104 CHAPTER 6 CYTOTOXICITY, ANTIBACTERIAL, ANTIFUNGAL, ANTICANCER ACTIVITY OF SILVER NANOPARTICLES SYNTHESIZED FROM WRIGHTIA TINCTORIA 6.1 INTRODUCTION Silver nanoparticles are being used in numerous technologies and incorporated into a wide array of consumer products that take advantage of their desirable optical, conductive, and antibacterial properties. Diagnostic Applications: Silver nanoparticles are used in biosensors and numerous assays where the silver nanoparticle materials can be used as biological tags for quantitative detection. Antibacterial Applications: Silver nanoparticles are incorporated in apparel, footwear, paints, wound dressings, appliances, cosmetics, and plastics for their antibacterial properties. Conductive Applications: Silver nanoparticles are used in conductive inks and integrated into composites to enhance thermal and electrical conductivity. Optical Applications: Silver nanoparticles are used to efficiently harvest light and for enhanced optical spectroscopies including metal-enhanced fluorescence (MEF) and surface-enhanced Raman scattering (SERS).
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
104
CHAPTER 6
CYTOTOXICITY, ANTIBACTERIAL, ANTIFUNGAL,
ANTICANCER ACTIVITY OF SILVER NANOPARTICLES
SYNTHESIZED FROM WRIGHTIA TINCTORIA
6.1 INTRODUCTION
Silver nanoparticles are being used in numerous technologies and
incorporated into a wide array of consumer products that take advantage of
their desirable optical, conductive, and antibacterial properties.
Diagnostic Applications: Silver nanoparticles are used in biosensors and
numerous assays where the silver nanoparticle materials can be used as
biological tags for quantitative detection.
Antibacterial Applications: Silver nanoparticles are incorporated in apparel,
footwear, paints, wound dressings, appliances, cosmetics, and plastics for
their antibacterial properties.
Conductive Applications: Silver nanoparticles are used in conductive inks
and integrated into composites to enhance thermal and electrical conductivity.
Optical Applications: Silver nanoparticles are used to efficiently harvest
light and for enhanced optical spectroscopies including metal-enhanced
fluorescence (MEF) and surface-enhanced Raman scattering (SERS).
105
Wrightia tinctoria (veppallai) with its rich source of polyphenolic
compounds was exploited for the reduction and capping of silver
nanoparticles (Ag NPs), making it a complete green chemical route. The
reduction of Ag+ to Ag0 was observed by the color change from pale yellow to
dark yellow.
6.2 MATERIALS AND METHODS
6.2.1 Synthesis of Silver Nanoparticles
The silver nitrate (AgNO3) was purchased from Sigma-Aldrich
chemicals. Wrightia tinctorialeaves were collected from chengalpet. The leaf
extract was used for the reduction of Ag+ ions to Ag°. 50g of finely cut leaves
were thoroughly washed, dried and immersed in 100 ml of distilled water
contained in a 500 ml Erlenmeyer flask. The mixture was boiled for 15 to 20
minutes in a hot plate. Then the extract was further filtered through Whatman
No. 1 filter paper and stored at 4°C for further experiments. 1mM aqueous
AgNO3 solution was prepared. 5 ml of the leaf extract was added to 45 ml of
1 mM AgNO3 (aqueous) solution and kept in dark for 48 hours for the
formation of silver nanoparticles.
6.2.2 Characterization of the Silver Nanoparticles
The nanoparticles were characterized by UV spectroscopy (Perkin
39.55 nm and 39.95 nm, respectively. Based upon the characteristic peak
e crystal was
calculated and this ranges from 50nm to 400 nm in diameter. The particles are
characterized using XRD, UV {visible, photoluminescence and Raman
spectroscopy.The absorption feature (peak) at480 nm which is considerably
116
blue-shifted relative to the peak absorption of bulk CdS indicating quantum
size effect. the photoluminescence (PL) spectra of nanoparticles of CdS for
different excitation wavelengths of 300, 240 and 230 nm (energies 4.1, 5.1
and 5.4 eV).The Raman spectrum of nanoparticles of CdS is in the range 180-
400 cm-1. The spectrum exhibits a strong but broadpeak at 302 cm-1
corresponding to the LO phonon mode (KaruppasamyKandasamy et al 2009).
X-ray diffraction patterns of CdS nanoparticles are characteristic of
the hexagonal phase and t values were found to be 4.124 and
6.686Å. The XRD peaks are very broad indicating the presence of very fine
grains of particles. The XRD pattern exhibits broad peaks of 26.778, 43.928
and 51.6780. The size of the particle increases with the concentration of the
capping agent increased (Manickathai et al 2008).
Figure 6.2 XRD analysis indicating the formation of silver nanoparticle
117
6.3.2.3 SEM EDAX analysis
The surface morphology of the formed silver nanoparticles was observed and was represented as nanoclusters. Besides, the elemental composition was checked by EDAX, which indicates unique peak for silver which constitutes major composition.Electron-diffraction patterns and high-resolution lattice images obtained with transmission electron microscopy (TEM) indicated that the CdS nanoparticles were crystalline with dspacings corresponding to the zinc blende structure ( dspacings, 0.336, 0.206, 0.176, 0.133, 0.118 nm) (Limin Qi et al 2001). Absorption, fluorescence spectroscopy, and transmission electron microscopy were employed for characterization, which revealed that the prepared silver nanoparticles had a well-resolved cubic structure and were monodisperse in size Figure 6.3. It was also found that the silver nanoparticles were dispersed in solution as single entities and showed a very good resistance against oxidation for months, according to their polymer shell. The particle size was controllable in the range between 2 and 4 nm by adjusting the polymer concentration and choice of the solvent (Rajeev Prabhu & Abdul Khadar 2005).
Figure 6.3 SEM image for the formation of silver nanoparticle
118
6.3.2.4 FTIR analysis
The FTIR spectrum of silvernanoparticles was shown in
Figure 6.4. The higher energy region peaks at 3784 cm-1 and 2426cm-1 are
assigned to O-H stretching of silver nanoparticles. Also a stretch of N-H
amine group was observed at 3399 cm-1 and 1615 cm-1 respectively. A strong
stretch of N-O nitro group was observed at 1384 cm-1. An alkene =C-H
bending was observed at 805 cm-1. Also a strong alkyl halide C-I stretch was
observed at 463cm-1 .Upon verification with the present database, the
spectrum matches with that of silver and partially with the spectrum of Al2O3.
Figure 6.4FTIR analysis for the formation of silver nanoparticle
Chick embryonic cells and bovine bone marrow cells does not
124
viable cells started decreasing. This was proved by the MTT assay, wherein
proportional to the decrease in the cellular proliferation at 24 hrs and 48 hrs of
incubation respectively. With reference to the assay performed, it was
concluded that
concentration for the silver nanoparticles (AgNps) derived using Wrightia
tinctoria plant extracts offered the cell viability Figure 6.8(a) and
Figure 6.8(b).
CYTOTOXIC EFFECTS OF SILVER NANOPARTICLES DERIVED FROM WRIGHTIA TINCTORIA ON THE CELLULAR
PROLIFERATION
0
0.5
1
1.5
2
2.5
0 100 200 300 400 500 600
Conentration ( M)
Wrightia tinctoria derived AgNps activity on chick
embryonic cell proliferation
24 hrs48 hrs
0
0.5
1
1.5
2
2.5
3
0 200 400 600
Concentration ( M)
Wrightia tinctoria derived AgNpsactivity on bovine bone marrow
cell proliferation
24 hrs
48 hrs
Figure6.8 (a) Cytotoxic effects of
silver nanoparticles derived from wrightia tinctoriaon the cellular proliferationof chick embryonic cd45-/cd14- stem cells
Figure6.8 (b) Cytotoxicity effects of silver nanoparticles derived from wrightiatinctoriaon thecellular proliferation of bovine bone marrow cells cd45-/cd14- stem cells
125
6.3.3.5 Anticancer activity of silver nanoparticles on A549 cell line
(Adenocarcinomic human alveolar basal epithelial cells)
With respect to biological and clinical applications, the ability to
control and manipulate the accumulation of nanoparticles for an extended
period of time inside a cell can provide sensitivity towards diagnosis and
therapeutic efficiency.. In general, silver nanoparticles should serve as one of
the best ways of treating diseases that involve cell proliferation and cell death.
Measurements were performed and the concentration required for a
50% inhibition (IC50) was determined graphically (Table 6.2). The % cell
viability was calculated using the following formula,
%cell viability = A570 of treated cells / A570 of control cells × 100
Graphs were plotted using the % of Cell Viability at Y-axis and
concentration of the sample in X-axis. Cell control and sample control was
included in each assay to compare the full cell viability in Cytotoxicity and
assay confirmed the anti cancer potential of the silver nanoparticles.
126
Table 6.2 Anticancer effect of silver nanoparticles derived from Wrightia tinctoria on A549 cell line
S.No Concentration (µg/ml)
Dilutions Absorbance (O.D)
Cell viability (%)
1 1000 Neat 0.12 21.05
2 500 1:1 0.17 29.82
3 250 1:2 0.23 40.35
4 125 1:4 0.27 47.36
5 62.5 1:8 0.32 56.14
6 31.2 1:16 0.40 70.17
7 15.6 1:32 0.45 78.94
8 7.8 1:64 0.49 85.96
9 Cell control - 0.57 100
ANTICANCER ACTIVITY OF SILVER NANOPARTICLE ON A549 CELL LINE (ADENOCARCINOMIC HUMAN ALVEOLAR BASAL
EPITHELIAL CELLS)
IC50 values of AgNps derived from Wrightia tinctoria on A549 tumour cell line
Figure 6.9 IC50 values of AgNps derived from Wrightia tinctoriaon A549 tumour cell line
127
IN VITRO CYTOTOXICITY ASSAY (MTT ASSAY) FOR THE EVALUATION OF THE ANTICANCER ACTIVITY OF SILVER
NANOPARTICLE DERIVED FROM WRIGHTIA TINCTORIA ON A549 CELL LINE
Normal A549 cell line
Toxicity- 250µg/mlToxicity- 1000µg/ml
Toxicity- 125µg/ml Toxicity- 62.5µg/ml
Figure 6.10 In vitro cytotoxicity assay (MTT assay) for the evaluation of the anticancer activity of silver nanoparticle derived from wrightia tinctoria on A549 cell line
6.3.3.6 Evaluation of the anti apototic activity of the silver
nanoparticles by DNA fragmentation assay
It was observed that there was no cleavage in the control, where as
the DNA isolated from the cancer cell A549 was found to be cleaved into two
to three fragments on the well treated with various concentration of silver
nanoparticles .This investigation depicted the anti apoptotic behavior of the
silver nanoparticles derived from Wrightia tinctortia (Figure 6.11).
128
Programmed cell death was also be resulted by the activation of
caspase 3, which leads to to the cleavage of caspase substrates, resulting in
the fragmentation of DNA. This induces the apoptotic signal of the cell by the
trafiicking of the nanoparticle.
EVALUATION OF THE ANTI APOPTOTIC ACTIVITY OF THE SILVER NANOPARTICLES DERIVED FROM WRIGHTIA TINCTORIA
BY DNA FRAGMENTATION ASSAY
Lane M: Marker(100bp DNA Ladder) Lane 1: ControlLane 2: Con 125µg/mlLane 3: Con 250µg/ml Lane 4: Con 62.5µg/ml
M 1 2 3 4
Figure 6.11 Evaluation of the anti apoptotic activity of the silver
nanoparticles derived from wrightia tinctoria by DNA fragmentation assay
6.3.3.7 Evaluation of antioxidant activity of the silver nanoparticles by
DPPH (1, 1-Diphenyl-2-Picrylhydrazyl)
Based on the DPPH assay performed, it was found that, a good free
radical scavenging potential was observed for the silver nanoparticles derived
using the plants extracts of Wrightia tinctoria.
129
The percentage antioxidant activity of the silver nanoparticle
derived using wrightia tinctoria leaf extract was found to be 30.55%
Figure 6.12
Figure 6.12 Evaluation of % antioxidant activity of the silver nanoparticles derived from Wrightia tinctoriaby DPPH assay
6.3.3.8 Evaluation of antioxidant activity of the silver nanoparticles by
frap (Ferric ion reduction potential) assay
The FRAP value of the silver nanoparticle was found to be
nanoparticles derived form Wrightia tinctoria was justified Figure 6.13.
0
20
40
60
80
100
% of antioxidant activity
130
Figure 6.13 Evaluation of % antioxidant activity of the silver nanoparticles derived from Wrightia tinctoriaby FRAP assay
6.3.3.9 Evaluation of wound healing activity of silver nanoparticles on
vero cell lines
Silver nanoparticles afford a good wound healing potential and this
was manipulated by scratch wound healing assay on Vero cell line. After
24hrs and after 48hrs of incubation with silver nanoparticles Figure 6.14,
migration of cells occurred which facilitate the wound healing activity of the
cells was justified. Silver nanoparticle exhibit to increase the
polymorphonuclear cell apoptosis, but he activity of matrix metalloproteinase
(MMP) remains low, conferred an anti-inflammatory potency.
0
50
100
150
200
250
AgNPS 50 g/ml AgNps
100 g/ml control
FRAP Value(mM/L)
FRAP Value(mM/L)
131
ASSESSMENT OF WOUND HEALING ACTIVITY OF THE SILVER NANOPARTICLES DERIVEN FROM WRIGHTIA TINCTORIA ON
VERO CELL LINES
Wound created on vero Cell lineWound healed after 24 hrs of
incubation with silver nanoparticle
(a) (b)
Figure 6.14 Assessment of wound healing activity of the silver nanoparticles derived from wrightia tinctoria on vero cell lines(a) scratched cells (b) migrated cells
6.3.3.10 Evaluation of cell viability of silver nanoparticle /BSA scaffold
on mesenchymal stem cells by in vitro cytotoxicity assay
from Wrightia tinctoria was blended with BSA 10mM (Sigma Aldrich)
Bovine Serum Albumin . The contents were then lyophilized at -80 C for
the formation of scaffold. MTT assay was perfomed by adding the
nanoparticle solution on the mesenchymal stem cells seeded on th 96 well
microtitre plate.The absorbance were read at 570nm.It was observed that after
12 hr, 24 hrs and 48 hrs of incubation , the bio scaffold blended with silver
132
nanoparticle and BSA showed celluar proliferation and supports cell viability
(Figure 6.15).
Figure 6.15 Effect of Wrightia tinctoria AgNps /BSA scaffold on mesenchymal stem cells by invitro cytotoxicity assay
6.4 CONCLUSION
From this summary, it was concluded that plant mediated synthesis of
silver nanoparticles possess potential antimicrobial applications. The
characterization analysis proved that the particle so produced in nanodimensions
would be equally effective as that of antibiotics and other drugs in pharmaceutical
applications. The use of Wrigthia tinctoria derived silver nanoparticles in drug
delivery systems might be the future thrust in the field of medicine. It was
concluded that the silver nanoparticles can serve as a potential drug with various
clinical and pharmacological properties, thereby demonstrating enhanced
characteristic anti cancer activity, anti apoptotic activity, anti oxidant activity,
wound healing activity and antimicrobial activity.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
12 hrs 24 hrs 36 hrs 48 hrs 60 hrs 72 hrs
AgNps/BSA scaffold showing cell viability on mesenchymal stem cells