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Biomedical potential of silver nanoparticles synthesized from calli cells of Citrullus colocynthis (L.) Schrad
© K et al; licensee BioMed Central Ltd. 2011
Received: 12 May 2011
Accepted: 26 September 2011
Published: 26 September 2011
An increasingly common application is the use of silver nanoparticles for antimicrobial coatings, wound dressings, and biomedical devices. In this present investigation, we report, biomedical potential of silver nanopaticles synthesized from calli extract of Citrullus colocynthis on Human epidermoid larynx carcinoma (HEp -2) cell line.
The callus extract react with silver nitrate solution confirmed silver nanoparticles synthesis through the steady change of greenish colour to reddish brown and characterized by using FT-IR, AFM. Toxicity on HEp 2 cell line assessed using MTT assay, caspase -3 assay, Lactate dehydrogenase leakage assay and DNA fragmentation assay.
The synthesized silver nanoparticles were generally found to be spherical in shape with size 31 nm by AFM. The molar concentration of the silver nanoparticles solution in our present study is 1100 nM/10 mL. The results exhibit that silver nanoparticles mediate a dose-dependent toxicity for the cell tested, and the silver nanoparticles at 500 nM decreased the viability of HEp 2 cells to 50% of the initial level. LDH activities found to be significantly elevated after 48 h of exposure in the medium containing silver nanoparticles when compared to the control and Caspase 3 activation suggested that silver nanoparticles caused cell death through apoptosis, which was further supported by cellular DNA fragmentation, showed that the silver nanoparticles treated HEp2 cells exhibited extensive double strand breaks, thereby yielding a ladder appearance (Lane 2), while the DNA of control HEp2 cells supplemented with 10% serum exhibited minimum breakage (Lane 1). This study revealed completely would eliminate the use of expensive drug for cancer treatment.
Citrullus colocynthis (Bitter cucumber) belongs to the family of cucurbitaceae, which are abundantly grown along the arid soils of Southeast coast of Tamil Nadu. It has a large, fleshy perennial root, which sends out slender, tough, angular, scabrid vine-like stems. The therapeutic potentials viz., antimicrobial , anti inflammatory , anti diabetic  and anti oxidant  effect of Citrullus colocynthis have reported in our laboratory. For conservation of this potent medicinal plant we have micro propagated and transplanted to the coastal region of Parangipettai.
Nanoparticles usually referred as particles with a size up to 100 nm. Nanoparticles exhibit completely new properties based on specific characteristics such as size, distribution and morphology. As specific surface area of nanoparticles is increased, their biological effectiveness can increase in surface energy . Silver has long been recognized as having an inhibitory effect towards many bacterial strains and micro organisms commonly present in medical and industrial processes . The most widely used and known applications of silver and silver nanoparticles are include topical ointments and creams containing silver to prevent infection of burns and open wounds . Production of nanoparticles can be achieved through different methods. Chemical approaches are the most popular methods for the production of nanoparticles. However, some chemical methods cannot avoid the use of toxic chemicals in the synthesis protocol. Biological methods of nanoparticles synthesis using micro organisms , enzyme , and plant or plant extract have been suggested as possible ecofriendly alternatives to chemical and physical methods. Using plant for nanoparticles can be advantageous over other biological processes by eliminating the elaborate process of maintaining cell culture . If biological synthesis of nanoparticles can compete with chemical methods, there is a need to achieve faster synthesis rates. The exact mechanism of silver nanoparticles synthesis by plant extracts is not yet fully understood. Only participation of phenolics, proteins and reducing agents in their synthesis has been speculated. Recently nano-encapsulated therapeutic agents such as antineoplastic drugs had been used to selectively targeting anti tumor agents and obtaining higher drug concentration at the tumour site . Nanotechnology could be very helpful in regenerating the injured nerves. For biological and clinical applications, the ability to control and manipulate the accumulation of nanoparticles for an extended period of time inside a cell can lead to improvements in diagnostic sensitivity and therapeutic efficiency. This when revealed completely would eliminate the use of expensive drugs for cancer treatment . The callus and leaf extract of Citrullus colocynthis reported moderate antimicrobial activity against biofilm forming bacteria  and harmful human pathogens . Therefore the present study, we evaluated, biomedical potential of silver nanopaticles synthesized from calli extract of Citrullus colocynthis on Human epidermoid larynx carcinoma (HEp -2) cell line.
Results and Discussion
Cell viability and LDH Leakage in control and SNp, treated HEp2 cells after 48 h of exposure
Percentage of inhibition
(μmol of NADH/per well/min.)
0.10 ± 0.004
DMSO 1% (v/v)
0.12 ± 0.005
0.14 ± 0.006*
21.98 ± 1.47*
0.20 ± 0.01*
50.14 ± 1.24*
0.38 ± 0.02*
67.60 ± 1.42*
0.46 ± 0.02*
91.84 ± 1.28*
0.57 ± 0.02*
However, when compared as a function of the Ag+ concentration, toxicity of AgNP appeared to be much higher than that of AgNO3. The cytotoxic effects of silver are the result of active physicochemical interaction of silver atoms with the functional groups of intracellular proteins, as well as with the nitrogen bases and phosphate groups in DNA . Regular green tea and decaffeinated green tea exhibit dose-dependent inhibitory activity in (H1299 cell line) human lung carcinoma cell line. Also the apoptosis mechanism is induced in the presence of polyphenols concentrations were less .
This may be due to their inhibitory activities in several signaling cascades responsible for the development and pathogenesis of the disease which are as yet not understood. Taken together, our data suggest that silver nanoparticles can induce cytotoxic effects on HEp -2 cells, inhibiting tumor succession and thereby effectively controlling disease progression without toxicity to normal cells and these agents an effective alternative in tumor and angiogenesis-related diseases.
In conclusions, plant based sliver nanoparticles possess considerable anticancer effect compared with commercial nanosilver. The reduction of the metal ions through the callus extracts leading to the formation of silver nanoparticles of fairly well defined dimensions. Use of AgNPs should emerge as one of the novel approaches in cancer therapy and, when the molecular mechanism of targeting is better understood, the applications of AgNPs are likely to expand further .
Materials and Methods
Plant material and preparation of the extract
Fresh Citrullus colocynthis leaves were collected from the Southeast coast of Parangipettai (Tamil Nadu) India. The specimen was certified by Botanical Survey of India (BSI) Coimbatore, and documented in the Herbaria of C.A.S. in Marine Biology, Annamalai University, India, during 2010. The experimental chemicals were purchased from Sigma Chemicals (Mumbai).
Sample preparation for synthesis of Silver Nanoparticles
One month old compact, hard greenish white callus derived from stem explants was used to obtain the callus extract in our lab . The callus was washed twice with sterile distilled water to remove medium components before grinding. Approximate 20 g of callus was grinded in 100 ml of sterile distilled water in mortar and pestle. The resulting extract was filtered through filter paper (What man No.1) and used for the synthesis of silver nanoparticles. 10 ml suspension of callus culture was added to 90 ml aqueous solution of silver nitrate (1 mM) solution separately
for reduction in to Ag+ ions and incubated at room temperature (35°C) for about 24 hours. The primary detection of synthesized silver nanoparticles was carried out in the reaction mixture by observing the colour change of the medium from greenish to dark brown. The silver nanoparticles were isolated and concentrated by repeated (4-5 times) centrifugation of the reaction mixture at 10, 000 g for 10 min. The supernatant was replaced by distilled each time and suspension stored as lyophilized powder for the optical measurements.
Atomic Force Microscope
Purified SNP in suspension was also characterized their morphology using a VEeco diNanoscope 3D AFM (Atomic Force Microscope). A small volume of sample was spread on a well-cleaned glass cover slip surface mounted on the AFM stub, and was dried with nitrogen flow at room temperature. Images were obtained in tapping mode using a silicon probe cantilever of 125 μm length, resonance frequency 209-286 kHz, spring constant 20-80 nm-1 minimum of five images for each sample were obtained with AFM and analyzed to ensure reproducible results.
Fourier Transform Infra Red Spectroscope
To identify Silver nanoparticles associated biomolecules, the Fourier transform infra red spectra of washed and purified Silver nanoparticles powder were recorded on the Nicolet Avatar 660 FT-IR Spectroscopy (Nicolet, USA) using KBr pellets. To obtain good signal to noise ratio, 256 scans of Silver nanoparticles were taken in the range of 400-4000 cm-1 and the resolution was kept as 4 cm-1
Determination of Nanoparticles concentration
Accurate determination of the size and concentration of nanoparticles is essential for biomedical application of nanoparticles . The concentration of nanoparticles to be administered at an nM level of determination by Marquis method .
Toxicity Study of SNp on Human Epidermoid Larynx Carcinoma (HEP -2) Cell Line
HEp-2 cell line was purchased from National Cell Centre, Pune (India). Cancerous cells were seeded in flask with MEM medium with 2-10% Fetal Calf Serum (FCS) and incubated at 37°C in a 5% CO2 atmosphere. After 48 h incubation period, the attached cells were trypsinated for 3- 5 mints and centrifuged at 1, 400 rpm for 5 mints. The cells counted and distributed in 24 well micro titer plates with 10, 000 cells in each well and incubated 48 hrs at 37°C in a 5% CO2 atmosphere for the attachment of cells to bottom of the wells.
Cell Treatment with silver nanoparticles
The amount of different concentrations of stabilized silver nanoparticles was added to each well in duplicates. The different silver nanoparticles concentrations (15, 30, 62, 125, 250, 500, 1000 μg/ml) were inoculated in to grown cell (1 × 10 4 cells/well) and the cell population was determined by optical microscopy at 24 and 48 hrs.
Cell viability was evaluated by MTT colorimetric technique . 200 μl of the yellow tetrazolium (MTT (3-(4, 5-dimethylthiazol-2)-2, 5 diphenyl tetrazolium bromide) without phenol red, are yellowish in color (Sigma) solution (5 mg/mL in PBS) was added to each well. The plates were incubated for 3-4 h at 37°C, for reduction of MTT by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan solubilized the MTT crystals by adding and quantified by spectrophotometric mean and then the supernatants were removed. For solubilization of MTT crystals, 100 μl DMSO was added to the wells. The plates were placed on a shaker for 15 mints for complete solubilization of crystals and then the optical density of each well was determined. The quantity of formazan product was measured by the amount of 545 nm absorbance is directly proportional to the number of living cells in culture. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles as a vechicle was calculated by [A]test/[A]control×100. Where [A]test is the absorbance of the test sample and [A]control is the absorbance of control sample
Lactate Dehydrogenase (LDH) leakage assay
Intracellular lactate dehydrogenase (LDH) leakage, a well known indicator of cell membrane integrity and cell viability was performed by the method of Borna et al., (2009) . 100 ∞ l of silver nanoparticles was added to a 1 ml cuvette containing 0.9 ml of a reaction mixture to yield a final concentration of 1 mM pyruvate, 0.15 mM NADH and 104 mM disodium hydrogen phosphate. After mixing thoroughly, the absorbance of the solution was measured at 340 nm for 45 seconds. LDH activity was expressed as moles of NADH used per minute per well.
Caspase 3 assay
Caspase-3 is an intracellular cysteine protease that exists as a proenzyme, becoming activated during the cascade of events associated with apoptosis. Caspase-3 cleaves a variety of cellular molecules that contain the amino acid motif DEVD such as poly ADP-ribose polymerase (PARP), the 70 kD protein of the U1-ribonucleoprotein and a subunit of the DNA dependent protein kinase . The presence of caspase-3 in cells of different lineages suggests that caspase-3 is a key enzyme required for the execution of apoptosis . The cells were lysed with the lysis buffer provided in the caspase 3 assay kit (Sigma, USA) and kept on ice for 15-20 minutes. The assay is based on the hydrolysis of the peptide substrate, Ac-DEVD-pNA, by caspase 3, resulting in the release of Ac-DEVD and p nitroaniline (pNA) which absorbs light significantly at 450 nm. Briefly, for 1 mL of the reaction mixture, 10 mL of the cell lysate from treated samples was added along with 980 mL of assay buffer, followed by addition of 10 mL of 20 mM caspase 3 colorimetric substrate (Ac-DEVD pNA). The cell lysates of the SNp-treated Hep-2 cells were then incubated at 37°C with the caspase 3 substrate for two hours and the absorbance was read at 450 nm in a double-beam UV- spectrophotometer (Shimadzu, Japan). The assay was also performed with noninduced cells and in the presence of caspase 3 inhibitor for a comparative analysis.
DNA fragmentation assay
DNA fragmentation has long been used to distinguish apoptosis from necrosis, and is among the most reliable methods for detection of apoptotic cells. When DNA strands are cleaved or nicked by nucleases, 3'-hydroxyl ends are exposed. 1 × 106 cells were lysed in 250 μL cell lysis buffer containing 50 mM Tris HCl, pH 8.0, 10 mM ethylenediaminetetraacetic acid, 0.1 M NaCl, and 0.5% sodium dodecyl sulfate. The lysate was incubated with 0.5 mg/mL RNase A at 37°C for one hour, and then with 0.2 mg/mL proteinase K at 50°C overnight. Phenol extraction of this mixture was carried out, and DNA in the aqueous phase was precipitated by 25 μL (1/10 volume) of 7.5 M ammonium acetate and 250 μL (1/1 volume) isopropanol. DNA electrophoresis was performed in a 1% agarose gel containing 1 μg/mL ethidium bromide at 70 V, and the DNA fragments were visualized by exposing the gel to ultraviolet light, followed by photography.
All experiments were done in duplicate and then values were expressed as mean ± standard deviation (SD). Statistical significance (5%) was evaluated by one-way analysis of variance (ANOVA) followed by Student's t-test (p < 0.05, SPSS 11 version).
The authors are gratefully acknowledge to the Director & Dean, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India for providing all support during the study period.
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