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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 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 refer 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 have a large number of application 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. Poly meric nanoparticles are prepared using hydrophilic and hydrophobic biodegredable and biocompatible polymers for drug and gene delivery, respectively. For lipid based oil-in-water nanoemulsions 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.
Importance of Nanomedicine
Nanomedicine is the medical application of nanotechnology. Nanomedicine ranges from the medical applications of nanomaterials, to nanoelectronic biosensors, and even possible future applications of molecular nanotechnology. Two forms of nanomedicine that have already been tested in mice and are awaiting human trials are using gold nanoshells to help diagnose and treat cancer, and using liposomes as vaccine adjuvants and as vehicles for drug transport. Similarly, drug detoxification is also another application for nanomedicine which has shown promising results in rats. A benefit of using nanoscale for medical technologies is that smaller devices are less invasive and can possibly be implanted inside the body, plus biochemical reaction times are much shorter. These devices are faster and more sensitive than typical drug delivery.
Types of Nanomedicine
Nanomedicine is of basically two types:
1.Organic/Biopolymer based: Here various organic substances are used for nanomedicine synthesis.Oil may be used for oil particle synthesis of <100 nm diameter and sometimes gelatine which is nothing but a biopolymer.
2.Inorganic based: Here inorganic substances such as gold,silver etc. are used.
Toxicity of Nanomedicine
Among the limitations of nanomedicine, its toxicity is very much important.The toxicity parameter depends on the materials used for synthesis.Organic substances have low toxicity whereas inorganic substances have high toxicity.Our body may be affected by various ways by the use of nanomedicine,sometimes even genotoxicity.
Genotoxicity of Nanomedicine
Genotoxicity means toxic effect on cell's chromosome.Chromosome may be affected in various ways like chromosomal aberrations,ring chromosome formation etc.
Current work and aim
Nanomedicine is still a growing field,so lots of works are going on in this field.Scientists are working to make it less toxic and more effective.Works are going on to use nanomedicine for treating cancer in the near future.
Collection of plant materials
The dried fruit bodies of the Juglans regia was collected from local market in Chennai, India. The fruit bodies were rinsed with water thrice followed by Milli-Q water to remove the fine dust materials and then, the fruit bodies were dried under direct sun light for 1 week to completely remove the moisture.
Preparation of fruit extracts
The dried fruits were pulverized well with mortar and pestle to make a powder. Five grams of powder sample was mixed into 100 ml of deionized water and the mixture was boiled for 10 min. After cooling the fruit extract was filtered with Whatman No. 1 filter paper. The filtrate was stored at four degree centigrade for further use.
Synthesis of silver nanoparticles
The 100 ml of aqueous filtrate extract of Juglans regia was taken into 250 ml of Erlenmeyer 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 con-dition until the colour change was arisen. The reaction solution colour changes have observed for the characterization of silver nanparticles.
Characterization of nanoparticles
The synthesized silver nanoparticles were characterized by UV-vis spectroscopy periodically for a week in order to observe rapid reduction silver nanoparticles by the action of plant extracts.
The biologically reduced brown colour solution mixture was scanned by Perkin Elmer preclsley, Lambda 25 instrument operated at a resolution of 1 nm. In this analysis the fruit extract without adding the silver nitrate was used as a control. The FTIR is per-formed to the extract which was exposed before and after addition to the silver nitrate solution. The samples were mixed with KBr to make a pellet and it was placed into the sample holder. The spectrum was recorded at a resolution of 4 cm âˆ’1 . X-ray diffraction pattern was obtained from lyophilized silver nanoparticles by powder diffraction method, where it gives grain size and shape of the particles by h, k and l index value.
The morphological analysis of the particle was done with trans-mission electron microscopy (TEM). The drop of aqueous silver nanoparticle sample was loaded on carbon-coated copper grid and it was allowed to dry for an hour. The TEM micrograph images were recorded on JEOL instrument 1200 EX instrument on carbon coated copper grids with an accelerating voltage of 80 kV. The clear micro-scopic views were observed and documented in different range of magnifications. The synthesized silver nanoparticles structure morphology and size of the nanoparticle were further character-ized by the atomic force microscopy (AFM) images. The microscopic images were recorded with silicon cantilever with force constant 0.22-0.77 N/m, tip height 10-12 nm in the contact mode.
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 slide 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.
Work in progress. Result is awaiting.