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Copper–copper oxide coated nanofibrillar cellulose: a

promising biomaterial3
Cite this: DOI: 10.1039/c3ra42209g
Published on 25 June 2013. Downloaded by University of York on 15/07/2013 22:12:07.

Shaswat Barua,a Gautam Das,b Lipika Aidew,c Alak K. Buragohainc

and Niranjan Karak*a

Received 4th May 2013,
Accepted 24th June 2013
DOI: 10.1039/c3ra42209g
www.rsc.org/advances
Nanocellulose is gaining impetus as a hierarchical material in many advanced applications. The isolation of
nanocellulose from easily available bio-resources is an area to be delved into thoroughly. This article
highlights the isolation of nanofibrillar cellulose from an abundant natural source, Colocasia esculenta, by
a chemical method. The nanofibrils were coated with copper–copper oxide nanoparticles through a
‘green’ reductive technique using the alcoholic extract of Terminalia chebula fruit. The prepared fibrils and
the nanohybrid were characterized by Fourier transformed infrared spectroscopy, X-ray diffraction and
transmission electron microscopic studies. The coated nanofibrils showed promising antimicrobial activity
against Staphyllococcus aureus, Escherichia coli and Candida albicans. The nanohybrid was quite
compatible with peripheral blood mononuclear cells (PBMC) as well as mammalian red blood cells
(RBCs). The structural integrity of bovine serum albumin (BSA) was unaltered upon interaction with the
nanohybrid. The biocompatible and antimicrobial nanohybrid presented here possesses high potential to
be used as a biomaterial in a suitable niche of modern biomedical fields.

Introduction
Availability and sustainability are the signature attributes of
renewable resources. The tailoring of innovative products from
renewable resources for materials science and technology, as
well as for the biomedical domain, generated a global interest
on cellulose – the most abundant organic polymer.1 The
plant’s cellulose generally forms a native composite with
lignin and other carbohydrates (e.g. hemicelluloses) from
which it can be isolated by various physico-chemical techni-
ques.1 A scrutiny of the literature reveals a limited number of
reports on the isolation of micro and nanofibrillar cellulose
(NFC) from natural and industrial wastes.2,3 Recently, Mandal
and Chakrabarty reported the isolation of nanocellulose from
waste sugarcane bagasse by the acid hydrolysis method.4 Apart
from plants, certain non-pathogenic bacteria, algae and fungi
found in fruits, vegetables etc. have been reported to be used
in the isolation of cellulose.5,6 However, the crystallinity and
fiber size are dependent on the isolation conditions as well as
a
Advanced Polymer and Nanomaterial Laboratory, Department of Chemical Sciences,
Tezpur University, Napaam-784028, Assam, India.
E-mail: karakniranjan@yahoo.com; Fax: +913712267006; Tel: +91-3712-267009
b
Department of Environment & Energy Engineering, Gachon University, 1342
Seongnam Daero, Seongnam, Gyeonggi, 461-701, Korea
c
Department of Molecular Biology and Biotechnology, Tezpur University, Napaam-
784028, Assam, India
3 Electronic supplementary information (ESI) available: EDX images of NFC and
Cu NFCs and UV-visible spectra of Cu NFC are given here. See DOI: 10.1039/
c3ra42209g
the post- and pre-treatments.7 Another vital factor that dictates
these features is the original source from which it is isolated.
Again, Colocasia esculenta is a plant that grows abundantly
in the tropical regions across the globe. The nutrient
composition and the cellulose content of this plant have
already been evaluated thoroughly.8 In the present work, we
report the isolation of nanofibrillar cellulose from C. esculenta,
by a chemical method using alkali and acid treatments.
Furthermore, copper nanoparticles have been associated
with remarkable properties including an excellent antimicro-
bial activity.9 Greener approaches for the reduction of metal
salts have been a continuous endeavor,10–14 however, reports
on the reduction of copper salts are still limited in the
literature.15 Herein, we wish to report the reduction of a
copper acetate salt by ethanolic extracts of the Terminalia
chebula fruit. Again, the synthesis of pure metallic copper
nanoparticles is still a challenge for researchers, because
copper is highly susceptible to oxidation in the atmosphere.16
Thus, the present endeavor was to prepare copper–copper
oxide (Cu–CuO) nanoparticle-coated cellulose nanofibrils.
Recently, Jia et al. described the preparation of copper
nanoparticle-coated cellulose films.17 However, they used toxic
reductants like NaBH4 for the reduction of the copper salt.17
Green nanomaterials with potential bioactivity have been
gaining attention recently.11 The interaction of such nanoma-
terials with living systems is of paramount importance for
their potential biomedical applications. The role of protein–
nanoparticle interactions in nanomedicine and nanotoxicol-
ogy is a promising field. Reports suggest that distortion of the

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protein may occur upon interaction with nanosurfaces.18
Lynch and Dawson recently studied a collection of proteins
that associate with nanoparticles in biological fluids.19 The
most abundant protein found in plasma is serum albumin,
which plays a key role in many physiological functions such as
transport and delivery of fatty acids, porphyrins, bilirubin and
steroids, etc. Bovine serum albumin (BSA) was selected as our
protein model due to its water-soluble nature, which is
important for the study of the interaction with nanomater-
ials.20
Again, cellulose has extensive utility in the biomedical
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domain, especially in bandage applications due to its porous
nature.21 A surgical bandage material demands compatibility
with blood cells, especially with peripheral blood mononuclear
cells (PBMC), a fibroblast cell line that helps in the contraction
and healing of wounds.22 To achieve a better comparison with
in vivo conditions and to evaluate whether the nanohybrid
system shows any toxic effect, studies were conducted on
mammalian PBMC, which consist mainly of lymphocytes (e.g.
T-cells) and monocytes, and also on red blood cells (RBCs). In
this endeavor, the aim was to prepare Cu–CuO coated
nanofibrillar cellulose with promising antimicrobial potency
and biocompatibility. This nanohybrid may be a potential
material for biomedical applications.
Experimental
Materials
Colocasia esculenta stems were collected from the Tezpur
University (Assam, India) campus and were cut, sun dried and
washed. The dried sample was ground with a domestic
blender. The chemicals used for the extraction of the fiber
and the preparation of the nanofibrillar cellulose were sodium
hydroxide, glacial acetic acid and hydrogen peroxide (MERCK,
India). Mueller Hinton agar, potato dextrose agar (Himedia,
India) and dimethyl sulfoxide (DMSO) (MERCK, India) were
used for the antimicrobial tests. Trypan Blue, a diazo stain,
RPMI-1640, the cell culture media and BSA (Sigma Aldrich,
India) were used for the compatibility assay with PBMC and
the interaction study of the nanohybrid with the protein.
Method of isolation of the cellulose nanofibrils from the C.
esculenta stems
The ground mass of C. esculenta was bleached with a 7%
hydrogen peroxide solution (pH 5, adjusted with glacial acetic
acid), maintaining a fiber to solution ratio (1 : 50) for 2 h at 45
uC. This was followed by successive washings with double
distilled water to maintain a neutral pH. The treatment
facilitated the removal of lignin from the fibers and helped in
the partial removal of hemicelluloses. For the complete
delignification of the fibers, a 5% sodium hydroxide solution
was used. Fibers were soaked in 250 mL alkaline solution for 2
h at 45 uC. The residue was collected by filtration through a
micro filter and washed several times with double distilled
water. The residue was then air dried and treated with 50%
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DMSO, at 80 uC for 2 h.4 This was again filtered and washed
with distilled water.
The delignified cellulose fibers were treated with 50% glacial
acetic acid for 2 h at 45 uC. The acid treated fibers were washed
with water until the medium reached neutral pH. The acid
hydrolysis was quenched by pouring water to the reactor and
allowed to cool at room temperature. The fibrils were collected
by centrifugation and washed several times with distilled
water. Then, fibrils were mechanically dispersed in water to
obtain a suspension of nanofibrillar cellulose (NFC). The NFC
suspension was frozen by lowering the pressure under
vacuum, followed by sublimation of the water using a
lyophilizer (Labtech Freeze dryer, Daihan Labtech Co.
L-DF5512). This resulted in a dry nanofibrillar cellulose
powder.
Preparation of copper-coated nanofibrillar cellulose
About 2 g of Terminalia chebula fruit was washed and ground
in a domestic blender. It was stirred for about 30 min in 100
mL of ethanol at 40 uC. The alcoholic extract was filtered
through a muslin cloth. The pH of the extract was 7.4, which is
near neutral. A copper acetate solution was prepared in
ethanol with varying amounts of the salt: 5, 10 and 15% (w/v),
by stirring 15 min at room temperature. Nanofibrillar cellulose
(4 g) was dispersed in 10 mL ethanol. The copper acetate
solution was poured onto the dispersed nanofibrillar cellulose
and stirred at room temperature for 2 h. The T. chebula extract
(1 mL) was added to the above solution and stirred for another
6 h at room temperature. The color of the solution changed
from blue to dark brown and finally to faint black. The
solution was centrifuged and the fibers were washed several
times with ethanol, followed by water. The reaction was carried
out at pH 7.4 and the samples were encoded as Cu NFC 1, Cu
NFC 2 and Cu NFC 3 for 5, 10 and 15% (w/v) of the copper
acetate solution, respectively. The copper–copper oxide coated
fibrils were mechanically dispersed in water and freeze dried
under vacuum using a lyophilizer (Labtech Freez dryer,
DaihanLabtech Co. L-DF5512). For comparison, another
sample (Cu NFC 4) was prepared in which copper acetate
was reduced by NaOH according to a reported procedure,
where a 0.2 M ethanolic solution of copper acetate was
reduced with 0.01 mol of NaOH.23
Antioxidant activity
The antioxidant activity of the prepared samples was deter-
mined by the modified DPPH (19,19-diphenyl picryl-hydrazyle)
method as reported previously.10 Then, 0, 50, 100, 150 and 200
mL of all the samples were mixed with 2 mL of a 100 mM DPPH
(HiMedia, India) solution and vortexed. DPPH scavenging was
measured in the dark by measuring the absorbance of the
solutions at 515 nm, using a UV-visible spectrophotometer.
Measurements were done in triplicate. The percentage of
scavenging was calculated using the formula:
DPPH scavenging = [(AC 2 AS)/AC] 6 100 (1)
where, AC and AS are the absorption of blank DPPH and the
sample-mixed DPPH at 517 nm, respectively.
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Antimicrobial assay
NFC and Cu NFC were tested against the following microbial
strains: Escherichia coli (ATCC 10536, Gram negative),
Staphylococcus aureus (ATCC 11632, Gram positive) and
Candida albicans (ATCC 10231). The bacterial strains were
cultured on Mueller Hinton agar and the fungal species (C.
albicans) was cultured on potato dextrose agar. The agar well
diffusion method was employed as a preliminary test to find
out if the samples had antimicrobial activity.10 The fibrils were
dispersed in sterile 1% DMSO (MERCK, India) to a concentra-
tion of 30 mg mL21. Wells with 8 mm diameter were punched
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in the media into which 50 mL samples were loaded.
Streptomycin and nystatin (8 mg mL21) were used as the
positive controls for bacteria and C. albicans respectively and
1% DMSO was used as the blank. Prepared plates were
incubated at 37 uC for 16 h. The antimicrobial activity was
examined by measuring the diameter of the zone of inhibition
using a zone scale (HIMEDIA). The tests were carried out in
triplicate.
Hemolysis assay
The hemolysis assays were performed to investigate the lysis (if
any) of the RBC membrane by the prepared fibrils. Goat blood
was collected from a slaughter house in a heparinized tube
containing 4% sodium citrate. It was then centrifuged (MPW)
at 1006 6 g (3000 rpm) for 20 min at 4 uC. The erythrocytes
were washed twice with phosphate buffer saline (PBS). After
washing, the packed erythrocytes were resuspended in PBS (10
mM at pH = 7.4) to obtain a 5% haematocrit. Varying
concentrations of the samples (0.39, 0.78, 1.56, 3.12, and
6.25 mg mL21) were prepared. 100 mL of the test samples along
with 1900 mL of haematocrit were added in each microfuge
tube (Eppendorf) and incubated at 37 uC for 30 min. Cells were
kept in an ice bath for 60 s for post-incubation and then
centrifuged for 5 min at 1006 6 g (3000 rpm) at 4 uC. The
haemoglobin concentration, as a measure of haemolysis, was
determined by taking the supernatants and reading the
absorbance at 540 nm.24,25 The results were taken with three
replicates and analyzed with one way ANOVA.
Compatibility with PBMC
In vitro cytotoxicity assays of the samples were carried out on
PBMC.22 Goat blood was collected in citrated containers,
transported to the laboratory and diluted to 1 : 1 PBS, of which
9 mL was layered onto 6 mL of Ficoll-Paque. The suspension
was then centrifuged for 15 min at 400 6 g (1891 rpm). The
plasma PBS was carefully removed without disturbing the
interface. The interface was collected with a syringe and
diluted to 20 mL in RPMI-1640 (serum free cell culture
medium). PBMC were cultured in a 6 well plate (1 6 105 cells/
well) in RPMI-1640. The samples (0.1 mg mL21) were then
added to the cells and the culture was incubated for 24 h in a
CO2 incubator at 37 uC. Cells were stained with Trypan Blue
(diazo stain) and counted on a hemocytometer. Cells without
the treated sample were used as the control. The experiment
was done in triplicate.
A MTT assay was performed with the cultured PBMC to
evaluate the cell viability.11 Twenty microlitres of a sterile
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filtered 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT, Sigma Aldrich) stock solution in PBS at pH
7.4 (5 mg mL21) were added to each well. After 4 h of
incubation, the insoluble formazan crystals were dissolved in
DMSO (MERCK, India) and measured spectrophotometrically
in an ELISA reader (Thermo Scientific) at a wavelength of 570
nm. The relative cell viability (%) with respect to the control
wells containing cell culture medium without nanoparticles
was calculated by the absorbance of (test sample/control) 6
100.
Interaction with bovine serum albumin
To study the interaction of the fibrils (both pristine and
copper–copper oxide nanoparticle-coated nanofibrillar cellu-
lose) with BSA, 10 mg of the samples were incubated with 500
mL of BSA (1 mg mL21) for 16 h at 37 uC.20 After incubation,
samples were treated with buffer containing 60 mM Tris (pH
6.8), 25% glycerol, 2% sodium dodecyl sulfate (SDS), 14.4 mM
2-mercaptoethanol, 0.1% bromophenol blue (HIMEDIA,
INDIA) and boiled for 5 min, followed by centrifugation at
7155 6 g (8000 rpm) for 1 min at 4 uC. Electrophoresis was
performed using an Amersham Biosciences Gel system at a
constant voltage of 80 kV for 240 min. After electrophoresis,
the gel was stained with the Coomassie Brilliant Blue dye and
observed in a gel imaging system (Bio-Rad, USA).
Characterization
FTIR spectra of samples were recorded in a FTIR spectro-
photometer (Impact-410, Nicolet, USA). The dried fibers and
prepared nanohybrid were mixed with KBr pellets and pressed
in a compressor to obtain a transparent film, which was
analyzed with the spectrophotometer. UV-visible spectra of the
diluted suspensions of the nanohybrid were analyzed by a
Hitachi (U-2001, Tokyo, Japan) UV spectrophotometer. The
weight percentage of dried Cu–CuO loaded in the nanohybrid
was investigated by electron dispersive X-ray (EDX) JSM-
6390LV. An X-ray diffractometer (powder XRD), ‘Miniflex’
(Rigaku Corporation Japan), was used for the analysis of the
dry fibrils (cast on a glass slide) at room temperature (approx.
23 uC). The scanning rate of 5u min21 over the range of 2h =
10–80u was used. The size and dispersion of the nanoparticles
on the cellulose nanofibrils (dispersed in water and dried)
were determined by using a JEOL JEMCXII transmission
electron microscope (TEM) at an operating voltage of 200 kV.
Results and discussions
Isolation of NFC and formation of Cu NFC
Hydrogen peroxide is used extensively for bleaching lignocel-
lulosic materials.26 The lignin was partly removed from the
cellulosic fiber in this step. The alkali treatment accelerated
the removal of lignins whereas hemicellulose was partially
removed. Finally, the paracrystalline regions of the fibers got
hydrolyzed preferentially by the acid treatment, while the
crystalline regions were resistant to the acid attack.27 The use
of T. chebula extract is a greener trial to reduce the copper salt.
The alcoholic extract was well reported to have many
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Fig. 1 Probable mechanism of formation of the Cu–CuO nanoparticle-coated
NFC.
polyphenol compounds, where gallic acid is the major
component.28 These polyphenol compounds have a tendency
to form complexes with metal ions29 and, during the
complexation process, high amounts of electrons and protons
are released. This is schematically presented in Fig. 1, where
gallic acid was taken as the active component for the reduction
of copper acetate. The released electrons are responsible for
the reduction of Cu2+ to Cu0 but again, copper is oxidized in
the presence of atmospheric oxygen.30 Thus this mechanism
may demonstrate the formation of Cu–CuO nanoparticles, via
the mentioned greener route. The interaction of the nanopar-
ticles with the cellulose matrix was previously shown by Jia
et al.17
The percentage of Cu–CuO loaded in the prepared hybrid
was characterized with EDX analysis (ESI3 Fig. 1). The
percentage of copper was the highest in Cu NFC 3, while the
lowest was in Cu NFC 1 (Table 1). The O/C ratio in Table 1
indicates the presence of both Cu and CuO as with the
increase in the loading (%) of copper acetate, the oxygen
percentage also increased.
UV-visible absorption bands for Cu NFC were observed at
around 281 and 348 nm (ESI3 Fig. 2), which indicates a mixed
phase of Cu and CuO in the dispersion.30 The spectra were
taken after successive washings of the nanohybrid with
distilled water and ethanol. No significant decrease in the
absorption was found after this rinsing process.
FTIR studies are a potential tool in cellulose research.31 The
chemical changes in the cellulose fibers during the different
treatments are shown in Fig. 2 a. The peak observed for all the
samples at 1051 cm21 is assigned to the C–O–C stretching
vibration of the pyranose ring. The glycosidic ether compo-
nents seemed to be diminished gradually in the NFC because
of the loss of molecular weight during hydrolysis, which was
Table 1 EDX data and O/C ratio of the samples
Samples C (weight%) O (weight%) Cu (weight%) O/C ratio
NFC 44.09 55.91 0 1.26
Cu NFC 1 36.13 57.39 6.48 1.58
Cu NFC 2 30.01 59.18 10.18 1.97
Cu NFC 3 22.69 61.08 16.23 2.69
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Fig. 2 FTIR spectra of (a) base treated fibers (1), acid treated fibers (2) and acid
and base treated fibers (NFC) (3), (b) Cu NFC; and XRD patterns for (c) NFC and
(d) Cu NFC.
clearly indicated by the band sharpness at 1159 cm21. Only
NaOH treated fibers were showing the methoxy (O–CH3)
groups associated with the lignin at 1427 cm21, which
disappeared gradually during the acid treatment and finally
became negligible in the case of NFC.2 The absorption peak at
1610 cm21 is associated with the symmetrical stretching (CLC)
of the aromatic rings present in lignin.2 Thus, hemicellulose
was removed predominantly under the base treatment as
compared to the samples subjected to the acid treatment. The
characteristic C–H stretching vibration around 2919 cm21 was
common for all the three samples.
Mandal et al. recently reported that the absorption band at
902 cm21 continually increased during alkaline and acid
hydrolysis.4 A slight broadening of the peak associated with
the b-glycosidic linkages between glucose units in cellulose
can be observed in Fig. 2 a, from 898–912 cm21 in the base
treated cellulose to 878–927 cm21 in nanocellulose. This
indicates the formation of cellulose II.26
The FTIR study was also carried out for Cu NFC Fig. 2 b. A
broad band ranging from 3200–3600 cm21 is a clear indication
of adsorption of the polyphenolic compounds of the extract on
the surface of the nanohybrid.31 Bands at 1640, 1551 and 1419
cm21 correspond to the stretching vibrations of the carbonyl
groups and the aromatic ring of gallic acid, the major
component of the extract.32 The bands for Cu–O were observed
at around 461, 521 and 668 cm21.30
The diffractogram in Fig. 2 c shows well-defined peaks at
around 2h = 16.46u and 22.25u for the {110} and {200}
characteristic planes of cellulose.4 The X-ray diffractogram of
Cu NFC showed distinct peaks at 2h values of about 30.49u,
36.12u, 47.26u, 52.27u, 56.66u representing the {110}, {111},
{112}, {020} and {021} Bragg reflections of the monoclinic
structure (JCPDS data file 80-1917) of CuO (Fig. 2 d).33 Besides,
the formation of Cu nanoparticles was evident from the peaks
at 40.60u for the {111} plane of Cu (fcc) (JCPDS data file 85-
1326).
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Fig. 3 TEM images of NFC (a,b) and Cu NFC 2 (c,d).
TEM micrographs of the diluted suspensions of both NFC
and Cu NFC are shown in Fig. 3 a–d. The diameters of the
pristine fibers, in the range of 12–36 nm, confirmed the
formation of nanofibrillar cellulose Fig. 3 a,b.34 In the case of
the Cu NFC, the copper–copper oxide nanoparticles were
distributed on the surface of NFC. The size of the Cu–CuO
nanoparticles in the matrix varied in the range of 3–12 nm
(Fig. 3 c,d).
Antioxidant activity
It was interesting to probe the antioxidant potency of the
prepared nanohybrids. The antioxidant activity of the green
nanohybrides was profound (Fig. 4), while Cu NFC 4 showed
poor DPPH scavenging potency. This clearly indicated that the
polyphenol compounds present in the T. chebula extract were
adsorbed on the surface of the nanohybrids and that the
antioxidant activity was due to the interaction of such
Fig. 4 DPPH scavenging assay.
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polyphenols with DPPH.10 In the case of sample Cu NFC 4,
prepared by the alkali method, such surface activity was absent
and this is reflected in the results of the scavenging activity.
Thus, Cu NFC 4 was not taken under consideration for further
experiments.
Antimicrobial efficacy
Metal nanoparticles have a strong antimicrobial activity, as
reported earlier.35–39 Copper and copper oxides are common
antimicrobial agents in a large number of commercial
products like Nordox, Super KL K90 Red etc.9 The antimicro-
bial efficacy of NFC and Cu NFCs is presented in Fig. 5. NFC
did not show any effect against both bacteria and fungi,
whereas Cu NFC 2 exhibited a remarkable effect against both
Gram positive and Gram negative bacteria as well as for the
fungus C. albicans (Table 2). Cu–CuO nanoparticles were
distributed on the surface of the nanofibrillar cellulose. These
nanoparticles basically impart antimicrobial activity to the
nanohybrid system, as evident from Table 2. The activity
increases with the increase of the Cu–CuO loading. The
mechanism of action of copper on microbes is basically due to
the adsorption of Cu on the surface of the bacterial cells.40
Moreover, Cu ruptures the bacterial cell membrane, which
leads to the solidification of the structural proteins.40
However, a different effect was observed for Gram positive
and Gram negative bacterial species. This may be ascribed to
the differential expression of molecular moieties at the surface
of the respective groups.10
The nanohybrid showed more antimicrobial activity than
the bare copper nanoparticles. This may be due to the
stabilization of the nanoparticles in the cellulose matrix,
which prevented the agglomeration of the nanoparticles.
Hemolytic assay
The biocompatibility of NFC and Cu NFC was investigated by
the RBC hemolysis protection assay (Fig. 6). The RBC
protection assay showed that NFC, Cu NFC 1 and Cu NFC 2
possessed good compatibility with the erythrocytes. However,
lysis of the RBC membrane was observed for Cu NFC 3. As
evident from the reported literature, an excess of copper is
cytotoxic as it interferes with various cellular events and
initiates oxidative damage.41 Tween 20 showed multifold
destruction of the RBC membrane against the negative control
haematocrit, as evident from the comparatively large value of
hemoglobin absorbance. Cu NFC 1 and Cu NFC 2 showed
excellent compatibility with the RBCs, vouched by the
absorbance of hemoglobin almost parallel to that of haema-
tocrit. This supports that the bio-interfacial action of the
nanomaterials is a concentration and surface chemistry
dependent feature. This calls for a compilation of toxicity
profiles of the nanomaterials (including those prepared
through greener routes) prior to their commercialization.
Compatibility with PBMC
Peripheral blood mononuclear cells (PBMC) are blood cells
with a round nucleus, such as lymphocytes, monocytes,
macrophages etc.22 These blood cells have a critical role in
the immune system and wound contraction.42 Thus, it is
desirable to undertake a compatibility assessment of the
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Fig. 5 Representative antimicrobial activity of Cu NFC 2 against Staphylococcus aureus, Escherichia coli and Candida albicans.

Table 2 Antimicrobial activity of Cu NFC performed to confirm this observation. It was found that the
cell survival rate for Cu NFC 2 is equivalent to that of the
Test organisms S. aureus E. coli C. albicans control (Fig. 8). The aforementioned bioassays ascertained
that Cu NFC 2 possesses good antimicrobial activity as well as
Cu 0 14.8 ¡ 0.3 18.16 ¡ 0.2
NFC 0 0 0 excellent biocompatibility. Thus, this nanohybrid is a good
Cu NFC 1 20.2 ¡ 0.18 24.4 ¡ 0.3 22 ¡ 0.1 candidate for its application in biomedical bandages.
Cu NFC 2 21.93 ¡ 0.06 25.96 ¡ 0.15 22.9 ¡ 0.17
Cu NFC 3 22.86 ¡ 0.15 26.06 ¡ 0.2 25 ¡ 0.17 Interaction with BSA
Antibiotic 31.9 ¡ 0.2 31.8 ¡ 0.1 30 ¡ 0.15
Protein–nanoparticle interaction studies are an emerging field
in nanomedicine and nanotoxicology.19 Nanomaterials with a
high surface area can absorb the dynamic layer of proteins
nanomaterials with PBMC, which may reveal their potential to when they interact with living systems. Kathiravan et al.
be used in advanced biomedical applications. The biocompat- recently studied the interaction of BSA, a physiologically
ibility of the prepared nanohybrids (Cu NFC) was examined important protein, with metal nanoparticles.20 In yet another
through their interaction with PBMC. It is quite clear from report, Ravindran et al. concluded that the interaction of BSA
Fig. 7 that there is no change in the morphology of the cells with silver nanoparticles prevented aggregation, which was
after treating with Cu NFC 1 and Cu NFC 2. Cell survival was confirmed by spectroscopic methods.43 In this study, we
found to be almost the same as compared to the control but delved into the interaction of the same protein with NFC and
cell death was observed for Cu NFC 3. A MTT assay was Cu NFC 2 by gel electrophoresis (Fig. 9). BSA gives a band at 66
KDa and single bands were observed in all the three lanes for
both the samples and the control. This clearly indicated that
the surface charge of BSA is not disturbed by its interaction

Fig. 6 Anti-hemolytic activity of NFC and Cu NFCs. Fig. 7 Microscopic images of Trypan Blue stained PBMC treated with Cu NFCs.

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Fig. 8 Cell survival test by the MTT assay.
Fig. 9 SDS-PAGE images of control, Cu NFC 2 and NFC.
with NFC and Cu NFC 2, which is evident from their similar
migration pattern on the gel.
Conclusions
Nanofibrillar cellulose was isolated from Colocasia esculenta
stems by a chemical method and copper–copper oxide
nanoparticles were coated on the surface of the nanofibrils.
The best hybrid system (Cu NFC 2) exhibited antimicrobial
activity against Gram positive and Gram negative bacteria as
well as against a fungal species. Cytotoxicity tests proved that
the nanohybrid was quite compatible with RBCs and PBMC.
No degradation of BSA, the most abundant protein, was
observed upon interaction with the hybrid system. Thus, this
bio-resource based nanohybrid system holds potential for
utilization in various biomedical applications including as an
advanced wound dressing material. Further detailed studies
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with animal models would be able to establish the exact
toxicity profile for the reported nanohybrid.
Acknowledgements
The authors express their gratitude to NRB for financial
assistance through the grant no. DNRD/05/4003/NRB/251
dated 29.02.12, SAP (UGC), India through the grant no. F.3-
30/2009(SAP-II) and FIST program-2009 (DST), India through
the grant no. SR/FST/CSI-203/209/1 dated 06.05.2010. RSIC,
NEHU, Shillong is acknowledged for the TEM imaging. The
authors are thankful to Mr. Dipankar Kalita, Food Engineering
technology, Tezpur University for technical support.
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