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Better comprehension of molecular and cellular biology has lead to anticancer therapies that are able to overcome the biophysical, biomedical and cellular barriers and expected to give lesser side effects and better antitumor efficacy. Nanoparticles are unique due to their small size and surface modifiability which paves the way for development of exciting new methods of nanoparticle synthesis with variable composition and a great numbers of functions. They can be used as drug delivery system, imaging, as well as therapeutic agents [1-3]. Surface modification allows for better and more accurate targeting of cancer cells compared to conventional methods and there is a great potential that nanoparticles would meet the pressing need for better cancer therapy. Silver nanoparticles were therefore tested in vitro against two cancer cell lines from ATCC for their potential anticancer activity.
Materials and Methods
All chemical used in this work were analytical grade Silver nitrate and starch were purchased from Fluka and were used without any purification. All the solutions were prepared in double distilled and deionized water.
Synthesis of Ag nanoparticles
20mM of AgNO3 was dissolved in 100mL deionized water to get approximately 2% (w/v) of Ag+1 aqueous ionic solution. Similarly (2% w/v) starch solution was prepared by dissolving 2g of it in 100mL of deionized water and heated at 100Â°C to get transparent solution then it was cooled to room temperature. To this starch solution, 1mL of silver ionic solution was dissolved to get 0.2mM Ag aqueous solution. The resultant solution was stirred for over night at room temperature. During preparation, the solution turned to the characteristic yellowish brown color, then to grayish black, indicating the formation of silver nanoparticles. The colloidal solution of silver nano-particles were allowed to characterized for different techniques for example UV-visible, TEM, XRD and etc The silver nanoparticles thus prepared in aqueous starch solution were stable for more than two months without any change in the surface plasmon resonance as indicated from the absorption spectra at room temperatures, (Figure 1.) showing that the starch was not only a good reducing as well as stabilizing agent for the silver nanoparticles.
UV-visible spectra of colloidal solution of silver nanoparticles was collected by using UV-1700 UV-visible spectrophotometer (SHIMADZU Japan)
XRD (Phillips PW 3040/60 X Pert Pro) powder diffractometer was used for analysis of thin film of silver nanoparticles using nickel filtered copper K alpha radiations with generator setting of 40 KeV. The diffraction pattern were recorded in the range, 50 ~700. Thin film of silver nanoparticles was made by evaporating small amount of their colloidal solution on glass slide.
FT-IR analysis of the silver nanoparticles and starch was performed by Thermo Scientific Nicolet 6700 FT-IR 3.
HRTEM images of the silver nanoparticles were acquired by using FEI Company's 300 keV TEM equipped with a field-emission electron gun. The images were digitally recorded by using a 4k Ã- 4k pixel Charged Couple Devices (CCD) camera manufactured by Gatan Inc. (Model: Ultrascan 4000). The TEM magnification calibrations were performed prior to the image
Results and discussion
The reaction solution became red and its UV/vis absorption spectrum show a characteristic absorption band at 420 nm , indicating the formation of metallic Ag nanoparticles.
Figure 1: UV-vis absorption spectra of (a) freshly prepared colloidal AgNPs [Ag]aq = 0.2mM, while [Starch]aq 2% by (w/v), (b) after 15 days, respectively
By the analysis electron diffraction pattern and XRD, the resultant particles were confirmed to be pure Ag with a face centered cubic (fcc) structure. From the HRTEM analysis, it was found that the mean diameter of Ag nanoparticles first decreased and then approaches to a constant value after overnight constant stirring. In addition, the starch concentration had significant influence on the size of Ag nanoparticles. It was suggested that starch polymer cover the surface of Ag nanoparticles to prevent them to agglomeration.
Figure 2: XRD spectra of Ag nanoparticles as synthesized, [Ag]aq = 0.2mM, while [Starch]aq 2% by (w/v) and deride with acetone
X-ray diffraction patterns reveals that all silver nanoparticles are crystalline, with face centered cubic (fcc) packing arrangements of bulk metals. In figure 2, the four distinct XRD peaks at 2Î¸ values of 38.4â-¦, 44.3â-¦, 64.4â-¦ and 77.4â-¦ represent the (111), (200), (220) and (311) crystalline planes of the face centered cubic structure [5-7] The sizes of the nanoparticles were estimated to be 10-15nm, by analysing the XRD spectra using Scherrer's formula. The silver particle size distribution estimated from the XRD spectra shown in figures 2 which has been further confirmed by TEM images. A typical TEM image of the ultra thin film made from solution shown in figure 3. An examination of figure (a) depicts that the nanoparticles are nearly spherical and the size distribution lies in the range 5-20 nm, with average size 10nm. The electron diffraction rings shown in the figure (b) could be indexed to face-centred cubic phase with (111), (200), and (220) planes of silver crystals . These results are consistent with those of the XRD peaks shown in figure 2.
Figure 3: Typical HRTEM images of Ag nanoparticles. as synthesized, [Ag]aq = 0.2mM, while [Starch]aq 2% by (w/v) and deride with acetone
Role of Starch
In this work starch is performing two roles it not only function as reducing Ag+1 to Ag0 but also stabilizes silver in to nano size. Starch consists of two types of molecules, amylose and amylopectin. Both consist of polymers of Î±-D-glucose units in the 4C1 conformation forming long chains of single helical structure  This single helix is responsible for the characteristic binding of starch molecule to the negatively charged iodine molecules in characteristic iodine test (for example, the polyiodides; chains of [I3]- and [I5]- forming structures such as [I93]- and [I153]-; note that neutral I2 molecules may give polyiodides in aqueous solution and there is no interaction with I2 molecules except under strictly anhydrous conditions) similarly the surfaces of many metallic nanoparticles of Au, Ag, Pt and Cu are negatively charged , rich in free electron. Likewise negatively charged surface of silver can be caged by helix of starch polymer where each turn of the helix holds silver nano-cluster to stabilized silver in nanosize.
Since starch is polymer containing a hydroxyl groups as capping agents. Hence, it was suggested that Ag nanoparticles were capped by starch molecules after they were formed. To investigate process of capping we have to analyze the FT-IR spectra of starch and Ag nanoparticles.
The infrared spectra of the dried nanoparticles of silver using starch as a template presented in figure 9. An extremely broad band due to hydrogen bonded hydroxyl groups (O-H) appears at around 3425 cmâˆ’1 for starch, which get disappear in case of silver nanoparticles. It can be found that the IR spectra of silver nanoparticles as well as that of starch are more or less similar. Some minor changes in the fingerprint region of starch such as the peaks at 1083 and 1022 cm-1 attributable to the anhydro-glucose ring O-C stretch in the case of starch appears to be broad in the case of silver, indicating a possible coating of the silver nanoparticles with starch. Absence of a band at 1384 cm-1 indicates the complete reduction of Ag+ to Ag0.
Figure 4. FT-IR spectrum of: (a) pure starch and (b) starch caped silver nanoparticles
On the basis of our findings we propose the following mechanism presented in figure 7 for the formation of Au nano particles using the above synthesis procedures.
Figur 4. The postulated mechanism of synthesized starch caped silver nanoparticles.
Antibecterial Activity Test
Now a days nanotechnology has expended its application in biomedical field including fighting and preventing of diseases using atomic scale funuctional materials. Interestingly, silver nanoparticles have been considered to be antibacterial for wide verity of stains. A recent study indicates that the bactericidal effect of silver nanoparticles mostly depends on size of particles and smaller is the better i.e. 1-10 nm which have direct interaction with the bacteria . We have studied the antibacterial effect of starch caped silver nanoparticles by varying their concentrations. Different loadings of AgNP in different bacterial stains show remarkable variation in its effect. It is observed that increasing the concentration of AgNP (from 6.25ppm to 200ppm) in crease in width of inhibition zone concludind that an increase in concentration of AgNP increase the antibacterial effect of AgNP. Pseudomonas shows maximum vulnerability to AgNP even at lower concentration (Figure 7) but approximately all test stains show inhabitation above 50ppm AgNP concentration. While antibacterial activity in case oflower concentration, Staph aurous does not show remarkable effect of AgNPs, similarly E. coli also has no effect at 6.25ppm concentration of AgNP. Both lower and higher concentration of AgNP show remarkable antifungal activity against Candida sp.
Figure 6: Antibectrial effect of Ag nanoparticles. as synthesized, [Ag]aq = 0.2mM, while [Starch]aq 2% by (w/v) and deride with acetone
Table 1: Zone of inhabitation(mm) of Ag nanoparticles with different concentration.
Zone of inhibition (mm)
Figure 7.Effect of Ag nanoparticle on different bacterial stains
Anticancer, activity test.
Synthesized nanoparticles were diluted with deionized water from a concentration of 200ppm to 20ppm and then filter sterilized from 0.2Âµm filter. A hundred fold dilution of the filtered stock was then made with RPMI 1640 medium, and then a serial dilution was made down to 0.024ppm. Human cancer cell lines HT144 (malignanat melanoma of skin) and H157 (squamous cell lung carcinoma) from ATCC were grown in 96 well tissue culture grade plates. Cytotoxicity was assayed by Sulfrhodamine B (SRB) test  and trypan blue exclusion test.
For both cell lines 10,000 cells, 100Âµl per well were seeded on day 0. The plates were incubated at 37Â°C, 5% CO2 to allow the cells to form a monolayer. On day 1 the dilutions were added in quadruplicate by adding 100 Âµl of the each dilution with appropriate blanks as well as negative and positive controls. Cells were observed under the inverted microscope for any morphological change immediately after adding the dilutions. Dye development with Sulfrhodamine B was recorded after 48 hrs at 490nm.
Cancer Treatment Mechanism
Malignant cells have epidermal growth factor receptor (EGFR)
Immune system poduce Anti-bodies (anti- EGFR) that have -SH and -NH2 binding sites.
By conjugation of AuNPs with -SH and -NH2 sites of anti- EGFR, they can be specifically target the cancer cells.
anti- EGFR-AuNPs complex attached with affected tissue via EGFR and cause the damage to the affected tissue.
In vitro growth of American Type Tissue Culture cell lines (ATCC) H157 & HT144
A) Origin: lung carcinoma b) Cells laid on to 96 well plates
(a) Culture growth in flask, (b) unstained normal culture growth (c) controls (live cells stained, (d)Incubation with silvernanoparticles (0.1 ppm)
Cytotoxic activity was observed for both cell lines against the silver nanoparticles. 50% Growth Inhibition (ID50) was observed at 0.39ppm. It was also observed that the nanoprticles had an instant effect after treatment with the silver nanoparticles as observed under the inverted microscope. The cells clearly showed a morphological change as they lost their adherent nature and became rounded in shape. In contrast, normal blood lymphocytes from healthy individuals did not show any effect of nanoparticles and remained viable after 48 hrs of treatment. Cell viability was also confirmed on duplicate plates by Trypan blue exclusion test
Silver nanoparticles showed a clear cytotoxic effect on adherent cancer cell lines of skin and lung. The particles had an instant effect on cancer cell lines and were nontoxic towards normal cells. Further experiments are needed to be carried out to show a comparative result of adherent cancer as well as adherent non cancer cell. Toxicity studies need to be carried out thoroughly before they can be launched as anticancer agents in clinical settings.