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ORIGINAL ARTICLE
Chitin membranes containing
silver nanoparticles for
wound dressing application
Rita Singh, Durgeshwer Singh
Singh R, Singh D. Chitin membranes containing silver nanoparticles for wound dressing application. Int Wound J
2012; doi: 10.1111/j.1742-481X.2012.01084.x
ABSTRACT
Silver nanoparticles are gaining importance as an antimicrobial agent in wound dressings. Chitin is a biopolymer
envisioned to promote rapid dermal regeneration and accelerate wound healing. This study was focused on the
evaluation of chitin membranes containing silver nanoparticles for use as an antimicrobial wound dressing. Silver
nanoparticles were synthesised by gamma irradiation at doses of 50 kGy in the presence of sodium alginate as
stabiliser. The UV–Vis absorption spectra of nanoparticles exhibited an absorption band at 415–420 nm, which is
the typical plasmon resonance band of silver nanoparticles. The peaks in the X-ray diffraction (XRD) pattern are
in agreement with the standard values of the face-centred cubic silver. Transmission electron microscopy (TEM)
images indicate silver nanoparticles with spherical morphology and small particle size in the range of 3–13 nm.
In vitro antimicrobial tests were performed using Pseudomonas aeruginosa and Staphylococcus aureus to determine
the antimicrobial efficiency of the chitin membranes containing 30, 50, 70 and 100 ppm nanosilver. No viable
counts for P. aeruginosa were detected with 70 ppm silver nanoparticles dressing after 1-hour exposure. A 2-log
reduction in viable cell count was observed for S. aureus after 1 hour and a 4-log reduction after 6 hours with
100 ppm nanosilver chitin membranes. This study demonstrates the antimicrobial capability of chitin membranes
containing silver nanoparticles. The chitin membranes with 100 ppm nanosilver showed promising antimicrobial
activity against common wound pathogens.
Key words: Antimicrobial activity • Chitin • Silver nanoparticles • Wound dressing
INTRODUCTION
Wounds often provide a favourable environment for the colonisation of micro-organisms.
High bacterial levels interfere with the progression of wound healing (1). Infection is a leading
cause of morbidity and mortality in extensive
burn injuries, traumatic injuries and surgical
procedures. The control of infection remains
a major challenge in wound management. In
order to improve the opportunity for wound
healing, it is important to create conditions
Authors: R Singh, PhD, Defence Laboratory, Defence Research
& Development Organization, Jodhpur, India; D Singh,
MSc, Defence Laboratory, Defence Research & Development
Organization, Jodhpur, India
Address for correspondence: Dr Rita Singh, PhD, Defence
Laboratory, Defence Research & Development Organization,
Jodhpur 342 011, India
E-mail: [email protected]
that are unfavourable to micro-organisms and
favourable for the host repair mechanisms.
Consequently, broad-spectrum antimicrobial
agents are required to control infection and
facilitate the healing process.
Silver has been used for centuries in the
form of metallic silver, silver nitrate and silver sulfadiazine for the treatment of burns,
wounds and several bacterial infections (2).
The increasing level of bacterial resistance
to traditional antibiotics and the isolation of
organisms with minimal antibiotic sensitivity are driving the current interest in silver
products for chronic wound care. Ionic silver is a potent antimicrobial agent and its
use in health care has increased in recent
years as a consequence of escalating bacterial
resistance to antibiotics. The proven antimicrobial activity of ionic silver includes its
© 2012 The Authors
International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc doi: 10.1111/j.1742-481X.2012.01084.x
Key Points
• high bacterial levels interfere
with the progression of wound
healing
• control of infection remains
a major challenge in wound
management
• nanosilver dressings can play an
important role in the healing of
wounds due to their antimicrobial effects
• the present study was focused
on the preparation and evaluation of chitin membranes containing silver nanoparticles for
use as an antimicrobial wound
dressing
1
Chitin membranes for wound dressing application
Key Points
• silver nanoparticles were synthesized by gamma irradiation with sodium alginate as
stabilizer
• UV-Vis spectroscopy, X-ray
diffraction (XRD) and transmission electron microscopy (TEM)
was used to characterize the
silver nanoparticles
• chitin membranes with 30, 50,
70 and 100 ppm nanosilver
were prepared
• the antimicrobial activity of
nanosilver dressings was evaluated against Gram-negative
bacteria Pseudomonas aeruginosa and Gram-positive bacteria Staphylococcus aureus
broad-spectrum activity against Gram-positive
and Gram-negative micro-organisms (3,4) and
also its antibacterial effects on antibioticresistant bacteria, such as methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycinresistant enterococci (VRE) (1,5). In addition to
the recognised antimicrobial properties, silver
is also reported to promote wound healing (6).
The emergence of nanotechnology has provided a new therapeutic modality of silver nanoparticles for use in wounds. Silver
nanoparticles are reported to possess antibacterial (7), antifungal (8), antiviral (9), antiinflammatory (10), antiangiogenesis (11) and
antiplatelet activity (12). Nanoparticles play
a crucial role in inhibiting microbial growth
because of their high reactivity due to the large
surface to volume ratio. Several mechanisms
have been postulated for the antimicrobial
activity of silver nanoparticles. The adhesion of
nanoparticles to the surface causes deformation
of the membrane which leads to an increase in
membrane permeability. The nanosilver particles penetration into the bacterial cell results in
the DNA damages. The dissolution of nanosilver releases antimicrobial silver ions. The free
silver ion bind to the thiol (SH) groups of
cellular components, such as protein causing
inactivation of bacteria (13,14). Nanocrystalline
technology gives the highest, sustained release
of silver to a wound without clear risk of toxicity (15). The silver ion is released into the
wound for long-lasting bactericidal effect in
a controlled fashion. Hence, nanosilver has
found tremendous applications in the field of
antimicrobials and therapeutics (16). Nanosilver dressings can play an important role in the
healing of wounds due to their antimicrobial
effects. This study focuses on the preparation
and evaluation of chitin membranes containing
silver nanoparticles for use as an antimicrobial
wound dressing.
MATERIALS AND METHODS
Preparation of chitin-nanosilver
dressing
Silver nanoparticles were synthesised by
gamma irradiation using 10 mM AgNO3 .
Sodium alginate as stabiliser and isopropanol
as a scavenger of hydroxyl radicals were
used (17). The solution was irradiated with
60 Co Gamma rays to 50 kGy under ambient conditions. UV–Vis spectroscopy, X-ray
2
diffraction (XRD) and transmission electron
microscopy (TEM) was used to characterise
the silver nanoparticles. UV-Visible absorption
spectra were recorded using SPECORD S600
UV–Vis Spectrophotometer (Analytik Jena AG,
Jena, Germany). XRD patterns were recorded
on a Panalytical, X’pertPRO Diffractometer
with a Cu Kα source. TEM was used to characterise the morphology of the silver nanoparticles. TEM images were taken with a JEOL 2100
HRTEM (JEOL Ltd., Akishima, Japan) operating at 200kV. 30, 50, 70 and 100 ppm nanosilver
was added to the chitin in 5% lithium chloride
and dimethylacetamide solvent system. Dressing films were caste on flat glass plates.
Antimicrobial assay of chitin-nanosilver
dressing
The antimicrobial activity of nanosilver dressings was tested quantitatively by viable cell
count method. Two clinically relevant bacteriaGram-negative Pseudomonas aeruginosa (ATCC
No. 9027) and Gram-positive S. aureus (ATCC
No. 6538) were used. The bacteria were grown
in Nutrient Broth at 32◦ C for 18 hours. The inocula were prepared as a suspension representing 105 –106 CFU/ml. The nanosilver dressings
were immersed in Tryptone Soya Broth inoculated with 104 –105 CFU/ml of the test organism. The broths were incubated and samples
were withdrawn at periodic intervals. The solutions were serially diluted and plated for viable
counts using Soyabean Casein Digest Agar
medium. The number of surviving bacteria
was determined in the presence of chitin membranes containing 0, 30, 50, 70 and 100 ppm
nanosilver and without any membrane.
Statistical analysis
Results of antimicrobial assay were represented as mean ± SD (N = 4). Software SPSS
16.0 (SPSS Inc., Chicago, IL) was used for statistical analysis. One-way ANOVA followed
by Duncan post hoc test was used to identify
differences between groups.
RESULTS
Silver nanoparticles were prepared by gamma
irradiation method. The formation of the
silver nanoparticles was monitored using
UV–Vis absorption spectroscopy. The UV–Vis
absorption spectra of silver nanoparticles
© 2012 The Authors
International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc
Chitin membranes for wound dressing application
1.2
(A)
Absorbance
1
0.8
0.6
0.4
0.2
0
250
300
350
400
450
500
550
600
650
700
Wavelength (nm)
Figure 1. UV–Vis absorption spectrum of silver nanoparticles
prepared by gamma radiation.
16
12
8
4
0
1
3
5
7
9
11
13
15
Particle Size (nm)
Figure 3. (A) TEM image of silver nanoparticles, (B) histogram
of particle size distribution.
Key Points
Figure 4. Effect of chitin membranes containing nanosilver
on Pseudomonas aeruginosa. Data presented are mean ± SD,
N = 4.
400
Intensity (a.u.)
(B) 20
Frequ ency
e
(%)
prepared by gamma irradiation at doses of
50 kGy and stabilised by sodium alginate
is presented in Figure 1. The absorption
spectrum reveals the formation of silver
nanopartı́cles by exhibiting the typical surface
plasmon absorption maxima at 415–420 nm.
XRD analysis was also performed to confirm
the crystal phase of silver nanoparticles.
Figure 2 shows XRD pattern of the silver
nanoparticles supported on glass substrate.
The reflection peaks can be indexed to facecentred cubic silver, as indicated by diffraction
peaks (111), (200), (220) and (311). The average
size and morphology of silver nanoparticles
were determined by TEM. TEM photograph
(Figure 3A) indicates that the nanoparticles
with spherical morphology are well dispersed.
Histograms of size distribution were calculated
from the TEM images by measuring the
diameters of 100 particles. The particle size
histograms of silver particles (Figure 3B) show
that the particles range in size from 3–13 nm.
The antimicrobial efficacy of the chitin membranes containing nanosilver against Gramnegative bacteria, P. aeruginosa is presented in
300
200
100
0
30
40
50
60
70
2θ
Figure 2. XRD pattern of silver nanoparticles prepared by
gamma radiation.
Figure 4. The initial counts of P. aeruginosa in
the broth was 5·16 ± 0·25 log CFU/ml. About
1-log reduction in the counts was observed in
the presence of 30 ppm nanosilver dressing
after 1 hour of exposure. With increasing
exposure time, the counts were significantly
reduced (P < 0·01) and no viable counts were
detected after 4 hours. About 4-log reduction
in the counts were observed with 50 ppm
© 2012 The Authors
International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc
• the UV-Vis absorption spectra of nanoparticles exhibited an absorption band at
415–420 nm, which is the typical plasmon resonance band of
silver nanoparticles
• the peaks in the XRD pattern are
in agreement with the standard
values of the face-centred cubic
silver
• TEM images indicate silver
nanoparticles with spherical
morphology and small particle
size in the range of 3–13 nm
3
Chitin membranes for wound dressing application
Figure 5. Effect of chitin membranes containing nanosilver on
Staphylococcus aureus. Data presented are mean ± SD, N = 4.
Key Points
• no viable counts for Pseudomonas aeruginosa were
detected with 70 ppm nanosilver dressing after 1 hour exposure
• a 2-log reduction in viable cell
count was observed for Staphylococcus aureus after 1 hour
with 100 ppm nanosilver chitin
membranes
• the chitin membranes with
100 ppm nanosilver showed
promising antimicrobial activity against common wound
pathogens
nanosilver dressing after 1 hour and complete
killing was observed at 3 hours. No viable
counts for P. aeruginosa were detected at 70
and 100 ppm nanosilver dressing after 1-hour
exposure. The counts in the broth after 24 hours
in the absence of nanosilver dressings were
7·53 ± 0·27 log CFU/ml. However, no counts
were detected (P < 0·001) in the presence
of chitin membranes containing 30–100 ppm
nanosilver after 24 hours.
The effect of chitin nanosilver membranes on
Gram-positive bacteria S. aureus is presented
in Figure 5. Bactericidal effect was significant
(P < 0·01) in the presence of chitin nanosilver
dressings, whereas the counts progressively
increased in the absence of nanosilver dressings. About 3-log reduction in cell counts
was observed with 30 ppm nanosilver dressing after 6 hours as compared to counts in the
absence of chitin nanosilver membranes. Maximum bactericidal effect was observed with
100 ppm nanosilver dressing. A 2-log reduction in viable cell count was observed for
S. aureus after 1 hour and a 4-log reduction
after 6 hours with 100 ppm nanosilver membranes. More than 99·9% reduction (P < 0·001)
in the viable cell counts was observed after
24 hours in the presence of 100 ppm nanosilver
dressings. Comparison of the microbial reduction rates in this study reveals Gram-negative
bacteria P. aeruginosa to be more susceptible
to the antimicrobial effects of nanosilver than
Gram-positive S. aureus.
DISCUSSION
Antimicrobial dressings reduce wound bioburden, decrease the risk of infection and create an
4
environment that supports the normal healing process (1,18). Silver is commonly used
in wound dressings and topical formulations
to assist in the management of wounds that
are infected or at the risk of infection. They
provide potent broad-spectrum antimicrobial
activity (4,19). Percival et al. (19) have demonstrated the efficacy of silver alginate and silver carboxymethyl cellulose dressing against
planktonic S. aureus and P. aeruginosa. Silver dressings are used to remove or reduce
an increasing bioburden in burns and open
wounds, or to act as a barrier against cross
contamination of resistant organisms such as
MRSA. Silver-releasing dressings are reported
to have a positive effect on tissue management,
control of infection and inflammation, moisture balance, epithelial advancement and are
cost-effective (20). Nanotechnology is gaining
tremendous impetus in the present century due
to its capability of modulating metals into their
nanosize, which drastically changes the chemical, physical and optical properties of metals.
Silver nanoparticles have high reactivity due
to the large surface to volume ratio and have
emerged up with diverse medical applications
including wound dressings.
Silver nanoparticles were synthesised by
gamma radiation in the presence of sodium
alginate as stabiliser. A number of chemical and physical approaches are available
for the synthesis of silver nanoparticles. The
radiation technique offers many advantages
and has proven to be a convenient method
to prepare size-controllable metal nanoparticles (21,22). Hydrated electrons generated over
the course of irradiation reduce metal ions
to zero-valent metal particles. The growth of
silver nanoparticles by reduction of Ag+ to
Ag is stepwise (23). Silver atoms primarily
reduced by hydrated electrons rapidly combine with silver ions to form dimmer clusters. Continued reduction of the Ag+ solution
causes the aggregation of tetramer clusters
into nanoparticles. The metallic clusters and
the nanoparticles formed via gamma irradiation are capped with the sodium alginate
chain. Sodium alginate is a natural polymer
with excellent biocompatibility. The sodium
alginate stabilised Ag nanoparticles, therefore,
offer benefits of compatibility for biomedical
applications. The nanoparticles were examined using UV-Visible Spectroscopy, XRD and
TEM. The UV–Vis absorption spectra of silver
© 2012 The Authors
International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc
Chitin membranes for wound dressing application
nanoparticles exhibited an absorption band at
415–420nm, which is the typical plasmon resonance band of silver nanoparticles (24). The
narrow band range indicates a narrow size
distribution of the silver nanoparticles. The
synthesised nanoparticles were characterised
by XRD. The peaks in the XRD pattern are in
agreement with the standard values of the facecentred cubic silver. Strong reflections around
38◦ , 44◦ , 65◦ and 78◦ are characteristic of (111)
(200) (220) and (311) planes of face centred
cubic (FCC) of the silver nanoparticles (25). The
wide peaks indicate the small size of the silver nanoparticles (26). According to the Scherer
equation, the average size of the silver particles
calculated is 3 nm. TEM studies also suggest
the formation of silver nanoparticles of size
3–13 nm.
Wound dressings containing silver have
been in widespread use for many years. Topical
silver creams and solutions, and other topical antimicrobial preparations require frequent
application, are care intensive to apply and
remove, and are sometimes painful. In contrast
to the topical silver agents, the dressings control the release of silver to the wound and are
required to be changed with less frequency.
Therefore, wound dressings containing silver
as antimicrobial agent are being developed for
wound care in recent years. Silver dressings
are used extensively for wound management,
particularly in burn wounds, chronic leg ulcers,
diabetic wounds and traumatic injuries (27,28).
Caruso et al. (27) have reported Aquacel Ag
containing 1·2% w/w silver to be beneficial for the management of partial-thickness
burns. Karlsmark et al. (28) evaluated the clinical performance of sustained silver-releasing
dressing, Contreet Foam comprising of soft
hydrophilic polyurethane foam. The dressing
was found to be safe and performed well for
the treatment of chronic exuding venous leg
ulcers, combining effective antibacterial properties with excellent exudate management.
These dressings vary in containing compounds
of silver nitrate or sulphadiazine, to sustained
silver-ion release preparation and silver-based
crystalline nanoparticles. The dressing component also varies, as nylon, mesh, hydrocolloid
or methylcellulose. Chitin is a natural polymer that possesses excellent properties that
are advantageous for wound dressing (29).
The repeat unit N-acetyl-glucosamine occurs
in hyaluronic acid that is responsible for the
formation of fibrous networks for protein
attachment during wound healing. Chitin has
been found to have an accelerating effect on
the wound-healing process (30). The incorporation of silver nanoparticles in biopolymer
chitin is therefore envisioned to play an important role in the wound management. In the
present investigation, nanosilver-incorporated
chitin dressings were prepared and evaluated
for their antimicrobial activity. Chitin dressing containing 100 ppm nanosilver completely
inactivated viable cells of P. aeruginosa within
1 hour. S. aureus was reduced by about 2-log
units within the same period. The difference
of Gram-positive and Gram-negative bacteria
in terms of antibacterial activity of nanosilver can be attributed to the cell wall content.
The cell walls of Gram-positive bacteria are
known to contain 3–20 times more peptidoglycan than Gram-negative bacteria (31). As
peptidoglycans are negatively charged, they
probably bind to some portion of silver ion.
Therefore, silver is supposed to exhibit better antibacterial activity against Gram-negative
bacteria (32).
The results of this study demonstrate
the potential of the chitin membranes
containing 100 ppm silver nanoparticles for
use as an antimicrobial wound dressing. The
chitin dressings containing silver nanoparticles demonstrated in vitro antimicrobial
effect against wound-related micro-organisms
including Gram-positive and Gram-negative
bacteria.
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© 2012 The Authors
International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc