of intermolecular hydrogen
bonding interaction occurred between the two polymers
[23]. The peaks become weak in the XRD pattern of Chi-
tosan–Agar /ZnO Nanocomposites as shown in Fig. 1b–e.
All the XRD peaks of Chitosan–Agar /ZnO nanocomposites
corresponds to the hexagonal wurtzite structure. The peaks
for 2θ values at 36.3°, 34.4°, 31.8°, 47.6°, 56.67°, 62.93°,
68.0° and 69.15° were pertaining to ZnO which indicate the
reduction of ZnO nanoparticles in the polymer matrix. The
discernible peaks can be indexed to the planes (101), (002),
(100), (102), (110), (103). (112) and (220) and these are
perfectly matched with JCPDS Card No. (36-1451). From
Fig. 1b–e shows that, with an increase in the concentration of
Chitosan–Agar blend the peak becomes broader and weaker
which implies the possibility of changes in grain size. This
might be due to the lower crystallinity and the formation
of smaller nanoparticles. However, it confirms the role of
Chitosan–Agar blend as reducing and stabilizing agents. The
crystallite size and the strain analysis of the Nanocomposites
are carried out using De-bye Scherrer method and W–H plot
method which is discussed below [24].
3.1.1 Debye Scherer Method
The crystallite size and strain of the Chitosan–Agar blend
ZnO nanocomposites was calculated by Debye Scherer
Eq. (1).
Fig. 1  X-ray diffraction patterns a pure Chitosan–Agar blend and
b–e 0.1, 0.5, 1 and 3 g of Chitosan–Agar/ZnO nanocomposites (inset
shows peak broadening)
Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539 1531

k𝜆 is called W–H plot equations. Figure 2 shows the W–H

D= , (1)
𝛽 cos 𝜃
where D is the crystallite size (nm), λ is the wavelength of
the radiation (λ = 1.541 Å), k is a constant (0.94), β is the peak
width at half maximum (FWHM) in radian and θ is the peak
position. Micro strain is calculated using the Eq. (2).
𝛽 cos 𝜃
𝜀= . (2)
4
The calculated crystallite size and strain were shown in
Table 1. The unit cell volume, bond length and lattice param-
eters of the Nanocomposites are calculated from the equation
stated by Bindu and Sabu [25].
From Table 1 it can be found that the crystallite size was
found to be 17.07–12.98  nm. While increasing the Chi-
tosan–Agar concentration, the intensity of the peaks is found
to decrease gradually and becomes relatively broad suggesting
the decrease in the degree of crystallinity [26]. The decrease in
crystallite size (D) is mainly because of the Biopolymers (Chi-
tosan–Agar) which adsorbs to the surface and it restricts the
growth of ZnO nanoparticles [27]. The microstarin increases
with decrease in particle size. The calculated lattice constants
‘a’ and ‘c’ is very close to the standard values [36-1451]. There
is no significant change in lattice constants. The bond length
values of Chitosan–Agar /ZnO Nanocomposites are 1.973,
1.975, 1.976 and 1.979 Å respectively. The reduction of ZnO
in the biopolymer matrix may be the reason for changes on
Zn–O bond length.
3.1.2 Williamson–Hall (W–H) Method
In order to understand the strain associated with the sample
due to lattice deformation and line broadening analysis were
carried out by Williamson–Hall (W–H) method [28]. William-
son and Hall proposed a good method for separation of strain
and size effects on broadening by the peak width as a function
of diffraction angle. The addition of the Scherrer equation and
the volume-weighted average strain εstr = βhkl/4 tan θ results in
the following Eq. (3),
k𝜆
𝛽hkl = + 4𝜀 tan 𝜃 (3)
D cos 𝜃
plot of Chitosan–Agar blend ZnO nanocomposites. From
the linear fit data, the Y-intercept and slope represents the
crystallite size, strain respectively.
3.2 FESEM and EDX Analysis
The surface morphology (0.1, 0.5, 1, and 3  g) of Chi-
tosan–Agar/ZnO samples have been characterized by
FESEM. Figure 3a–d shows the well distributed ZnO in
biopolymer matrix. From the SEM images, it was clear that
they are in nanosize with uniform spindle shape morphol-
ogy. While increasing the concentration of Chitosan–Agar
blend the size distribution of the particles is different. There
was no agglomeration observed between the particles. It is
suggested that the Chitosan–Agar blend played a crucial
role in stabilizing and terminates the growth of the particle
which correlates with XRD. The elemental composition of
the (0.1, 0.5, 1 and 3 g) Chitosan–Agar/ZnO nanocomposites
were analyzed by EDX in the range between 0 and 20 Kev
binding energy. Figure 4a–d shows the quantitative atomic
and weight percentage (%) of Zn and O. The EDX analysis
confirms the presence of Zinc and Oxygen which indicates
the purity of the samples.
3.3 HRTEM and SAED Analysis
The structural morphology of the samples was character-
ized by HRTEM and SAED pattern. Figure 5 shows the
HRTEM morphology with different magnification of 0.1 g
of Chitosan–Agar /ZnO nanocomposites. It is observed that
at 0.1 g of Chitosan–Agar/ZnO nanocomposites (Fig. 5a,
b) were spindle in shape with well defined edges. Figure 5c
indicates the lattice fringes of interplanar distance d = 2.80 Å
that corresponds to miller indices of (200) crystal planes.
Figure 5d exhibits the SAED pattern and the particle size
distribution histogram. An average particle size of 15–20 nm
is found from the particle size histogram which is consist-
ent with the crystallite size obtained from XRD. The bright
spots in SAED pattern show the polycrystalline nature of the
sample with symmetrical orientation. The HRTEM image of
3 g Chitosan–Agar/ZnO nanocomposites (Fig. 6a, b) were

Table 1  Structural parameters of Chitosan–Agar/ZnO nanocomposites

Chitosan–Agar Debye Scherer method W–H plot method Lattice parameters Atomic packing Bond length
concentration (g) factor c/a ratio (Zn–O) L (Å)
Crystallite size ‘D’ (nm) D (nm) Strain ɛ × 10− 3 a = b (Å) c (Å)

0.1 17.07 17.34 0.5 3.241 5.197 1.6036 1.9730

0.5 16.87 16.31 0.9 3.245 5.200 1.6032 1.9750
1 15.08 15.23 1.1 3.246 5.201 1.6030 1.9755
3 12.98 13.34 3.2 3.252 5.214 1.6023 1.9795

Standard values: a = b = 3.498 and c = 5.2066 Å (JCPDS card no. 36-1451)

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1532 Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539

Fig. 2  a–d Williamson and Hall (W–H) plot of (0.1, 0.5, 1, and 3 g) Chitosan–Agar/ZnO nanocomposites

Fig. 3  a–d SEM images of (0.1, 0.5, 1 and 3 g) Chitosan–Agar/ZnO nanocomposites

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Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539 1533
Fig. 4  a–d EDX spectrum of (0.1, 0.5, 1 and 3 g) Chitosan–Agar/ZnO nanocomposites
in spherical and spindle shape morphology. Chitosan–Agar
blend formed a layer around the ZnO nanoparticles is clearly
visible as a layer of amorphous phase. Figure 6c indicates
the lattice fringes of interplanar distance d = 2.24 Å cor-
responding to the Miller indices of (101) crystal plane.
Figure 6d exhibits the SAED pattern and particle size dis-
tribution histogram. An average particle size of 14–15 nm
is found from the histogram which is consistent with the
crystalline size obtained form XRD. The rings with discrete
spots shows the polycrystalline nature of the sample. The d
spacing values calculated from the circular ring pattern cor-
responds to the lattice planes of (100), (002), (101), (102),
(110) and (103) which well agrees with the XRD results. In
this case, Chitosan–Agar biopolymer blend may enhance
the Ostwald ripening kinetics by forming the inter and intra
molecular interaction between metal oxides ­(Zn2+) ions and
the amine, hydroxyl group of the blends which terminate the
rate of particle growth [29].
3.4 UV–Vis Analysis (UV–Vis)
Figure 7 shows the room temperature UV–Vis spectra of
pure Chitosan–Agar blend and Chitosan–Agar/ZnO nano-
composite for various concentrations of (0.1, 0.5, 1 and 3 g)
Chitosan–Agar blend. Figure 2(insert) shows sharp peak at
220 nm for Chitosan–Agar blend which was attributed to the
n − π* transistion and semicrystalline nature of the sample
[30]. This peak comes from the presence of C=O strech-
ing bond which were observed in FTIR at about 1646 cm−1
[31]. From Fig. 7a–d, the absorption band is obtained at
367 nm for low concentration (0.1 g) of Chitosan–Agar/ZnO
Nanocomposites. By increasing the concentration (0.5, 1 and
3 g) of Chitosan–Agar, the absorption peak shifts to lower
wavelength 359, 354 and 349 nm which can be assigned
to the intrinsic bandgap absorption of ZnO due to electron
transistion from the valence band to the conduction band.
This indicates the formation of ZnO nanoparticles in the
biopolymer blend matrix.
The optical bandgap of the nanocomposites were deter-
mined by using the tauc relation (4) [32].
)n
(4)
(
(𝛼h𝜐) = 𝛽 h𝜐 − Eg .
The value of n = 1/2, 3/2, 2 or 3 depends on the nature of
the electronic transistion responsible for absorption. Here
n = 1/2 corresponds to the direct bandgap of semiconduc-
tors [33].
Where, α = absorption coefficient, hυ = photon energy,
­Eg = direct bandgap energy.
The bandgap energy of the Chitosan–Agar/ZnO nano-
composites were calculated by the extrapolation of a straight
line graph between the (αhυ)2 verses photon energy (hυ)
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1534 Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539

Fig. 5  HRTEM images a, b 0.1 g of Chitosan–Agar/ZnO nanocomposites for different magnification, c interplanar lattice fringes, d SAED pat-
tern and the particle size distribution histogram

Fig. 6  HRTEM images a, b
3 g of Chitosan–Agar/ZnO
nanocomposites for different
magnification, c interplanar
lattice fringes, d SAED pattern
and the particle size distribution
histogram

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Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539 1535

Fig. 7  a–d UV–Vis absorption spectra of (0.1, 0.5, 1 and 3  g) Chi- Fig. 8  a–d Tauc plot of (0.1, 0.5, 1 and 3  g) Chitosan–Agar/ZnO
tosan–Agar/ZnO nanocomposites (insert shows the UV–Vis spectra nanocomposites
of pure Chitosan–Agar blend)

as shown in Fig. 8a–d. The bandgap energy of the Nano-
composites was calculated as 3.14, 3.21, 3.23 and 3.26 eV
respectively. While increasing the concentration of Chi-
tosan–Agar blend the band gap also increases. The observed
red shift in bandgap is due to smaller crystallites and strain.
3.5 FTIR Spectrum
shifts to lower energy side from 3400 to 3366 and 1662 to
1646 cm− 1 with increase in Chitosan–Agar concentration.
This observation results confirm the reduction of ZnO nano-
particles in the Chitosan–Agar blend matrix.
3.6 Photoluminescence Spectroscopic
Characterization

Figure 9 shows the FTIR spectrum of pure Chitosan–Agar The effect of Chitosan–Agar/ZnO nanocomposites at vari-
blend and Chitosan–Agar/ZnO nanocomposites between ous concentrations (0.1, 0.5, 1 and 3 g) of Chitosan–Agar
4000 and 400 cm−1 using KBr pellet method at room tem- blend ratio was studied using PL under photon excitation
perature. Figure 9a exhibits a characteristics broadband at at 325 nm. Figure 10 shows the changes in the PL spec-
3400 cm−1 which is attributed to (–OH) stretching vibration trum of Chitosan–Agar/ZnO nanocomposites. The emission
modes of the adsorded water on the surface [34]. While the
absorption peak at 1647, 1559, 1417 and 1376 cm−1 are
ascribed to bending vibration of C=O, –NH2, C–C, and CH
(amide II) group. Based on the observation Chitosan–Agar
is perfectly compatible and miscible polymers by intramo-
lecular hydrogen bends between the amino and the hydroxyl
group of Chitosan and Agar [35]. The IR spectrum of nano-
composites contains all the characteristics peak of Chitosan,
Agar and ZnO (Fig. 9b–e). On comparision with blend a
new intense absorption peaks at 584–512 and 456–452 cm−1
are assigned to stretching mode of Zn–O bonding is due
to the inter molecular interaction between Amide group
of Chitosan–Agar blend and ZnO. The weak band around
898–877 cm−1 is ascribed to the Zn–O vibration frequency.
This absorption bands is well matched with the literature
[36, 37]. While increasing the Chitosan–Agar concentra-
tion, the intensity of the Zn–O vibration frequency become
weak and shifts to lower frequency region which is due to
the changes in the nanostructural features. The intensity of Fig. 9  FTIR spectrum of a pure Chitosan–Agar blend and (0.1, 0.5, 1
the characteristics band become broader and considerably and 3 g) Chitosan–Agar/ZnO nanocomposites (b–e)

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1536
spectrum exhibits a weak blue broadband which is observed
at 446 nm due to surface defects in ZnO, mainly due to Zn
vacancy [38]. A sharp emission peak is observed at 545 nm
in the green region of the spectrum which is due to the sin-
gly ionized oxygen vacancy in ZnO and recombination of
a photo generated hole in the single ionized charge state
of the particular defect [39]. On varying the concentra-
tions, we observe a small blue shift in the blue emission
along with a systematic blue shift in the green emission of
Chitosan–Agar/ZnO nanocomposites. On interaction with
3 g of Chitosan–Agar blend, a large blue shift of the green
emission from 547 to 535.5 nm is observed as shown in
Fig. 10. But in this case intensities of both blue emission and
green emission of Chitosan–Agar/ZnO nanocomposites are
quenched significantly while increasing the concentrations
of Chitosan–Agar [40].
Figure 10 inset shows concentration vs intensity graph
of Chitosan–Agar/ZnO nanocomposites. It is evident that at
545 nm, the PL peak intensity decreases with increase in the
concentration of Chitosan–Agar blend which might be due
to the decrease of particle size. These changes of emission
confirms the reduction of ZnO nanoparticles in the Biopoly-
mer blend matrix [41].
3.7 Antibacterial Activity
Figure  11 shows the antibacterial activity of Pure Chi-
tosan–Agar blend and (0.1, 0.5, 1 and 3 g) Chitosan–Agar/
ZnO nanocomposites against Gram-positive (B. subtilis
and S. aureus) and Gram-negative (P. aeruginosa and K.
pneumonia) bacteria respectively. Chitosan–Agar blend
(control) did not reveal any antibacterial activity, whereas
Fig. 10  a–d Photoluminescence spectra of (0.1, 0.5, 1 and 3 g) Chi-
tosan–Agar/ZnO nanocomposites [insert shows the PL peak intensity
(545 nm) variation with the concentration]
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Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539
the Chitosan–Agar blend loaded with ZnO exhibited potent
antibacterial activity against Gram-positive and Gram-neg-
ative bacteria. As the concentration of Chitosan–Agar blend
increases from 0.1 to 3 g the zone of inhibition also increases
(Fig. 12). Enhanced bioactivity is due to the generation of
­H2O2 molecules on the surface of ZnO which can penetrate
through the cell membrane and inhibit the growth of bac-
teria. In addition, it is clear that the bactericidal activity
increases with decrease in particle size which might be due
to the large surface to volume ratio [19]. While comparing
Gram-positive and Gram-negative bacteria, it has been fre-
quently observed that Chitosan–Agar/ZnO Nanocomposites
shows strong antibacterial activity towards Gram-negative
than Gram-positive which may be attiributed to the different
structure and thickness of peptide glycans in the cell wall
[42]. Ahmed et al. reported that CMC/PVA films loaded
with ZnO nanocomposites shows the respectable antibacte-
rial activity against Gram-positive and Gram-negative bac-
teria [43]. Based on the experimental data (Table 2) it was
observed that, the bactericidal activity of Chitosan–Agar/
ZnO nanocomposites was greater than Chitosan–Agar blend
which may attribute to the synergistic biological properties
of Chitosan, Agar and ZnO.
3.8 Anticancer Activity
3.8.1 MTT Assay
In vitro cytotoxic activity of Chitosan–Agar/ZnO nanocom-
posites (Control, 25, 50, 75, 100, 125 µg/ml) against MB-231
(cancer cell line) was evaluated and compared with L929
(normal fibroblast cell line) after 24 h incubation. Figure 13
shows the anticancer activity of the Nanocomposites treated
with MB-231 cell line. The highest cell inhibition 87% was
observed in the concentration of 125 µg/ml. Cytotoxicity
rate against MB-231 cell line increases with an increase in
the concentration of the sample. The L929 normal cell line
similarly treated with ZnO Nanocomposites shows low tox-
icity 17% at 125 µg/ml concentration as shown in Fig. 14a.
The cytotoxicity of the nanocomposites might be due to the
electrostatic interaction between negatively charged anionic
phospholipids on the cancer cells with positively charged
surface of the ZnO. The size of the nanocomposites plays
a great role in the cytotoxicity [44].The cytotoxicity of the
nanocomposites against the cancer cell line is due to the
generation of reactive oxygen species (ROS). The anticancer
activity of ZnO might be due to the functionalization of bio-
active compounds present in the Chitosan–Agar biopolymer
blend. Similar reports of cytotoxicity of ZnO nanoparticles
against A549 and normal cell line were discussed by Balraj
et al. [45] and Mahendran et al. [46].
Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539 1537

Fig. 11  Antibacterial activity
of pure Chitosan–Agar blend
­(B0), 0.5 g (­ B1), 1 g (­ B2), g (­ B3)
and 5 g ­(B4) Chitosan–Agar/
ZnO nanocomposites against
a, b Gram-negative bacteria (P.
aeruginosa and K. neumoniae)
and c, d Gram-positive bacteria
(B. subtilis and S. aureus)

25 Table 2  Antibacterial activity of Chitosan–Agar/ZnO nanocompos-

ites
20
Samples (g) Zone of inhibition (mm)
Zone of Inhibtion (mm)

15 P. aeruginosa K. pneumonia S. aureus B. subtilis

0.1 g

10
0.5 g Control – – – –
1g O 0.1 14 12 15 14
3g O
5 0.5 16 15 16 14
1 18 15 17 15
0 3 20 18 17 16
P.aeruginosa K.neumoniae S.aureaus B.subtillis
Gram Negative Gram Positive

Fig. 12  Graphical representation of zone of inhibition (mm) of (0.1, 4 Conclusion

0.5, 1 and 3 g) Chitosan–Agar/ZnO nanocomposites
The dose response curves for the MB-231 and L929 cell
lines are shown in Fig. 14b. The inhibitory concentration
IC(50) values for ZnO Nanocomposites against MB-231
cell line was 48.82 µg/ml, while it was found to be signifi-
cantly higher (> 100 µg/ml) for the normal cell line. The
IC(50) value of the MB-231 cancer cell line is lower than
L929 cell line indicating the ZnO Nanocomposites as a
potential dose dependent anticancer agent.
Chitosan–Agar blend ZnO Nanocomposites with varied
Chitosan–Agar blend (0.1, 0.5, 1 and 3 g) concentration
were prepared by simple, facile and green in-situ approach.
XRD result exhibits the Chitosan–Agar blend ZnO Nano-
composites were in the hexagonal wurtzite structure. On
increasing the concentration of Chitosan–Agar blend, the
crystallite size reduced from 17.07 to 12.98 nm. FESEM
and HRTEM images reveal the spindle shape morphology
and the size distribution of Chitosan/Agar blend nanocom-
posites. The presence of Zn, O and the composition of

13

1538 Journal of Inorganic and Organometallic Polymers and Materials (2018) 28:1528–1539

Fig. 13  a–f Cytotoxic activity

of Chitosan–Agar/ZnO nano-
composites on MB-231 cell line
at different concentrations

Fig. 14  a In vitro cytotoxic

effect of Chitosan–Agar/ZnO
nanocomposites (bar diagram).
b Percentage of viability versus
concentration Chitosan–Agar/
ZnO nanocomposites against
MB-231 (Cancer) and L929
(Normal) cell line

the nanocomposites were confirmed by EDX spectrum. References

FTIR spectrum reveals the intraction between the func-
tional group of ZnO and the polymer blend matrix. From
UV, the optical bandgap energy of nanocomposites was
found to vary from 3.14, 3.21, 3.23 and 3.26  eV. Chi-
tosan–Agar blend ZnO nanocomposites exhibit excellent
antibacterial activity against Gram-positive and Gram-
negative bacteria which may due to the synergitic biologi-
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from the above studies, the synthesized Chitosan–Agar
blend ZnO nanocomposites could be applicable for the
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