journal of the mechanical behavior of biomedical materials 65 (2017) 281–294

Available online at www.sciencedirect.com

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Research Paper

Designing of silk and ZnO based antibacterial

and noncytotoxic bionanocomposite films
and study of their mechanical and UV
absorption behavior

Anamika Kiro, Jaya Bajpai, A.K. Bajpain

Bose Memorial Research Laboratory Department of Chemistry Govt. Autonomous Science College, Jabalpur, Madhya
Pradesh, India

art i cle i nfo ab st rac t

Article history: Bionanocomposites of sericin and polyvinyl alcohol (PVA) were prepared by solution
Received 25 June 2016 casting method and zinc oxide nanoparticles were impregnated within the polymer blend
Received in revised form matrix through homogenous phase reaction between zinc chloride and sodium hydroxide
19 August 2016 at high temperature following an ex-situ co-precipitation method. The prepared bionano-
Accepted 21 August 2016 composites were characterized using Fourier Transform Infrared Spectroscopy, X-ray
Available online 26 August 2016 diffraction, Field Emission Scanning Electron Microscopy, Transmission Electron Micro-

Keywords: scopy and Atomic Force Microscopy techniques. The presence of characteristic groups of

ZnO sericin and ZnO nanoparticles was ascertained by the FTIR spectra. XRD analysis

Silk protein confirmed the impregnation of ZnO nanoparticles and sericin within the PVA matrix.

Bionanocomposites XRD and FESEM of the bionanocomposites provided information about their semicrystal-

UV absorption line nature, crystallite size of the particles, and irregular rough surfaces. The TEM

Microhardness confirmed the size of ZnO particles to be in the nanometer range. AFM confirmed the

Antibacterial platykurtic nature of the surface while the negative surface skewness shows the
predominance of valleys over peaks suggesting for the planar nature of the surface of
the bionanocomposites. UV absorption properties of bionanocomposite films were deter-
mined by UV absorption spectroscopy. UV absorption increased with increasing amount of
ZnO nanoparticles in the nanocomposites. Sericin was found to absorb UV-C radiations
between 200–290 nm which is mainly due to aromatic amino acids like tryptophan,
tyrosine and phenylalanine. The ZnO nanoparticles and sericin protein showed antimi-
crobial properties as evident from the inhibition zones obtained against Staphylococcus
aureus and Escherichia coli. The bionanocomposite was found to be noncytotoxic which was
proved by in vitro cytotoxicity test. Microhardness of bionanocomposite films increased
with increase in the amount of ZnO nanoparticles in the sericin and PVA matrix.
& 2016 Elsevier Ltd. All rights reserved.

n
Corresponding author. Fax: þ0761 2678737.
E-mail address: akbmrl@yahoo.co.in (A.K. Bajpai).

http://dx.doi.org/10.1016/j.jmbbm.2016.08.029
1751-6161/& 2016 Elsevier Ltd. All rights reserved.
282 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294
1. Introduction
Polymer bionanocomposites are the composite materials
containing at least one constituent of biological origin. These
materials have been the subject matter of immense curiosity
as they incorporate the characteristics of polymers and small
entities like nanoparticles (Peukert et al., 2003). These poly-
mer bionanocomposites can be prepared by in situ and ex situ
methods (Mbhele et al., 2003; Latif et al., 2012; Guo et al., 2010;
Ricciardi et al., 2004). The metal particles are formed inside a
polymer matrix by decomposition or chemical reduction of a
metallic precursor dissolved into the polymer in the in situ
method. In ex situ technique the nanoparticles are first
synthesized and then dispersed into polymeric matrices (Lu
et al., 1997; Dirix et al., 1999; Zimmerman et al., 1993; Harris
and Wilkinson, 1995). The ex situ techniques are most pre-
ferably used for the preparation of highly proficient polymer
bionanocomposites.
Nanoparticles are normally defined as those particles
which have at least one dimension in the range of
1–100 nm (Hussain et al., 2006; Jordan et al., 2005). The
impregnation of ZnO nanoparticles into PVA matrices can
improve the mechanical, optical and electrical properties to
an enormous level (Jordan et al., 2005; Senthil et al., 2012;
Hema et al., 2007). ZnO based polymeric bionanocomposites
have drawn immense interest of scientific society because of
their good transparency, high electron mobility (Heo et al.,
2004; Fortunato et al., 2005), wide band gap ( 3.3 eV, at
300 K) and excellent optical and electrical properties. ZnO is a
low cost and eco-friendly semiconductor materials finding
promising applications in optoelectronic and electronic
devices such as solar cells (Gledhil et al., 2009), transparent
conducting films, light emitting diodes (Alivov et al., 2003;
Kim et al., 2008), and chemical sensors (Major and Chopra,
1988; Shishiyanu et al., 2005; Gupta, 1990; Anas et al., 2007).
Moreover, incorporation of ZnO in PVA matrix has been
reported to improvise its photocatalytic and antibac-
terial activities that are all essential for environmental
applications.
Sericin is a macromolecular protein, primarily amorphous,
more water soluble and serves as gum adhesive to sustain
the structural strength of the cocoon (Chopra and Gulrajani,
1994; Cho et al., 2003). Sericin has been viewed as an unusable
by-product so far and generally discarded, but there have
been persistent efforts to regain and reprocess it as a natural
biopolymer for several applications. This protein can be
intermixed with other macromolecular synthetic polymers
to design materials with improved properties. Natural silk
protein sericin finds huge number of applications as biona-
nocomposites. The protein was used as a coating material for
cellulose fiber; the treated textile delineated reduced free
formaldehyde composition, resistance to electricity, skin
irritation and allergic reactions with enhanced water reten-
tion and an exiguous decrease in the textile tensile strength
(Kongdee et al., 2005). It can furnish beneficial and excep-
tional properties to polymer gels, membranes, foams, fibers,
and other composite materials (Zhang, 2002) and can be used
for manufacturing cryo-preservatives, anticoagulants, and
biocompatible materials. It has great moisture absorbing
and releasing properties. It also exhibits antibacterial and
UV radiation absorbing properties (Sarovart et al., 2003).
Polyvinyl alcohol (PVA) is a hydrophilic polymer mostly
used as a host matrix for embedding a wide variety of
nanoparticles. This polymer has been mostly desired due to
its good film forming ability, biocompatibility, good chemical
resistance and strong mechanical properties (Shao et al.,
2003). PVA has many applications in electronics, as well as
packaging textile and food products due to its high clarity,
lack of charge density properties, excellent durability and
biodegradability (Krumova et al., 2000). When fortified with
relevant nanoparticles, this polymer exhibited excellent
microhardness, thermal stability, high optical damage
threshold, etc. Various current studies have revealed that
PVA could work as a template for developing nanoparticles
(Liyanage and Lerner, 2012; Xu et al., 2003).
Ultraviolet radiation is an electromagnetic radiation
emitted by the sun. UV radiation spectrum can be divided
into UVA (320–400 nm), UVB (290–320 nm) and UVC region
below 290 nm. The factors affecting solar UV radiation
include ozone layer, geographical position, high altitude
areas, environmental and related conditions. UV radiations
are also emitted through artificial sources such as industrial
place lightening, arc welding, hardening plastics and resins,
different types of phototherapy lamps, sterilizations and
medical fields. The long term exposure of UV radiations
causes serious health hazards leading to inflammation of
human skin, cell damage, skin reddening, premature aging,
and tanning, cataract, photokeratitis and skin cancer (Cakur
et al., 2012). The present work is specifically designed for
army personals and civilians working in harsh environments
of high altitude areas getting constant and continuous expo-
sure of these harmful UV radiations. In many cases, serious
reports of skin cancers have been reported which threaten
their lives and also affect their working efficiency. Although
there are several preventive measures to stay protected from
UV radiations, however, still it is very important to explore
such composite materials that can potentially reflect or
absorb the incident radiation. Thus, realizing the vital role
of polymer based nanocomposites in various areas, the
present work deals with designing of PVA, sericin and ZnO
based bionanocomposite films and studying their structural
and morphological characterization. The work also focuses
on assessment of antibacterial and UV-absorption behavior of
the prepared bionanocomposite films.
2. Experimental
2.1. Materials
Zinc chloride and sodium hydroxide were purchased from
Merck, Mumbai, India and paraffin oil was used as an oil
phase in the synthesis of ZnO nanoparticles. Cocoons of B.
mori silkworms were collected from the Sericulture farm,
Daroli, Sihora Road, Madhya Pradesh, India. Polyvinyl alcohol,
PVA (98.6% hydrolyzed, M.Wt. 30,000 Da), sodium carbonate
(Merck, Mumbai, India), glutaraldehyde (crosslinking agent)
(Merck, India) were used as received. Distilled water was used
throughout the experiments.
 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294 283

2.2. Methods Table 1 – ZnO-Sericin-PVA nanocomposite films with

varying amounts of ZnO nanoparticles,sericin in PVA
2.2.1. Synthesis of Zinc oxide Nanoparticles matrix and their codes.
Zinc oxide nanoparticles were prepared by a homogenous S.no. PVA (in g) ZnO (in mg) Sericin (g) Code
phase reaction between sodium hydroxide and zinc chloride
at high temperature. In the first step, ZnCl2 (5.5 g) was 1. 1 0 0.124 PZ0 S1
dissolved in 200 mL of water at 90 1C in an oil bath 2. 1 0 0.236 PZ0 S3
3. 1 200 0.000 PZ1 S0
and 16 mL of 5 M NaOH aqueous solution was added drop-
4. 1 200 0.304 PZ1 S4
wise to the zinc chloride solution with a mild mixing upto 5. 1 250 0.124 PZ2 S1
10 min at 901 C. The particles were separated from the 6. 1 250 0.192 PZ2 S2
supernatant by sedimentation. The supernatant solution 7. 1 250 0.236 PZ2 S3
was discarded and the remaining suspension was washed 8. 1 250 0.304 PZ2 S4
five times with distilled water to lower the concentra- 9. 1 250 0.368 PZ2 S5
tion of NaCl. The so prepared and purified nanoparticles were 10. 1 300 0.304 PZ3 S4
11. 1 350 0.304 PZ4 S4
filtered and dried in hot air oven for overnight at 60 1C which
12. 1 350 0.000 PZ4 S0
leads to the formation of ZnO nanoparticles.

2.2.2. Preparation of silk sericin solution from Bombyx mori

cocoon
Silk sericin protein was extracted from cocoons of B. mori
following general extraction procedure with minimum mod-
eration. In brief, cocoons were cut into small pieces, boiled
with 0.02 M Na2CO3 for 60 min and washed several times with
distilled water for the removal of sericin protein from the silk
fibers and the degummed fibers were then dried and com-
pletely discarded. The extracted sericin solution was used
further for the preparation of bionanocomposite films follow-
ing solution casting method.
2.2.3. Sericin assay in the extracted solution
The amount of sericin present in the extract was quantified
using bichinchoninic acid (BCA) method reported elsewhere
(Çapar and Aygün, 2015). In brief, copper-BCA mixture was
added into the samples and the samples were incubated in
water bath at 37 1C for 30 min. Thus, in an alkaline environ-
ment, Cu2þ ion was reduced to Cuþ ion due to the reaction of
protein and copper. A characteristic color appears in the
sample solutions due to formation of BCA-Cuþ and its
absorbance was read at 562 nm.
2.2.4. Preparation of ZnO impregnated silk sericin-PVA
bionanocomposite films
In this experiment, 1.0 g PVA was dissolved in 20 mL distilled
water by heating at 70 1C for 1 h to get a homogeneous
solution. Now into 4 mL sericin solution containing 0.124 g
of sericin, 5.29 mM of glutaraldehyde was added as a cross-
linker and the solution mixture was kept at room tempera-
ture under stirring for 10 min leading to the formation of a
homogeneous reaction mixture. ZnO nanoparticles were
impregnated by direct dispersion method in which 350 mg
of ZnO nanoparticles prepared earlier were properly mixed
into the above solution mixture on a magnetic stirrer. The
whole suspension was transferred into a petri dish and kept
at 50–60 1C for 7 h in hot air oven. After complete drying, a
solid semi-transparent thin film was obtained. Following the
same procedure, films with increased amount of ZnO nano-
particles ranging from 200 to 300 mg were prepared. Likewise,
the amount of sericin was also increased to 0.368g. The films
so prepared were immersed in distilled water for 24 h so that
the unreacted chemicals were leached out. The so purified
films were dried at room temperature for one day. The
prepared bionanocomposite films appeared yellow, homoge-
nous and transparent. The dried bionanocomposites films
were cut into small equal pieces and stored in airtight
polythene bags. Different bionanocomposites films contain-
ing varying amounts of zinc oxide, sericin and polyvinyl
alcohol were prepared and coded as summarized in Table 1.
2.2.5. FT-IR Spectral Analysis
Infrared spectra of native PVA, PVA crosslinked with glutar-
aldehyde, and bionanocomposites films were obtained using
FTIR-8400 Shimadzu spectrophotometer by mounting a thin
film of respective materials of proper dimension on the sample
holder. A total of 25 cumulative scans were taken in the
range of 4000-400 cm  1 with a resolution of 2 cm  1 in tran-
smission mode.
2.2.6. X-ray diffraction (XRD)
The crystallinity of the bionanocomposite films was deter-
mined using an XRD instrument, (9 kW) Rikagu, Japan with a
Cu Kα (λ ¼ 1.541) radiation. The diffractograms were obtained
in 2θ range from 0 to 801, 40 kV, 40 mA. The crystallite size of
ZnO nanoparticles was estimated using Debye Scherer's
formula. The XRD spectra of native PVA and bionanocompo-
sites films were also obtained.
2.2.7. Field Emission Scanning Electron Microscopy (FESEM)
Surface morphology of the bionanocomposites film was
examined using the instrument SEM, Philips 515, fine coater,
Philips Eindhoven, the Netherlands Ireland operated at
5.00 kV. The FESEM images were obtained in four different
magnifications 500  , 5.00 K  , 25.00 K  and 30.00 K  .
2.2.8. Transmission Electron Microscopy (TEM)
The size and morphology of the bionanocomposites film was
determined by conducting Transmission electron microscopy
analysis (TEM) of the film on Morgagni 268-D Transmission
Electron Microscope with an accelerating voltage of 80.0 kV.
284 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294

2.2.9. Atomic Force Microscopy (AFM)
AFM analysis was performed to investigate the surface
morphology of the bionanocomposites film using AFM, NTE-
GRA Prima, NT-MOT Ireland. 2-D and 3-D AFM images were
obtained in three different scanning areas 3  3 mm2, 5  5 mm2
and 10  10 mm2. The comparative studies of statistical and
spatial parameters skewness, kurtosis, entropy and redun-
dance were done at different scanning areas.
The diameters of indentation were measured by a micro-
meter eyepiece and the Vickers microhardness was calcu-
lated using Eq. (1):
Hv ¼ 1:8544  L=d2 kg=mm2 ð1Þ
where L is the load (kg) and d is diagonal of indentation (mm).
The duration of indentation load was 30 s for each test.

3. Results and discussion

2.2.10. UV-absorption spectroscopy
UV-absorption studies of the bionanocomposites films were
3.1. Fourier Transform Infrared Spectroscopy (FT-IR)
performed in the range 200–400 nm using UV-Visible Spectro-
photometer, SHIMADZU, UV-2600, Japan for estimating the
FT-IR spectroscopy is a significant tool to seek structural
absorption of UV by ZnO nanoparticles and sericin protein in
information that includes presence of specific functional
the PVA matrix.
groups in the bionanocomposites along with the prevailing
interactions between polymers. In the present work this
2.2.11. Antibacterial activity
study was performed to understand the possible interac-
Antibacterial activity of bionanocomposite films was deter-
tions between PVA matrix, sericin protein and ZnO nano-
mined against a gram positive and gram negative bacteria
particles. The FTIR spectra of native PVA, PVA crosslinked
following Bauer Kirby method. The petri plates with Mueller
with 5.29 mM glutaraldehyde, PZ2S4 and PZ3S4 bionanocom-
Hinton agar were prepared for the growth of Staphylococcus
posite films are shown in Fig. 1a, b, c and d, respectively. A
aureus and Escherichia coli. Then similar colonies were trans-
strong broad absorption band in the range 3000–3600 cm  1
ferred to 5 mL nutrient broth with the help of loop and
may be attributed to O–H stretching vibrations of PVA. A
incubated at 37 þ 2 1C for 2–3 h until light to moderate
prominent peak is observed at 3440 cm  1 in the spectra (a),
turbidity was developed. A sterile non-toxic cotton swab
3655 and 3431 cm  1 in the spectra (b), 3471 cm  1 in the
was dipped into the standardized inoculums (turbidity so
spectra (c), and 3533 cm  1 in the spectra (d), which all
adjusted, as to obtain confluent growth on the petri plate).
confirm the presence of OH groups of PVA in bionanocom-
The entire agar surface of the petri plate was streaked with
posite films. The peak observed at 2950 cm  1 gets shifted to
the swab three times. The inoculums were allowed to dry for
2958 cm  1 in the spectra (b) that may be attributed to C–H
5–15 min. The discs of the bionanocomposites were applied
asymmetric stretching vibration. A peak observed at
on it using aseptic technique and incubated immediately at
1736 cm  1 is attributed to C ¼ O stretching vibration of
37 þ 0.2 1C. The plates were examined after 20–24 h of
unhydrolyzed vinyl acetate groups of PVA while the peak
incubation. The zones showing complete inhibition were
at 1453 cm  1 is due to C–H bending of –CH2 groups of PVA.
measured and the diameters of inhibition zones were
The peak observed at 852 cm  1 also corresponds to –CH2
recorded.
stretching mode.
There were noticed considerable shifts in the peak posi-
2.2.12. In vitro cytotoxicity test
tions due to C¼O stretch. A peak at 1049 cm  1 is assigned to
A direct contact method (ISO10993-5, 2009) was applied in
C–O–C stretching of acetyl groups present in PVA. The peaks
order to determine in vitro cytotoxicity of the prepared
bionanocomposites as described here. Briefly, a test sample
of the nanocomposite, negative and positive controls in
triplicate were placed with sub confluent monolayer of
L-929 mouse fibroblast cells. After incubation of cells with
test samples at 37 7 0.2 1C for 24–26 h, cell cultures were
examined microscopically for cellular response around and
under the test samples. In these experiments USP-grade low
density polyethylene RS was used as the negative control and
tin stabilized polyvinyl chloride as positive control.

2.2.13. Microhardness measurements

Vickers microhardness method was used to determine the
hardness of bionanocomposites using mhp-160 microhard-
ness tester with a Vickers's diamond pyramidal indenter
attached to a Carl Zeiss NU2 microscope. Vickers hardness
is a standard method for measuring hardness and in this
method the surface is subjected to a standard force for a
standard length of time by means of a pyramid-shaped
diamond.
The bionanocomposite films were indented at room tem- Fig. 1 – (a) FT-IR spectra of native PVA thin film, (b) PVA
perature. The indenting loads ranged from 0.01 kg to 0.1 kg. crosslinked with glutaraldehyde, (c) PZ2S4, (d) PZ3S4.
 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294 285

at 638, 592, and 528 cm  1 in the spectra (c) and 644, 574, and
532 cm  1 in the spectra (d) indicate the stretching vibrations
of ZnO nanoparticles. Due to inter-atomic vibrations below
1000 cm  1 absorption bands are observed for metal oxides in
fingerprint region. The physical interaction between ZnO
nanoparticles and hydroxyl groups of PVA was confirmed
due to C–O–C stretching vibrations as shown in Fig.1a, c and
d, respectively. The OH groups of PVA have a property of
formation of charge transfer complex with ZnO nanoparticles
through chelation. The crosslinking reaction was also con-
firmed by FT-IR spectroscopy. FT-IR spectra shown in Fig. 1b
represent spectral patterns of PVA crosslinked with glutar-
aldehyde. The peak observed at 2830 cm  1 of C-H stretching
is related to aldehydes, duplet absorption with peaks attrib-
uted to the alkyl chain. The intensity of O-H stretching
vibrational peak was found to be less than native PVA which
reveals that the hydrogen bonding becomes weaker when
PVA is crosslinked with glutaraldehyde. Due to acetal forma-
Fig. 2 – XRD pattern of native PVA film and bionanocomposite
tion, there is decrease in number of OH groups. It can also be
films (a) PZ2S1, (b) PZ1S4, (c) PZ2S4, (d) PZ4S4..
seen that there was no significant increase in the C¼O band
at 1749 cm  1 indicating that the aldehyde groups of glutar-
aldehyde were used up in the crosslinking reaction with O-H
indicates the hexagonal Wurtzite structure formation of ZnO
groups of PVA chain. Sericin protein shows absorption in the
nanoparticles. The strong peaks at 19.49, 31.60 and 34.28o in
range 1600–1700 cm  1 which confirms amide I. The absorp-
Fig. 2(a), 31.66 and 34.30 in Fig. 2(b), 31.70 and 34.36 in Fg. 2 (c),
tion occurs mainly due to C¼ O stretching vibration as shown
31.71, 33.48 and 34.34 in Fig. 2(d) show the presence of sericin
in Fig. 1c and d. It is very useful in the determination of
protein in the bionanocomposites films.
secondary structure of proteins. The peak at 1540 cm  1
belongs to amide II which appears due to the random coil
3.3. Field Emission Scanning Electron Microscopy (FESEM)
structure of sericin.
The proteins usually show characteristic vibration bands
The FESEM micrographs of PZ2S4 illustrate the surface mor-
in the range 1630–1650 cm  1 for amide I (C-O stretching) for
phology at four different magnifications (500  ; 5.00 K  ;
which a peak at 1633 cm  1 was observed in the spectra (d),
25.00 K  ; and 30.00 K  ) as shown in Fig. 3. The results
1540-1520 cm  1 for amide II (secondary N-H bending) and
clearly indicate the differences in the surface morphology of
1270-1230 cm  1 for amide III (C-N and N-H functionalities) for
PZ2S4. It is clearly observed from the image that the sample
which a peak at 1244 cm  1 was observed in the spectra (d).
has a rough surface (Fig. 3b). It exhibits various shapes in the
3D form; most of them have irregular type of morphologies
3.2. X-ray diffraction (XRD)
(figure c and d). The surface of the bionanocomposites film is
irregular in shape and the particles of the ZnO appear in the
XRD analysis is useful in determining the crystalline struc-
nanometer size range. The homogeneous dispersion of ZnO
ture of the polymeric materials. XRD spectral peaks enable to
nanoparticles in the PVA and sericin matrix was observed in
know whether a material is crystalline or amorphous. The
Fig. 3(a) and (b). The efficiency of ZnO nanoparticles in the
XRD patterns of the bionanocomposites films having differ-
improvement of properties of the bionanocomposites film
ent amounts of ZnO and sericin are shown in Fig. 2. It can be
clearly observed from the XRD spectra that PVA present in depends on the degree of dispersion in the PVA and sericin
the bionanocomposites films shows a broad and shallow matrix.
diffraction peak around 201, 2θ which indicates the semicrys-
talline nature of PVA. The semicrystalline nature of PVA 3.4. Transmission Electron Microscopy (TEM)
results from the strong intermolecular interaction within
PVA polymer networks. The strong Bragg reflections for PVA The TEM image was recorded in order to determine the
were observed in Fig. 2(a) at 19.49, 31.60, 34.28, 36.081, in Fig. 2 size of ZnO nanoparticles more precisely as shown in Fig. 4.
(b) at 31.70, 34.36, 36.18, 67.86, 69.03, and in Fig. 2(c) at 31.71, It is clear from the results that the size of ZnO particles
34.34, 36.20, 67.751, 2θ values. The crystalline nature of ZnO is in the range of 30–50 nm which is not a wide size
nanoparticles was confirmed by various sharp peaks distribution. The observed nanosize of ZnO nanoparticles
appeared in Fig. 2. The eight diffraction peaks were observed confirms the nanocomposite nature of the prepared
at the 2θ values of 31.71, 33.48, 34.34, 36.20, 56.48, 62.68, 67.75 bionanocomposite films.
and 68.931 for ZnO nanoparticles in Fig. 2(d). The sharp peaks
of ZnO at 2θ values of 31.601, 34.281, 36.081 appear as small 3.5. Atomic Force Microscopy (AFM)
peaks in the beginning and the intensity increased with
increase in content of ZnO nanoparticles. The XRD pattern The AFM experiments were performed in order to analyze the
of the ZnO nanoparticles show the diffraction peaks which surface morphology of the bionanocomposite films. The
286 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294

Fig. 3 – FESEM images of PZ2S4 at four different magnifications (a) 500  ; (b) 5.00 K  ; (c) 25.00 K  ; and (d) 30.00 K  .

formation of step-like structures with different heights was

observed in Fig. 5. The surfaces of bionanocomposites films
appear to have granular and nanocrystaline morphology as
shown in (Fig. 6 a–f).

3.5.1. Functional Parameters

The functional parameters listed in Table 2 give information
about the surface structure. Skewness and Kurtosis moments
are the functional parameters for measuring the asymmetry
and the flatness, respectively. Skewness can be defined as the
third moment of profile amplitude probability density func-
tion. It is used to measure the profile symmetry about mean
line. Skewness is zero, if the height distribution is symme-
trical. If the height distribution is asymmetrical, and the
surface has more peaks than valleys the skewness moment
is positive. If the surface is more planar and valleys are more
than peaks, the skewness is negative.
Kurtosis moment is the fourth moment of profile ampli-
tude probability function. When Kurtosis moment is 3, which Fig. 4 – TEM image of bionanocomposite film.
indicates a Gaussian amplitude distribution, the surface is
called Mesokurtic. When Kurtosis is smaller than 3 and the
surface is flat, it is called Platykurtic. If the Kurtosis is higher
than 3, the surface has more peaks than valleys. It can be are predominant and the surface is more planar in sample (a).
observed that the surface is Platykurtic for both the samples. On increasing the amount of sericin in the sample
Kurtosis is smaller than 3 and the surface is flat as summar- (b) skewness remains negative and the valleys are more than
ized in Table 2. The surface skewness is negative, the valleys the peaks making the surface planar.
 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294 287

Fig. 5 – AFM images of bionanocomposite films (a)3  3 lm2 2-D AFM image of PZ2S1,(b) 3  3 lm2 3-D image of PZ2S1,
(c) 5  5 lm2 2-D image of PZ2S1, (d) 5  5 lm2 3-D image of PZ2S1, (e)10  10 lm2 2-D image of PZ2S1, (f) 10  10 lm2 3-D image of
PZ2S1.

3.5.2. Spatial Parameters 3.5.3. Line Profile

The spatial parameter entropy measures the samples mor-
phology related to the degree of order or disorder in the grain
structure distribution. It is clearly observed that entropy
increases with increase in the amount of sericin in the
sample (b). It is also observed that there is an increase in
entropy in higher scanning area as compared to lower
scanning area as revealed by Table 3.
Fractal morphology can be observed by the parameter
redundance. The lower the values of the redundance, least
disorder in the morphology of the films is obtained. It is clear
from the Table 3 that redundance decreases in higher scanning
area for sample (a) whereas it increases in higher scanning area
for sample (b). In lower scanning area (3  3 mm2), redundance
decreases on increasing the concentration of sericin, indicating
less fractal morphology.
A line profile is a two dimensional profile, or cross section
extracted from an AFM image. It is an important parameter,
where surface topology combined with line profile, produces
results based on the roughness parameters. Figs. 7 and 8 show
the AFM images of two different bionanocomposites films. The
line profiles of all the images are represented by horizontal
white line projected in the AFM images. The maximum height
of the peak for PZ2S1 is about 490 nm and for PZ2S4 is about
690 nm for the scanning area 10  10 mm2. The results of
skewness have been previously discussed for both the films
under scanning areas 5  5 mm2 and 10  10 mm2 which are
negative thus suggesting for more valleys as compared to
peaks. This is further confirmed by the line profiles shown in
Figs. 7(b) and 8 (b). It is observed that the depths of the valleys
are greater in higher scanning area as compared to the lower
scanning area.
288 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294

Fig. 6 – AFM images of bionanocomposite films (a) 3  3 lm2 2-D AFM image of PZ2S4, (b) 3  3 lm2 3-D AFM image of PZ2S4,
(c) 5  5 lm2 2-D AFM image of PZ2S4, (d) 5  5 lm2 3-D AFM image of PZ2S4,(e) 10  10 lm2 2-D AFM image of PZ2S4, and
(f) 10  10 lm2 3-D AFM image of PZ2S4.

Table 2 – Comparative studies of statistical parameters of two different ZnO- Sericin- PVA nanocomposite film samples.

Parameter Sample-(a) PZ2S1 Sample-(b) PZ2 S4

Scanning area (in mm2) 33 55 10  10 33 55 10  10

Surface Skewness 0.395026  0.195811  0.427555 0.108025  0.113309  0.509698
Kurtosis 0.258491 1.57732 0.717884 2.04663 1.19534 0.880297

3.6. UV-absorption spectroscopy and humps at 276 nm and 280 nm, which are indicative of
high energy absorption. The first band is associated with the
3.6.1. Variation of ZnO nanoparticles presence of some residual acetate groups of PVA. Character-
UV-absorption spectra of all the bionanocomposite films istic peaks, due to the absorption of amino acids were
were recorded in the range of 200–400 nm as shown in observed at 276 nm and 280 nm. Proteins are known to absorb
Fig. 9. The spectra 9 (a) is of only sericin- PVA film which strongly in the ultraviolet region mainly due to peptide bonds
exhibits three absorption bands, an intense band at 210 nm and aromatic acids. The aromatic amino acids like
 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294 289

tryptophan, tyrosine and phenylalanine are known to absorb
UV radiation in the range of 260–290 nm due to π- πn
transition. The UV absorbance spectra in the region 200–
400 nm for spectra 9 (a) PZ0S3, (b) PZ4S0, (c) PZ1S4, (d) PZ2S4,
(e) PZ3S4, and (f) PZ4S4 bionanocomposites films are shown in
Fig. 9. It is clear from the Figure that the absorption by all
films decreased with increasing wavelength, while for the
sericin and ZnO doped PVA nanocomposite films (Fig. 9c, d, e
and f), the absorption increased with increasing concentra-
tion of ZnO. This is in agreement with the values reported by
earlier researchers, in accordance with Beer's law. The
absorption is proportional to the number of absorbing mole-
cules. Emergence of new peaks for the samples after addition
of ZnO and also broadening of those peaks with increasing
amount of ZnO indicate a considerable interaction between
PVA, sericin and ZnO.
3.6.2. Effect of amounts of ZnO
In Fig. 9(a), native PVA exhibits characteristic peak at 210 nm.
However, after incorporation of different amounts of ZnO
nanoparticles into the PVA matrix, the peak undergoes a
shifting in the range of 357-340 nm. At 350 mg content of ZnO

Table 3 – Comparative studies of spatial parameters of ZnO- Sericin- PVA nanocomposite film samples.

Parameter Sample-(a) PZ2S1 Sample-(b) PZ2S4

Scanning area (in mm2) 33 55 10  10 33 55 10  10

Entropy 10.2152 10.9778 11.9909 9.31572 10.9546 12.3158
Redundance  0.359907 0.234847  0.264704 0.24344  0.263183 0.269997

Fig. 7 – Line profile of PZ2S1 in scanning area (a) 5  5 lm2 (b) 10  10 lm2.
290 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294
(Fig. 9 b), there is an absorption peak observed at 357 nm. By
decreasing the amount of ZnO to 200 mg, 250 mg and 300 mg
(Fig. 8, curves c, d and e respectively), the absorption peaks
are shifted to 354, 350 and 340 nm, respectively. In samples b,
c, d, e and f, the absorption peak appears at 376 nm, which is
a characteristic UV peak of ZnO nanoparticle. ZnO nanopar-
ticles impregnated into PVA and sericin matrix increases the
absorption of UV radiation. The higher values of UV-
absorbance were obtained in the region between 300–
400 nm when ZnO content is increased.
Thus ZnO doped PVA nanocomposite films can be applied
to block the UV-A radiation. The results imply that the
effectiveness in shielding of UV radiation is due to the UV
absorption capacity of ZnO nanoparticles present in the PVA
matrix. The increase of absorption in the UV region of the
spectrum is due to the excitations of donor level electrons to
the conduction band at these energies.
3.6.3. Effect of amount of Sericin
In Fig. 10(a), sericin exhibits characteristic peak at 244 nm.
After incorporation of different amounts of sericin into the
Fig. 8 – Line profile of PZ2S4 in scanning area (a) 5  5 lm2 (b) 10  10 lm2.
PVA, this peak undergoes shifting in the range 242–261 nm. In
the curves (b), (c), (d) and (e), on increasing the amount of
sericin to 0.192, 0.236, 0.304 and 0.368 g the peaks were
shifted to 242 nm, 245 nm, and 261 nm, respectively. Sericin
added into the PVA matrix in combination with ZnO
increases the absorption of UV radiation. The higher values
of UV-absorbance are obtained in the UV-A region between
300–00 nm due to ZnO and then at UV-C region between
320-400 nm due to aromatic amino acids like tryptophan,
tyrosine and phenylalanine which are known to absorb UV
radiation in the range of 260–290 nm.
3.7. Antibacterial activity
Antibacterial properties of prepared bionanocomposites films
were determined by testing them against gram-positive
bacteria Staphylococcus aureus ATCC 35696 and gram-
negative Escherichia coli ATCC 23282. The inhibition zones of
samples (d) and (e), were maximum due to the largest
amount of ZnO nanoparticles and sericin as shown in
 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294 291

Fig. 9 – UV Spectra of bionanocomposite films of varying ZnO

nanoparticles (a) PZ0S3, (b) PZ4S0, (c) PZ1S4, (d) PZ2S4,
(e) PZ3S4, (f) PZ4S4. Fig. 11 – Antibacterial activity test of bionanocomposite films
against Staphylococcus aureus, (a) PZ1S4, (b) PZ2S4, (c) PZ3S4,
(d) PZ4S4.

Fig. 10 – UV Spectra of bionanocomposite films of varying

sericin protein (a) PZ2S1, (b) PZ2S2, (c) PZ2S3, (d) PZ2S4,(e) PZ2S5.
Fig. 12 – Antibacterial activity test of bionanocomposite films
Escherichia coli (a) PZ1S0, (b) PZ1S4, (c) PZ2S4, (d) PZ3S4,
(e) PZ4S4.

Figs. 11 and 12. The antibacterial activity of ZnO is conside-

Table 4 – Inhibition Zones showing the antibacterial
red due to the generation of reactive oxygen species (ROS) activity against Staphylococcus aureus.
2- 
O , OH and H2O2 from its surface by photocatalytic activity.
Antibacterial activity of bionanocomposites Inhibition Zone (in
Sericin present in the nanocomposite also shows the anti-
against Staphylococcus aureus mm)
bacterial activity. From Tables 4 and 5, it can be observed that
antibacterial efficiency increases due to the presence of (a) PZ1S4 17
sericin. (b) PZ2S4 19
(c) PZ3S4 21
(d) PZ4 S4 24

3.8. In vitro cytotoxicity

The cytotoxic reactivity were graded based on zone of lysis,

vacuolization, detachment and membrane disintegration as
0, 1, 2 ,3 and 4 representing none, slight, mild, moderate and
severe, respectively and the microscopic observations are
depicted in Fig. 13. It was found that the test sample showed
none reactivity to fibroblasts cells after 24 hours of contact.
The zinc oxide nanocomposite material in the present work
achieved a reactivity grade of zero and, therefore, the mate-
rial may be considered as non cytotoxic.
3.9. Microhardness measurements
Hardness of nanocomposites is defined as the resistance to
deformation, particularly permanent deformation, indenta-
tion or scratching. Vickers hardness value were found to be
15.11, 16.46, 17.39, 18.71 and 20.09 kg/mm2 on varying the
amount of ZnO nanoparticles to 200, 250, 300 and 350 mg,
respectively in sericin and PVA matrix. Microhardness of
292 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294

bionanocomposites films increased with increase in the confirms that the sizes of ZnO particles are in the nanometer
amount of ZnO nanoparticles. range. The AFM confirms the platykurtic surface and the
The microhardness results are shown in Fig. 14 and Table 6 negative surface skewness shows the predominant valleys
which clearly indicate a slow growth in hardness at lower suggesting the planar surface of the nanocomposites. It is clearly
amount of ZnO nanoparticles. The hardness of PZ0S1 nanocom- observed that entropy increases on increasing the amount of
posites at 0.124 g sericin without ZnO nanoparticles increases
sericin. UV absorption properties of bionanocomposite films
from 9.371 kg/mm2, 10.27 kg/mm2 for PZ1S4, 11.18 kg/mm2 for
were confirmed by UV absorption spectroscopy. The absorption
PZ2S4, 12.35 kg/mm2 for PZ3S4 and 13.69 kg/mm2 for PZ4S4 on
increases with increasing amount of ZnO nanoparticles in the
applying 0.01 kg load. The maximum hardness of 20.09 kg/mm2
was observed as shown in the Fig. 14(e) on applying a load of
0.1 kg. A remarkable and substantial increment in microhard-
ness is observed at the highest content of ZnO nanoparticles
due to uniform distribution of nano- ZnO within the sericin and
PVA matrix. This clearly suggests for the superior interfacial
bond strength and consequently large value of hardness
achieved at the largest content of ZnO nanoparticles.

4. Conclusions

We have demonstrated an easy method of synthesizing biona-

nocomposite films by solution casting method. The ZnO nano-
particles were synthesized by the co- precipitation method and
impregnated into the PVA-sericin matrix by ex-situ method. The
nanocomposites were characterized by various analytical tech-
niques, such as FTIR spectroscopy, FESEM, TEM, AFM and XRD.
Fig. 14 – Vickers hardness of bionanocomposite films
The prepared bionanocomposites clearly show the presence of
(a) PZ0S1, (b) PZ1S4, (c) PZ2S4, (d) PZ3S4, (e) PZ4S4..
characteristic groups of sericin and ZnO nanoparticles as con-
firmed by their respective FTIR spectra. XRD analysis confirms
the impregnation of ZnO nanoparticles and sericin into the PVA Table 6 – Hardness values of bionanocomposite films for
matrix. XRD and FESEM of the bionanocomposites provide varying amount of ZnO nanoparticles.
information about their semicrystalline nature, crystallite size S. No. Load Hardness (in kg/mm2)
of the particles, and surface morphology, respectively. TEM (in kg)
(a) PZ0S1 (b) PZ1S4 (c) PZ2S4 (d) PZ3S4 (e) PZ4S4
Table 5 – Inhibition Zones showing the antibacterial
1. 0.01 9.371 10.27 11.18 12.35 13.69
activity against Escherichia coli.
2. 0.02 10.91 11.85 12.77 13.94 14.91
Antibacterial activity of bionanocomposites Inhibition Zone 3. 0.03 12.42 13.36 14.24 15.42 16.42
against Escherichia coli (in mm) 4. 0.04 13.54 14.54 15.48 16.75 17.86
5. 0.05 14.46 15.54 16.41 17.79 18.71
(a) PZ1S0 0 6. 0.06 14.87 15.87 16.74 17.86 18.96
(b) PZ1S4 16 7. 0.07 14.94 15.99 16.96 18.14 19.34
(c) PZ2S4 18 8. 0.08 15.01 16.32 17.29 18.45 19.56
(d) PZ3S4 20 9. 0.09 15.09 16.41 17.37 18.64 19.89
(e) PZ4 S4 25 10. 0.1 15.11 16.46 17.39 18.71 20.09

Fig. 13 – Microscopic images showing L-929 cells around (a) negative control, (b) positive control, and (c) polymer network,
respectively.
 journal of the mechanical behavior of biomedical materials 65 (2017) 281 –294
bionanocomposite films. Thus, the prepared bionanocomposites
films can be applied to block the UV-A radiation. Sericin also
absorbs UV-C radiation between 200–290 nm due to the presence
of aromatic amino acids like tryptophan, tyrosine and phenyla-
lanine. After incorporation of different amounts of sericin into
the PVA matrix, the shifting of characteristic peak in the range
242–261 nm is observed. The ZnO nanoparticles and protein
sericin show good antimicrobial properties. They inhibit the
bacterial growth as confirmed by the occurrence of inhibition
zones against Staphylococcus aureus and Escherichia coli in the
antibacterial studies. Microhardness of bionanocomposite films
increases with increase in the amount of ZnO nanoparticles due
to the uniform distribution of nano-ZnO within the sericin and
PVA matrix.
Funding
This work was supported by Defense Research and Development
Organization (DRDO), Directorate of Extramural Research and
Intellectual Property Rights (ER&IPR) funded project (No. ERIP/ER/
1204712/M/01/1492).
Acknowledgements
The authors wish to acknowledge the authority of Defence
Research and Development Organization (DRDO) for providing
financial assistance to one of the authors, Ms. Anamika Kiro, in
the form of a Junior Research Fellow in a Directorate of
Extramural Research and Intellectual Property Rights (ER&IPR)
funded project (No. ERIP/ER/1204712/M/01/1492). The author
would also like to acknowledge the Center of Nano-Science
and Nanotechnology, Sathyabama University, Chennai, India,
for extending XRD, FESEM and AFM facilities to characterize the
prepared bionanocomposite films.
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