Preparation, Characterization
and Anti-Inflammatory Activity of Chitosan Stabilized
Silver Nanoparticles
S. Ram
Prasad*, K. Elango, S. Daisy Chellakumari,
S. Dharani
Department of Pharmaceutics, College of Pharmacy,
Madras Medical College, Chennai-03.
ABSTRACT:
The present study deals with preparation of silver nanoparticles using green approach such as polysaccharides
method. (Chitosan was used as reducing and
stabilizing agent). The prepared silver nanoparticles
were characterized by UV Visible Spectroscopy, Fourier Transform Infra- red
spectroscopy, Photon Correlation Spectroscopy, Scanning Electron Microscope and
ICPOES. UV- Visible spectrum shows the SPR band at 420nm which confirms the
formation of silver nanoparticles. The average
particle size of chitosan stabilized silver nanoparticles was found to be 127.3nm. The amount of silver
present in the solution was found to be 1.259mg/L using ICPOES. Chitosan stabilized silver nanoparticles
were studied for anti-inflammatory activity. In vitro toxicity studies were carried out in 3T3/NIH fibroblast
cell line. Using the natural environmental benign polymer chitosan,
the silver nanoparticles were prepared and
characterized.
KEYWORDS: Chitosan, Silver nanoparticles,
SPR band, SEM, ICPOES, Membrane stabilizing activity
INTRODUCTION:
Several methods are available for preparation of silver
nanoparticles such as polysaccharide, Tollens, Irradiation (Microwave, UV, Gamma etc.,),
Biological and Polyoxometalates etc. Based on this
approach polysaccharides method was chosen. In this method single
polysaccharide plays a dual role as reducing and stabilizing agent.
5Chitin is known biodegradable natural
polymer based on polysaccharides, which is obtained from crustacean shell (e.g.
crab, shrimp, and lobster), some insect (e.g. true fly, Sulphur
butterfly) and fungi like yeasts and plants. Chitosan
is obtained from partial deacetylation of chitin. Chitosan is a linear polysaccharide comprising copolymers
of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated
unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan
a natural polysaccharide that has unique polycationic,
chelating, film forming, reducing, stabilizing properties and various
biological properties such as anti-inflammatory, anti-proliferative,
anti-oxidant, antimicrobial and anticholesterolemic
etc., Chitosan is an abundant, naturally occurring
polymer with versatile functionalities. 6
7Chitosan
is a cationic polyamine with high charge density. It is a linear
polyelectrolyte with reactive hydroxyl groups and amino groups. The presence of
number of amino groups allow chitosan to react
chemically with anionic systems, which results in alteration of physicochemical
characteristics of such combinations.
8Nanocrystalline silver dressings were introduced commercially as
antimicrobial dressings in 1998 and these have found to improve wound healing,
which may result from potent anti-inflammatory activity. The treatment of murine infected burns with silver nanoparticles
was found to increase the rate of healing and decrease the scarring in
comparison with silver sulfadiazine. This was accompanied by increased
expression of IL-10, vascular endothelial growth factor, and interferon-γ,
with reduced IL-6 expression. In a porcine infected wound model, nanocrystalline silver treatments enhanced tissue
regeneration while decreasing erythema and edema
relative to silver nitrate (AgNO3) treatments. Nanocrystalline
silver treatments were also found to increase the polymorphonuclear
cell apoptosis while the matrix metalloproteinase (MMP) levels remained low,
suggesting an anti-inflammatory effect.
In animal models nanocrystalline silver
alters the expression of matrix metalloproteinase’s (proteolytic
enzymes that are important in various inflammatory and repair processes),
suppresses the expression of tumor necrosis factor, Interleukin and induces
apoptosis of inflammatory cells. AgNPs modulates
cytokine involved in wound healing.
The induction of apoptosis by nanocrystalline
silver at the dermal cells suggests a highly discriminatory process, related to
the unique silver species released (e.g., Ag0), that is different from the
indiscriminate activity of Ag+. Silver ions can interfere with the respiratory
chain at the cytochromes and can interact with the
electron transport chain to activate the intrinsic signaling pathway to
apoptosis through the activation of downstream pro-caspases.
Nano crystalline silver has
anti-inflammatory activity and this is due to the induction of apoptosis in
inflammatory cells, and suppression of MMP activity. Nano
crystalline silver suppresses the production of proinflammatory
cytokines TNF-α, IL-8, and TGF-β, and may impact others, including
IL-12. Over expression of MMPs includingMMP-2 and MMP-9 contributes to tissue
injury and inflammation. Therefore, MMP inhibition has been suggested as a
therapeutic approach to controlling inflammation and the similarities between
the model system and human skin suggests the use of silver nanoparticles
for as better anti-inflammatory agent. Therefore it is of interest to prepare
silver nanoparticles using chitosan
to treat inflammation.
EXPERIMENTAL METHODS:
Materials:
Chitosan 85% deacetylated was obtained as gift sample from Central
Institute of Fisheries Technology, Kochi. Silver Nitrate (99%) was purchased
from Sigma Aldrich (Analytical Grade). Deionized
water was used in this experiment. All the other chemicals were of analytical
grade.
Synthesis of Silver Nanoparticles: 9,
10
50 ml of 0.1% chitosan solution was
preheated at 80ºC±2ºC for 30 minutes. 10ml aqueous solution of 0.1 M Silver
nitrate was added to the above solution. The mixture was transferred to a
magnetic stirrer and the temperature was maintained at 60ºC±2ºC, 70ºC±2ºC, and
80ºC±2ºC and any colour change was noted. Colloidal silver nanoparticles
exhibit absorption at wavelength from 380-420nm due to Mie scattering Theory.
The colour change was noted and analyzed by UV
Visible spectrophotometer.
In order to reduce Silver nitrate to NanoSilver,
various concentrations of silver nitrate (1mM, 2mM, 3mM, 4mM, 5mM & 0.1M)
were added to 0.1% chitosan
solution. The optimal concentration of silver nitrate to form silver nanoparticles was determined.
CHARACTERIZATION:
UV- Visible Spectroscopy:
Synthesized chitosan stabilized silver nanoparticles were scanned in the range of 200-800nm using
UV –Visible Spectrophotometer , UV 1800, Shimadzu, Japan.
Fourier Transformation Infrared Spectroscopy:
FTIR spectrum of Chitosan and Chitosan Stabilized Silver nanoparticles
spectrum were recorded using Nicolet
Fourier transformation Infrared spectroscopy (FT-IR) combined to PC (with
spectrum 2000 analysis software) in the range of 4000 cm-1 to 400 cm-1 by
Potassium Bromide Press Pellet technique .The pellet was placed in light path
and the spectra were analyzed.
Malvern particle size analyzer:
The sample of the optimized chitosan
stabilized nanoparticle was analyzed for particle size using Malvern Zetasizer. The particle size, their range and degree of
distribution were studied using this analyzer. The nanosuspension
was diluted 10 fold with millipore water. The diluted
solution was used for the analysis. The size of the particle was measured based
on the scattering of laser light by the particle. The angular intensity of the
scattered light was then measured by a series of photosensitive detectors. The
map of scattering intensity versus angle is the primary source of information
used to calculate the particle size.
Inductively Coupled Plasma Optical Emission Spectroscopy (ICPOES):
The amount of silver
content present in the nanosuspension was analyzed by
Perkin Elmer Optima 5300 DV ICP-OES. It is a most widely used analytical
technique for measuring concentrations of major, minor and trace elements. It
is a robust analytical method capable of providing analyzes for a wide range of
elements in a diversity of sample matrices.
Scanning electron microscopy:
The particle size of the optimized nanoparticles
was viewed and photographed using scanning electron microscope. The nano suspension of AgNPs was
diluted 10 fold with millipore water and transferred
to a glass slide which was cut in the diameter of 20×20mm. The slide was
mounted on an aluminum stub using double sided carbon tape. The solution was
slowly evaporated at room temperature. The completely dried sample was coated
with gold by sputter coating unit at 10 Pascal vacuum for 10 second to a
thickness of 100 A° using HITACHI evaporator. The image was captured on SEM
mode at desired magnification.
MEMBRANE STABILIZING ACTIVITY: 11, 12
Preparation of Human Red Blood Cells (HRBC) Suspension:
Fresh whole human blood was collected and mixed with equal volume of
isotonic saline (0.85%). The blood was
centrifuged at 3000 rpm for 10 min and packed cells were washed three times
with isotonic saline (0.85%, pH 7.2). The volume of the blood was measured and
reconstituted as 10% v/v suspension with isotonic saline.
Heat Induced Hemolysis:
The principle involved here is stabilization of human red blood cell
membrane by hypo tonicity induced membrane lysis. The
reaction mixture contains 1ml phosphate
buffer [pH 7.4], 2 ml hypo saline [0.36 %], 0.5 ml HRBC suspension [10 % v/v]
with 0.5 ml of test solutions(Chitosan Stabilized AgNPs) and standard drug Diclofenac sodium (50mg) and
control (distilled water instead of hypo saline to produce 100 % Hemolysis). They were incubated at 56ºC for 30 minutes. The
tubes were cooled under running tap water for 20 minutes. The mixtures were
centrifuged and the absorbance of the supernatants read at 560 nm.
The percentage of Hemolysis of HRBC membrane
is calculated as follows:
100- (O.D. of test – O.D. of product control)
Percent Stabilization = x 100
O.D. of
Control
In vitro Toxicity Studies of Silver Nanoparticles in 3T3 NIH Fibroblast cell line: 13, 14
Cell line:
3T3 NIH Fibroblast cell lines were obtained from National Centre
for Cell sciences, Pune (NCCS). The cells were
maintained in Minimal Essential Media supplemented with 10% FBS (Fetal Bovine
Serum), penicillin (100 U/ml), and streptomycin (100 μg/ml)
in a humidified atmosphere of 50 μg/ml CO2
at 37 °C.).
Reagents:
MEM was purchased from Hi Media Laboratories Fetal bovine serum (FBS)
was purchased from (Cistron laboratories) Trypsin, methylthiazolyl diphenyl- tetrazolium bromide
(MTT), and Dimethyl sulfoxide
(DMSO) were purchased from (Himedia).
In vitro assay for Cytotoxicity
activity (MTT assay):
The Cytotoxicity of samples on 3T3 NIH
fibroblast was determined by the MTT assay.
Cells (1 × 105/well) were placed in 100 μl
of medium/well in 96-well plates (Costar Corning, Rochester, NY). After 48
hours of incubation the cell reaches the confluence. Then, cells were incubated
in presence of various concentrations of the samples (Silver nanoparticles) in 0.1% DMSO for 48h at 37°C.The sample
solution was washed with phosphate-buffered saline (pH 7.4), 20µl/well (5mg/ml)
of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl--tetrazolium
bromide cells(MTT) in phosphate- buffered saline solution was added. After 4
hours of incubation, 1ml of DMSO was added. Viable cells were determined by the
absorbance at 540nm. Measurements were performed and the concentration required
for a 50% inhibition of viability (IC50) was determined graphically. The effect
of the samples on the proliferation of fibroblast cells was expressed as
the % cell viability.
% cell viability = A540 of treated cells / A540
of control cells × 100
RESULTS & DISCUSSION:
Preparation
of Silver nanoparticles:
Silver nanoparticles were prepared using
various concentration of silver nitrate at different temperatures. The optimum
concentration of was found to be 0.1M silver nitrate solution. During reduction
of silver nitrate colour change was noted. Initially
it was pale yellow colour and turns to yellowish
brown colour at the end of 2 hours which shows the
formation of AgNPs at 80ºC ± 2 ºC and the resulting
solutions were scanned by UV –Visible spectrophotometer. The observation is
shown in table: 1.
Table: 1 Optimization of temperature for preparation of silver nanoparticles
|
Volume of
0.1% Chitosan |
Volume of
0.1M Silver Nitrate |
Temperature |
Observation |
|
100 ml |
10 ml |
60ºC ± 2ºC |
No Change
in colour |
|
70ºC ± 2ºC |
No Change
in colour |
||
|
80ºC ± 2
ºC |
Yellowish
Brown colour |
UV –Visible
Spectroscopy:
Surface Plasmon Resonance band during reduction of Silver nanoparticles: 15
Silver nanoparticles were prepared by
controlled heating technique samples were withdrawn at half an hour interval
& analyzed in UV Visible Spectrophotometer. Colloidal silver nanoparticles exhibit absorption at wavelength from
380-420nm due to Mie scattering Theory. The absorption maxima at 420 nm which
confirms the formation of silver nanoparticles. The
overlay spectrum of absorption maxima of silver nanoparticles
are shown in the Figure: 1.
Figure: 1 Overlay spectrum of AgNPs (λmax at 60minutes, 90minutes & 120minutes)
ICPOES:
The amount of silver in
the pellets obtained after centrifugation is estimated by ICPOES. The amount of
Silver in colloidal silver solution was found to be 1.259mg/L.
Particle Size of Silver nanoparticles:
The Particle size distribution of chitosan
stabilized silver nanoparticles is shown in the
Figure: 2. The spectrum shows that the maximum number of silver nanoparticles in the range of 127.3 nm.
Scanning
Electron Microscopy:
The SEM image of chitosan stabilized silver nanoparticles was shown in Figure: 3. The silver nanoparticles were spherical in shape and uniform in size.
Fourier Transform Infrared Spectroscopy: 16
FTIR spectra of chitosan and chitosan Silver nanoparticles are
shown in the Figures 4 & 5. A peak at 3440 cm-1 indicates the
combined peaks of OH and NH2 group (stretching vibrations of chitosan). In Chitosan stabilized
silver nanoparticles a shift from 3440 cm-1 to
3325 cm-1 show the reduced hydrogen bonding. A peak at 1651 cm-1
disappears and a new peak appears at 1632 cm-1 shows the
attachment of silver with Nitrogen atoms, which reduces vibration intensity of
the N-H bond due to the molecular weight becoming greater after silver binding.
In vitro Toxicity of Silver Nanoparticles in 3T3 NIH fibroblast Cells:
From the morphology of cells and MTT assay concentration of upto 7.8 μg/ml indicates
91.4% cells are viable. Hence the prepared AgNPs were
found to be biocompatible and non-toxic as shown in Table 2.
Figure: 2 Particle size distributions of AgNPs
Figure: 3 SEM image of Silver nanoparticles
Figure: 4 FTIR spectra of Chitosan
Figure: 5 FTIR spectrum of Silver Nanoparticles
Table: 2 Percentage of Cell viability of
fibroblast cell on addition of AgNPs
|
S. No. |
Concentration µg/ml |
Dilution |
Absorbance 540nm |
% cell Viability |
|
1 |
1000 |
Neat |
0.18 |
25.7 |
|
2 |
500 |
1:1 |
0.22 |
31.4 |
|
3 |
250 |
1:2 |
0.29 |
41.4 |
|
4 |
125 |
1:4 |
0.35 |
50.0 |
|
5 |
62.5 |
1:8 |
0.43 |
61.4 |
|
6 |
31.2 |
1:16 |
0.49 |
70.0 |
|
7 |
15.6 |
1:32 |
0.56 |
80.0 |
|
8 |
7.8 |
1:64 |
0.64 |
91.4 |
|
9 |
control |
- |
0.70 |
100 |
Figure:6
Percentage Cell Viability at various concentrations μg/ml
Figure 6 represents that
percentage cell viability against concentration of silver nanoparticles.
Figure 7 shows that surface morphology of Fibroblast Cells with various
concentrations of Chitosan Stabilized Silver nanoparticles
Figure: 7 In vitro Toxicity of Silver Nanoparticles in 3T3 NIH fibroblast Cells
MEMBRANE STABILIZING ACTIVITY
The prepared Chitosan stabilized silver nanoparticles were tested for membrane stabilizing
activity. Percentage stabilization was 62.74 for AgNPs
and 78.21 for standard drug Diclofenac Sodium (Figure: 8). The percentage
stabilization of silver nanoparticles was good when
compared with the standard drug.
Figure: 8 Membrane Stabilizing activity (T1 – AgNPs, S- Standard Drug)
CONCLUSION:
The silver nanoparticles were prepared using
environmental benign natural polymer Chitosan (85% deacetylated) using green chemistry. The optimum
concentration of silver nitrate required to form silver nanoparticles
was found to be 0.1M AgNO3. In controlled heating technique, the
stable silver nanoparticles were formed at 80ºC±2ºC
for 2 hours. The in vitro toxicity studies normal fibroblast cells show
the biocompatibility and non toxicity of AgNPs. As
the chitosan and silver have multifunctional
properties, the AgNPs reduced with chitosan may be a better therapeutic agent to treat
inflammation/inflammatory sites. Silver nanoparticles
have anti-inflammatory and antibacterial property. Chitosan
is uused as anti-inflammatory, antioxidant, reducing
and stabilizing agent. The chitosan stabilized silver
nanoparticles produce synergistic effect to treat
inflammation.
ACKNOWLEDGEMENT:
I acknowledge my special thanks to Central Institute of Fisheries
Technology, Kochi, for providing Chitosan as gift
sample. It’s my privilege to thank The Director, National Centre for Cell
sciences, Pune for their material assistance of 3T3
/NIH fibroblast cell line .I extend my sincere thanks to my Professor and Head,
Staff Members, Friends and Family members for their support and encouragement.
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Received on 03.04.2013
Modified on 03.05.2013
Accepted on 12.05.2013
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Journal of Pharmaceutical Dosage Forms and Technology. 5(3): May- June, 2013, 161-167