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Published in IET Nanobiotechnology
Received on 22nd July 2013
Revised on 24th October 2013
Accepted on 29th October 2013
doi: 10.1049/iet-nbt.2013.0052

ISSN 1751-8741

Biosynthesis and wound healing activity of copper

nanoparticles
Mradul Tiwari, Kasinathan Narayanan, Mitali B. Thakar, Hitesh V. Jagani,
Josyula Venkata Rao
Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal
576104, India
E-mail: rao.josyula@gmail.com

Abstract: Nanotechnologies reinvented the utilities of various substances in healthcare. Copper in its native form (copper ion) has
been well studied for its antimicrobial and anti-inflammatory activities. Copper in its nano form could have better biological
profile and finds many applications in healthcare. There were reports on synthesis of copper nanoparticles by physical and
chemical methods and their biological activities, although these methods have limitations. Biosynthesis of nanoparticles using
microbes is an ecofriendly approach helping in the synthesis of biocompatible and stable nanoparticles. With this background
in mind, the present study was designed to synthesise copper nanoparticles by Pseudomonas aeruginosa and testing their
efficacy in enhancing the pace of wound healing. Culture supernatant was used to synthesise copper nanoparticles. Optimum
conditions were selected to maximise the biosynthesis of nanoparticles. Biosynthesised copper nanoparticles (BNCPs) were
characterised by Malvern zeta sizer and scanning electron microscopy. Average particle size, polydispersivity index and zeta
potential of BNCPs were found to be 110.9 nm, 0.312 and (−) 18.3 mV, respectively. BNCPs was evaluated for its wound
healing activity by excision wound model in rat. The pace of wound healing was enhanced by BNCPs compared with copper
in native form.

1 Introduction reducing agents and/or reductase-type enzyme present

Copper is well known for its antimicrobial activity and finds
application in wound healing, skin remodulation and
anti-inflammatory therapies [1]. Antimicrobial activity of
copper helps to reduce the microbial load at the site of
wound and enhances the pace of healing. Copper serves as
a cofactor for enzymes such as superoxide dismutase and
cytochrome oxidase [2]. It also augments the immunity by
stimulating the production of interleukin-2 [3]. Copper
induced expression of vascular endothelial growth factor
(VEGF) is known to stimulate angiogenesis in wounds [4].
These factors help in wound healing by enhancing
angiogenesis, anti-inflammatory and immunity power [5].
To enhance and for betterment of copper activity in wound
healing, it would be a viable approach to convert native form
(copper ion) into nano form. Copper nanoparticles (CNPs)
may have more catalytic activity and better bioavailability
compared with its native form. Metal nanoparticles
synthesised by chemical or physical methods are not
economic and safe for ‘in vivo’ use [6]. Biosynthesis of
metal nanoparticles using microorganisms is a new
emerging nanobiotechnology technique. It is a green,
ecofriendly, single-step bottom–up approach for synthesis
of metal nanoparticles (Fig. 1). It gives stable, mono-
dispersible metal nanoparticles with exquisite morphology
at ambient conditions [7, 8]. Biosynthesis of metal
nanoparticles in living system is catalysed by various
intracellularly or extracellularly [8, 9]. Metal nanoparticles
synthesised by microorganisms are stabilised by coating
of low-molecular weight biomolecules around the
nanoparticles [10].
Biosynthesis of metal nanoparticles using cell-free
supernatant of culture is economic, easier and beneficial
compared with whole cell. Here, cell-free supernatant of
culture with maximum extracellular biological active
component is needed to be used in this process [11].
Biosynthesis of nanoparticles depends on the enzyme
activity and can be improved by selecting optimum
conditions.
Although many reports are available in literature on the
biosynthesis of gold and silver nanoparticles, only a few
works have been reported on biosynthesis of CNPs.
Serratia species [12] Morgenella species [13],
Pseudomonas stutzeri [14] and Fusarium oxysporum [15]
are the microorganisms reported for the biosynthesis of
CNPs. The present study was taken up after the previous
work, initiated in our lab 5 years before on CNPs
synthesised by physical and chemical methods, had shown
promising antimicrobial activity. The main objective of the
present study is to synthesise CNPs by green synthesis
approach and testing their efficacy in enhancing the pace of
wound healing. For this, different industrial strains and soil
isolates were screened for selecting a suitable bacterium.
Apart from selecting a suitable bacterium for biosynthesis

230 IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 230–237

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Fig. 1 Steps involved in biosynthesis of metal nanoparticles
Biosynthesis of stable metal nanoparticles involves two types of
biomolecules; one is a reducing agent which reduce ion into atom and
another is capping agent to stabilise them at nano level
of CNPs, an attempt was made to optimise the conditions for
enhancing the enzyme activity involved in the synthesis of
CNPs. In the present study, biosynthesis of CNPs was
carried out using cell-free supernatant of culture of
Pseudomonas aeruginosa. Extracellular enzyme activities
were measured and optimum conditions required for the
biosynthesis of CNPs were selected. Biosynthesised CNPs
(BNCPs) were subjected for antimicrobial and wound
healing activity.
2 Materials and methods
2.1 Materials
All chemicals and media were obtained from Merck Ltd.
Mumbai (India) and Himedia lab Pvt. Ltd., Mumbai
(India). All chemical used were of analytical grade.
2.2 Cultures and cell lines
Escherichia coli (NCIM 5011), Bacillus subtilis (NCIM
2063), P. aeruginosa (NCIM 2036) and Staphylococcus
aureus (NCIM 2079) were procured from National
Collection of Industrial Microorganisms (Pune, India). The
cultures were grown, maintained and subcultured on Muller
Hinton Agar and stored at 4°C. HaCaT (human skin
keratinocytes) and Hep-G2 (human hepatocellular liver
carcinoma) cell lines were purchased from the National
Centre for Cell Sciences (Pune, India).
2.3 Animals
Animal care and handling were according to the Committee
for the Purpose of Control and Supervision of Experiments
on Animals guidelines. Approval for animal experiments
was obtained from the Institutional Animal Ethics
Committee (Clearance certificate no. IAEC/KMC/51/
2010-2011). Five- to- six-month-old Wistar albino rats of
either sex weighing 150–200 g were selected from an
inbred colony maintained under the controlled conditions of
temperature (23 ± 2°C), humidity (50 ± 5%) and light (12 h
each of light and dark cycle) at Central Animal Research
Facility, Manipal University, Manipal, India. The animals
were provided with standard rat food and water ‘ad
libitum’. Each animal was housed in separate polypropylene
cage containing paddy husk as bedding.
2.4 Preparation of cell-free supernatant
P. aeruginosa was cultured in Luria–Bertani (LB) broth for
36 h at 150 rpm and 37 °C in orbital shaker (Scigenics
Biotech Pvt. Ltd. Chennai, India). Cell-free supernatant of
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culture was prepared by passing the culture through 0.22
µm membrane filter [6].
2.5 Estimation of enzyme activity in cell-free
supernatant of culture
Extracellular nitrate reductase activity of cell-free supernatant
of culture was calculated by measuring the amount of nitrite
produced after 1 h reaction with potassium nitrate [11].
Similarly nitrate and nitrite reductase activities together
were calculated by measuring the amount of ammonia
produced [16, 17]. Ammonium molybdate was used in
growth medium to study the effect of molybdenum on
enzyme activity [18, 19].
2.6 Selection of optimum conditions for
biosynthesis
Biosynthesis of CNPs depends on various factors like pH,
temperature, rpm and concentration of the copper ion.
Cell-free supernatant of culture was exposed to different pH
(5.0, 7.0 and 9.0), temperatures (28, 37 and 45 °C) and rpm
(100, 150 and 200 rpm) by using 1 mM final copper
sulphate concentration to select optimum conditions for the
synthesis of CNPs. Similarly different copper sulphate
concentrations (0.5, 2.0 and 4.0 mM) were studied at
normal conditions [pH (7.0), temperature (37 °C) and rpm
(150)]. Change in the colour of reaction mixtures and
surface plasmon resonance (SPR) of synthesising
nanoparticles were considered for selecting the optimum
condition [6]. Change in colour was examined visually and
change in SPR was determined spectrophotometrically.
2.7 Biosynthesis of CNPs
P. aeruginosa was cultured in LB broth with ammonium
molybdate. The flask was harvested to get the cell-free
supernatant of culture after maximum growth. After
adjusting the pH of cell-free supernatant of culture at 6.5,
equal volume of 4 mM copper sulphate solution was added.
The flask was kept in orbital shaker at 45 °C and 200 rpm.
Synthesis of CNPs was monitored by measuring SPR at
different time intervals. Cell-free supernatant of culture
without copper was used as blank [11].
2.8 Characterisation of BNCPs
The mean particle size, particle size distribution and
polydispersivity index (PDI) of nanoparticles were
determined after re-dispersion of nanoparticles in water
treated with diethylpyrocarbonate (DEPC) by dynamic light
scattering using the photon correlation spectroscopy
technique at 25 °C on a Malvern Nano ZS (Malvern
Instruments Ltd., Worcestershire, UK) equipped with a 633
nm laser and 173° detection optics. The zeta potential of
the re-dispersed nanoparticles was measured via the LASER
Doppler electrophoresis technique at 25 °C using a Malvern
Nano ZS. Based on the measured conductivity of the
sample, the voltage used for zeta-potential measurement
was selected automatically. Malvern DTS v.5.00 software
(Malvern Instruments Ltd., Worcestershire, UK) was used
for data acquisition and analysis. The morphology of the
nanoparticles was determined using scanning electron
microscopy (SEM). To prepare smear, the nanoparticles
were suspended in DEPC-treated water (5 mg/ml) and dried
nanoparticles were visualised using a SEM with
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energy-dispersive X-ray spectroscopy (EDX) DS (JEOL,
Akishima, Tokyo, Japan). Potassium bromide pellet of the
BNCPs was made and Fourier transform infrared
spectroscopy (FTIR) spectrum was recorded using 8310
FTIR (Shimadzu, Japan). X-ray diffraction (XRD) pattern
measurement of dried powder of BNCPs was done by
MiniFlex 600 (Rigaku, Tokyo, Japan).
2.9 Antibacterial and cell viability assay of BNCPs
Standard inocula of B. subtilis, E. coli, S. aureus and
P. aeruginosa were prepared in saline having 25%
transmittance. Antibacterial activity of BNCPs was
compared with copper native by agar diffusion and broth
dilution method [20]. ‘In vitro’ cell viability assay was done
on HaCaT and HepG2 cell lines and compared with native
copper by MTT assay [21].
2.10 Formulation development
Gel formulations were developed using 1% carbopol 934P for
1% copper native and 0.1% BNCPs along with a control gel.
Developed gels were subjected to evaluation for pH,
homogeneity, viscosity, drug content and drug release
profile [22].
2.11 In vivo excision wound model
Animals were anaesthetised by ketamine HCl intraperitoneal
injection (3 ml/kg). The rats were shaved at the back just
below the neck. Stamp marks of 500 mm2 were placed to
give the excision by removing whole skin in border of the
stamp. Animals were divided into four groups containing
six animals each. Group I served as control (untreated) and
Group II served as control gel. Group III received copper
native gel (1.0%) and Group IV BNCPs gel (0.1%). The
wounds were covered with gel formulation in Groups II–IV
with 100 mg of respective gel. The formulations were
applied daily. The progressive changes in wound area were
monitored planimetrically by tracing the wound margin on
a graph paper every alternate day. Wound contraction was
expressed as percentage reduction of original wound size.
%Wound contraction = (Wound area on day 0
− Wound area on dayn ) × 100/Wound area on day 0
Falling of scab and leaving no raw wound behind was taken
Fig. 2 Nitrate and nitrite reductase activity
Enzyme activity(U/ml) in cell-free supernatant of P. aeruginosa culture with and without molybdenum for
a Nitrate reductase
b Nitrite reductase
**P < 0.01 when enzyme activities were compared with the presence of molybdenum from without molybdenum
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as the endpoint of complete epithelialisation and the days
required for this were taken as period of epithelialisation [23].
3 Results and discussion
3.1 Estimation of enzyme activity in cell-free
supernatant of culture
Nitrate reductase and nitrite reductase activities were measured
in cell-free supernatant of P. aeruginosa culture. It was found
that nitrite reductase activity was greater than the nitrate
reductase activity and the presence of molybdenum-enhanced
extracellular enzyme activities (Fig. 2).
Nitrate reductase is an important enzyme involved in the
biosynthesis of metal nanoparticle [11]. Molybdenum is an
important cofactor that enhances nitrate reductase activity.
However, the activity of nitrite reductase is independent of
the presence of molybdenum. In the present experiment,
when molybdenum was added at previously reported level
[18, 19], the activity of nitrate reductase increased by 96%
and there was significant increase in nitrite reducatase
activity.
3.2 Selection of optimum conditions for
biosynthesis
Factors affecting biosynthesis of CNPs were evaluated
(Fig. 3). Optimum conditions for biosynthesis are
represented in Table 1. Maximum SPR and change in
colour represents maximum synthesis of CNPs in cell-free
supernatant of culture [24]. Acidic condition decreases the
rate of reduction of copper ion into copper atom and results
only in slight change in the SPR and colour. Alkaline
condition precipitates copper and makes copper ion
unavailable for reduction. However, a neutral condition
favours maximum reduction of copper ions and provides
maximum synthesis of CNPs. Change in temperature and
rpm shows direct relation on synthesis of CNPs. Metal ion
concentration plays an important role in the biosynthesis of
nanoparticles. Reduction of metal ions does not occur at
low concentration (0.5 mM). However, at a high
concentration (4.0 mM) copper ion reduces at a faster rate
resulting in the formation of macroparticles which
ultimately precipitates out [6, 25].
3.3 Biosynthesis of CNPs
The SPR spectrums of synthesising CNPs at different time
intervals were recorded (Fig. 4a). Colour change because of
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300–310 nm in ultraviolet range [15, 26] and 570–630 nm
in visual range [13, 14]. In the current experiment, we
found that the magnitude of SPR absorption in ultraviolet
range was more as compared with visible range. Therefore
further experiments were carried out by measuring SPR in
ultraviolet range.

3.4 Characterisation of BNCPs

Using Malvern zeta sizer, mean particle size, PDI and zeta
potential were measured and found to be 110 nm, 0.314 and
(−) 18.1 mV, respectively, and the intensity graph of
particles showed narrow range of size distribution (Fig. 5c).
Fig. 3 Selection of optimum condition of a factor affecting SEM image (Fig. 5a) of synthesised CNPs showed that
biosynthesis most of the nanoparticles were spherical in shape. Typical
SPRs of synthesising CNPs were measured at three different pH (5.0, 7.0 and spot analysis (Fig. 5b) of CNPs confirms the presence of
9.0), temperatures (28, 37 and 45 °C), rpm (100, 150 and 200 rpm) and copper along with protein.
copper sulphate concentration (0.5, 2.0 and 4.0 mM) FTIR spectra of BNCPs was recorded and the presence of
protein was confirmed by a sharp peak of amide at 1642
cm−1, a broad peak of N–H stretching at 3415 cm−1 and a
peak of N–H bending at 1362 cm−1 (Fig. 5d ). In XRD
Table 1 Optimisation of conditions for biosynthesis of CNPsa spectra of BNCPs, four characteristic peaks at 2θ values of
31.398°, 45.155°, 56.197° and 75.062° corresponding to
S. no. Factor Optimum Colour SPR crystal facet (110), (111), (200) and (220) planes of copper
condition change
were observed (Fig. 6).
1 pH 6.5 green 1.391 ± 0.094 SEM image showed that even though the BNCPs appeared
2 temperature, °C 45 green 1.463 ± 0.101 as aggregates, a close examination revealed that they were
3 rpm 200 brownish 1.624 ± 0.134 uniform individual spherical particles separated from each
green other without any direct contact. This indicates stabilisation
4 metal ion 2.0 dark green 1.617 ± 0.136
concentration,
of nanoparticles with a capping agent [11]. EDX analysis of
mM the sample confirmed the presence of elemental copper [9].
control – – green 1.285 ± 0.086 As reported earlier, the presence of protein shows that these
proteins play a pivotal role in stabilising nanoparticles
a
Values are average of three determinations and prevent these nanoparticles from aggregating [27–29].
XRD spectrum contains face centric crystal pattern of
copper. Each crystallographic facet contains energetically
synthesis of CNPs was clearly visible from yellowish distinct sites based on atom density. CNPs contain high
supernatant to green (Figs. 4b and c). The presence of atom density facets such as (111) that are known to be
active biomolecules in cell-free supernatant of culture tends highly reactive [20]. Further studies on characterisation of
to reduce copper ion into copper atoms which aggregates to these proteins could help in developing a good stabilising
form nanoparticles [10, 15, 26]. Synthesis of nanoparticles agent.
is confirmed by change in SPR of reaction mixture. This PDI of BNCPs was meeting the uniformity criteria for
serves as indicator for the production of nanoparticles. particle size distribution. This shows that biosynthesis of
During the formation of nanoparticles, the colour changed CNPs by this organism had given good stable uniformly
from blue to green. Imax for the BNCPs is reported to be at distributed nanoparticle. Also, the evaluation of zeta

Fig. 4 Biosynthesis of CNPs in cell-free supernatant of culture of P. aeruginosa

a SPR of BNCPs in culture supernatant of P. aeruginosa at different incubation times
b Test tube with sterile medium (A), sterile medium and CuSO4 (B), cell-free supernatant of culture without CuSO4 (C) and cell-free supernatant of culture and
CuSO4 after 24 h (D)
c Flask with cell-free supernatant of culture and CuSO4 after 50 h

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Fig. 5 Characterisation of BNCPs

a SEM image of nanoparticles formed after exposing cell-free supernatant from P. aeruginosa to CuSO4
b Typical spot-profile EDX analysis for one of the CNPs shown in Fig. 5a
c Size distribution graph of BNCPs, where d.nm. is the diameter of nanoparticles in nanometre
d FTIR spectra of BNCPs

potential of BNCPs showed that these biosynthesised
nanoparticles have required potential charge needed for any
nanoparticle to be used ‘in vivo’ [30]. Besides, the negative
charge indicates that these particles will be stable in
solution and therefore problems related to stability during
reconstitution or during biodistribution would be avoided.
More than 94% of the particles were distributed in the
range of 50–300 nm. It is interesting to note that previous
works on BNCPs have reported a similar size distribution
[15, 26]. This shows that microbial synthesis of CNPs
would give a consistent size range, which is very essential
before proceeding with further purification and formulation
works. After 3 months of incubating BNCPs solution under
normal conditions, we did not note any precipitation in the
nanoparticles solution and no change in its SPR and
antimicrobial activity.
3.5 Antibacterial and cell viability assay of BNCPs
BNCPs (100 µg/ml) had better antimicrobial efficacy as
compared with native copper (200 µg/ml) by agar diffusion
(Table 2) and broth dilution (Table 3) methods. BNCPs was
more active against Gram-positive bacteria when compared
with Gram-negative bacteria. B. subtilis was found to be
highly sensitive whereas E. coli was least sensitive. The
difference in susceptibility between Gram-positive and
Gram-negative organism could be because of the basic
difference in the cell wall structure between these two
classes. Copper induces cytoplasmic leakage by rupturing

Table 2 Antimicrobial activity by agar diffusion method

Organism Zone of inhibition, mm

Native copper BNCPs Ciprofloxacin (2

(200 µg/ml) (100 µg/ml) µg/ml)

B. subtilis 10 19 29
P. aeruginosa 10 15 28
S. aureus 12 18 29
E. coli 8 12 23
Fig. 6 XRD pattern of BNCPs

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Table 3 Antimicrobial activity by broth dilution method are widely used for treating wound and skin infections [21].
Only few studies on possible application of native copper
Organism Range of minimum inhibitory concentration,
µg/ml and CNPs as topical application have been reported even
though documented evidence shows that copper enhances
Native copper (200 µg/ml) BNCPs (100 µg/ml) the pace of wound healing. In the current study, the amount
of native copper to be used in the formulation was decided
B. subtilis >100, but ≤200 >25, but ≤50 based on the currently available silver ointments as there
P. aeruginosa >100, but ≤200 >50, but ≤100 were no reports on copper ointment. Copper nanoparticles
S. aureus >100, but ≤200 >25, but ≤50
E. coli >100, but ≤200 >50, but ≤100 were used at one-tenth of native copper dose as it was
expected that BNCPs will have a better bioavailability and
better absorption [33]. This will also ensure that copper
toxicity would be avoided. In the current study, a higher
release profile was observed with native copper. This could
Table 4 Cell viability assaya be because of the higher solubility of copper ions in buffer.
Drug CTC50, µg/ml In case of BNCPs, the copper is coated with a capping
agent. Therefore, when compared with native copper
HaCaT HepG2 distributed within a gel, BNCPs is subjected to two rate
step limitation, that is, first diffusion from gel into the
copper native 10.22 ± 0.27 9.87 ± 0.34 buffer and second from the capping agent into the buffer.
BNCPs 17.55 ± 0.45 11.94 ± 0.41
However, once BNCPs was released from the formulation,
a the same capping agent would help in transportation across
Values are average of three determinations
the biological membrane.

cell wall [20]. Copper have higher affinity towards protein

when compared with lipids. Peptidoglycan and protein
content in the cell wall of Gram-positive bacteria are higher 3.7 Excision wound model
when compared with Gram-negative bacteria. Therefore
Gram-positive organisms would be easily destroyed when BNCPs showed a significant role in wound healing process in
copper is added. Biosynthesised metal nanoparticles have the earlier stage as represented in Fig. 7. On the second day of
better diffusion compared with native ions. Once reaching drug application, percentage healing in the BNCPs gel-treated
the cell surface, CNPs would be diffused from protein coats animals was twice than in the control group of animals and
to the bacterial cell interior resulting in interlinking of cell significantly better than in the control gel- and the copper
surface proteins and ultimately cell lysis [31, 32]. In the native gel-treated animals. As the days progressed, the pace
present experiment, a Gram-negative organism, that is, of wound healing with control gel was similar to control
P. aeruginosa was used in the biosynthesis of CNPs. This animal and lesser in native copper gel-treated group of
itself shows that Gram-negative organisms are either animals. After tenth day of excision, BNCPs treatment
inherently resistant to CNPs or could protect themselves
from copper toxicity by coating the copper particles with a
suitable material rendering them intoxic against them.
However, still such CNPs could be toxic for other class of
organisms viz. Gram positive. Further experiments on the
role of biosynthesis of CNPs by Gram-positive bacteria
could help in understanding this hypothesis.
CTC50 (concentration of the sample required to kill 50% of
the cells) of the native copper was found to be less than the
BNCPs in case of normal (HaCaT) as well as in cancerous
(HepG2) cell line (Table 4). A total 30 µM (7.5 µg/ml)
copper sulphate is required for stimulation of VEGF for
wound healing process [4]. Any formulation to be used ‘in
vivo’ should be safe. Studies on HaCaT, a normal cell line,
showed that BNCPs had higher margin of safety when
compared with native copper [22]. However, on HepG2, a
liver carcinoma, the cytotoxicity of both BNCPs and native
copper was close to each other. Further studies are needed
to explore the findings.

3.6 Formulation development

All the gels prepared were transparent and free from

particulate matter. Spreadability, pH, viscosity and
homogeneity were found to be optimum for topical
application. Drug content in copper native and BNCPs gel
was found to be 98 and 97%, respectively. Release of
copper after 24 h was found to be 68.19% for copper native
and 51.96% for BNCPs gel. Silver as topical formulations
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Fig. 7 Pace of wound healing in rats treated with BNCPs and
native copper
Effect of BNCPs and native copper on enhancement of pace of wound
healing in rats was checked by excision wound model. n = 6 and the values
are expressed as mean ± SEM. Statistical analysis of data was carried out
by one-way analysis of variance followed by Dunnett’s test. *P < 0.05,
**P < 0.01, ns = not significant, when other groups were compared with
Group I
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reduced the wound size by 92%. After 15th day of excision,
no significant difference was observed.
Wounds created in both normal control and gel control
healed at the same pace. The pace of wound healing in
BNCPs-treated animals was faster compared with the
control group. In case of native copper-treated animals,
wound healing was slow. Concentration of native copper in
native gel was 1%. This was prepared based on the
marketed silver containing ointments as there were no
previous reports on copper-based topical formulation. In
case of gel formulation containing BNCPs, the
concentration of BNCPs was 0.1%. This was based on the
assumption that generally nanoparticles have better
pharmacokinetic properties compared with their native form
and therefore the therapeutic efficacy could be achieved at a
lower dose than its native form. Therefore in the present
experiment BNCPs was included in the formulation at
one-tenth of native form dose. During the course of the
study it was found that the native copper had actually
delayed the wound healing even though BNCPs had
remarkably enhanced wound healing compared with the
control. This could be because of the toxicity of native
copper at the dose used even though this amount appears to
be safe for silver. In case of BNCPs, the pace of wound
healing was faster compared with other. Copper plays an
important role in angiogenesis [4]. Also they have good
anti-inflammatory activities [2]. Besides, the present study
showed promising antimicrobial activity especially against
Gram-positive organisms. These properties along with the
ability of copper to stimulate interleukin-2 production ‘in
vivo’ ensure that damaged tissues are taken care of and the
wound is healed in a shorter time [5]. Further experiments
are needed for determining the exact dose of native copper
for treating wounds which would help in comparing the
effectiveness of BNCPs in wound healing.
4 Conclusion
In the field of nanobiotechnology, it is needed to synthesise
biocompatible, uniform and stable metal nanoparticles to
increase their application in the medical science. In the
present work, CNPs were synthesised by green synthesis
method using P. aeruginosa. The BNCPs were uniform in
size distribution and had exquisite morphology. These
BNCPs were safe on normal cell lines and gel-containing
BNCPs enhanced wound healing in rats. These BNCPs
could be further evaluated for pharmacokinetic properties
and could be studied for possible application in other
pathological conditions.
Conflict of interest
The authors declare that there is no conflict of interest with
any financial organisation regarding the materials discussed in
this manuscript.
5 Acknowledgment
We wish to acknowledge the authorities of Manipal College
of Pharmaceutical Sciences, Manipal University, DST-FIST
and AICTE for providing necessary facilities to carry out
the research work.
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