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Nanotechnology is a field that is growing day by day, making an impact in every part of human life. Biological methods of "greener synthesis" of nanoparticles have proven in various experiments to be better methods due to slower kinetics, they offer better manipulation and control over crystal growth and their stabilization. Biosynthesis of nanoparticles by plant extracts is currently continuously going on. Plant extracts are very cost effective and eco-friendly and thus can be economic and efficient alternative for the large scale synthesis of nanoparticles.
In this project, biosynthesis of silver nanoparticles (AgNPs) will be carried out using aqueous extract of Juglans regia fruits outer shell and fruit body of Tribulus terrestris as agriculture waste and silver nitrate (AgNO3) at ambient temperature. For characterization of nanoparticles UV/Vis Spectrophotometer, Fourier Transform Infra-Red Spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) will be analyzed. Genotoxicity is a one type of toxicity which is cause of chromosomal aberration. In this project we will analyze the silver nanoparticles synthesized by Juglans regia fruits outer shell for genotoxic activity on human leukocyte cell culture and therefore the result will follow.
NANOMEDICINE, GREEN SYNTHESIS, UV- ANALYSIS, EDS, SEM, FTIR, GENOTOXICITY
Nanobiotechnology is a term that refers to the interection of nanotechnology and biology. This discipline allows scientists to create systems that can be used in various fields of biology. The medical application of nanotechnology is the nanomedicine. Nanomedicine has a large number of applications like, it can be used to diagnose and treat cancer, drug, detoxification, drug delivery, surgery etc. The nanoparticles used for nanomedicine are of three different types: polymeric, lipid-based and metal based. Polymeric nanoparticles are prepared using hydrophilic and hydrophobic bio degradable and biocompatible polymers for drug and gene delivery, respectively. For lipid based oil-in-water nano emulsions are used, where the oil droplets are reduced to less than 100nm in diameter. For metal based various metals such as gold, silver and iron are used in various shapes like, spheres, rods etc. The nanoparticles which are used for medical purpose have toxic effects on the living system. The toxic effects depend largely on the type of materials used and accumulation behaviour or the organs in which they accumulated. Sometimes the nanoparticles have toxic effect on cell's chromosome breakage, chromosomal mutation etc. The genotoxic effect of nanoparticles can be reduced to a large extent by the use of organic based nanoparticles . so, in the present study, we concentrated upon nanomedicine synthesis by green synthesis method by using outer shell powder of walnut (Juglans regia) and the dried fruit bodies of Tribulus terrestris which are nothing but an agricultural waste.
Material and method
Collection of plant materials
The outer shell of the Juglans regia dried fruit bodies of Tribulus terrestris was collected from local market for the synthesis of nanoparticles.
Preparation of fruit extracts
The outer shell of Juglans regia was grinded well with mortar and pestle to make a powder. Five grams of powder sample was mixed into 100 ml of deionized water then the mixture was boiled for 10 min. After cooling the fruit extract was filtered with Whatman No. 1 filter paper. The filtrate was stored for further use. The Tribulus terrestris extract was prepared separately by using the above method.
Synthesis of silver nanoparticles
The 100 ml of aqueous filtrate extract of Juglans regia was taken into 250 ml of conical flask. Then the extract was mixed into Silver nitrate (AgNO3) to make the final volume concentration of 1 mM solution. The reaction mixture was kept into dark room condition until the colour change was occurred. Then after occurrence of colour characterization was done of the silver nanoparticles reaction solution colour changes have observed for characterization of silver nanoparticles.
The above procedure is repeated for synthesizing silver nanoparticles form Tribulus terrestris plant extract.
Genotoxicity testing of the nanoparticles
5ml of media (RPMI) 1640- ready mix was mixed with 0.5ml of blood. Then the RPMI mixed blood is incubated for 72 hrs. Every 24 hours the CO2 is released by taking it out of the incubator and mixed well. Now at 71st hour it is treated with previously prepared nanoparticle solution and was kept in incubator again. After incubation for 1 hour 2 drops of colchicines were added. Then it was kept for 15 min in incubator.
After incubation it was centrifuged at 2000 rpm for 5 min and then the supernatant was discarded. After that 6ml of hypotonic solution (0.075M KCl) was added and it was incubated again for 6-7 min. The mixed solution was then centrifuged at 2000 rpm for 5 mins and supernatant was discarded. Then 6ml of freshly prepared fixative (Methanol: Acetic Acid= 3:1) was added and mixed well and kept for overnight in the incubator.
Then it was centrifuged at 2500 rpm for 7 min. The process was repeated until the white pellet was obtained. Then the supernatant was removed and slides were prepared with the white pellet. The slides was then stained with giemsa solution (4ml giemsa+ 46ml double distilled water) for 4 min and again in double distilled water for 1 min. Then the slides were air dried and observed under fluorescence microscope for any chromosomal aberration.
Standardizing protocol in progress. Previously we worked with a specific concentration of silver nanoparticles for genotoxicity testing, but we did not get any result out of it. So, we are continuing our work with more silver nanoparticles solutions of different concentrations of and the result is awaiting.
As a agriculture waste outer shell of walnut (Juglans regia) and dried fruit bodies of Tribulus terrestris is used for the synthesis of silver nanoparticles and its genotoxicity testing is done in human leukocyte culture. Different concentrations of silver nitrate solution is being used to prepare the nanoparticles. Gentoxicity testing of prepared nanoparticles are also in the process to watch out the expected DNA damage and other chromosomal abnormalities and its severances. Previously this genotoxicity testing was done (Acharya and Panda, 2010). Which provided evidence of severe DNA damaging potential of silver nanoparticles in a dose dependent manner.
In conclusion, the result of the above work is still waiting as the standardization of the protocol is on the process. The awaited result of the current study expected to reveal some important findings on various effects of introducing silver nanoparticle synthesized from agricultural waste as a nanomedicine in human leukocyte cell culture. Furthermore the result will show the feasibility of using AgNP-P in a large scale manner for producing nanomedicines.