Almost 2 years since I finished my bachelors degree in engineering... Those good old days, which I miss and talking about those days, there are a lot of things which I regret doing and there are few which i wish I had done. There is one thing which I dont regret for doing, and that will be the research project carried out by us. 'Us' includes myself along with 2 more friends-Charan & Siddarth. After a long time I got a chance to revive those memories when we decided to present a paper on that project in a National level conference on Chemical Engineering- 'CHEMCORE-2011' held at MVJCE on 7th & 8th of Apr 2011.
Nanotechnology is one of the fastest growing sectors in the field of science and technology. Nanotechnology deals with the study and manipulation of particles/ materials having the dimensions in terms of nanometers. As we all know 1 nanometer is 1 billionth of a meter. The minute sizes of these particles impart some special properties to these materials which are likely to disappear as the size of the material increases. Some of these properties are desirable which makes nanotechnology a very important part in various industries. Among them we can find cosmetics and healthcare industries, these are huge benefactors of nanomaterials. The usage of Nanoparticles ranges from sunscreen compounds to drug delivery techniques. In these foresaid applications, the intimate contact between the Nanoparticles and living tissues is inevitable. Even though there are a lot of advantages of nanotechnology and Nanoparticles, the ill effects of these cannot be overlooked. This is where Nanotoxicology comes into picture.
Nanotoxicology is a field of science which deals with the study of the toxic effects of Nanoparticles on living entities. Toxicity can be defined as the degree to which a substance can cause damage to a living organism. There are many features of these Nanoparticles which impart toxicity to them. Among them is the quantum size, its greater surface area to volume ratio increases its chemical reactivity as well as biological activity. The shape, size, aggregation, surface charge and solubility influence its toxicity to a great extent.
Large number of Nanoparticles in the body may trigger the phagocytic mechanism, causing inflammation, overloading the body’s defenses, and leaving it susceptible to pathogen. The reactive oxygen species and free radicals formed due to high chemical reactivity leads to inflammation, damage to proteins, DNA and membranes etc. Also Nanoparticles penetrate the tissues and cells causing undesirable effects.
Incidentally there are few studies which have already been carried out regarding the toxic effects of the Nanoparticles on living organisms. Dr. Eva Oberdoster exposed the fishes to 0.5 ppm of fullerenes for a period of 48 hours to find it causing extensive brain damage in the fishes. Changes were observed in the gene markers in the liver as well hinting the affect on the entire physiology.
Similarly, Dr.Shosaku Kashiwada studied the accumulation and effects of fluorescent Nanoparticles on the Japanese killifish/ rice medaka and its eggs which are both transparent and has a short life cycle. It was found that the Nanoparticles were taken up by the blood stream and deposited throughout the body. Also there was a large accumulation of Nanoparticles in the yolk.
We carried out few experiments using ZnO, TiO2, Alumina and MWCNT to find out its toxic effects on plant cells, animal cells and on protein molecules. Some of the parameters of toxicity evaluated for cellular level are IC50, Cell viability and growth rate during seed germination. IC50 is the measure of the effectiveness of a compound to inhibit a biological or biochemical function. It indicates the amount of substance required to inhibit a given biological process by half. Here, the cell viability is measured in order to find the IC50 value. Cell viability is the determination of the living and dead cells in a sample. It involves selective staining to differentiate between living and dead cells in a sample. We have employed Non radioactive cell proliferation (MTS) assay to check the cell viability. It contains a novel tetrazolium compound (MTS) & an electron coupling reagent (phenazine methosulfate). MTS gets bioreduced by dehydrogenase enzyme living cells to form formazan, soluble in culture media. Absorbance of which is checked at 490nm.
Mice fibroblast culture is prepared and they were serially diluted, MTS was added and reading taken at 490nm using a microplate reader and the data plotted to get a standard graph. The ZnO & TiO2 Nanoparticles are added to the culture media in different concentrations, incubated for 4 & 24 hr periods then absorbance checked at 490nm. Graphs are plotted and the IC50 values are found out. It is found that the 4 hr exposure does not have any significant effect on cell viability but on 24 hour exposure, both have significant effects.
Phytotoxic effects was found by exposing the plant seeds namely, peas, groundnuts, chickpea and green gram to 2% Nanoparticles suspension and allowing it to germinate in a growth chamber for 5 days. The root length is measured, graphs are plotted. We also In order to confirm the results we kept onion bulbs immersed in Nanoparticle suspension for a period of 7 days and the growth of roots in observed.
To find the action of Nanoparticles on biomolecules, we chose to experiment with the effects on Alpha amylase enzyme. The enzyme activity was analyzed using DNS assay. Amylase breaks down starch into glucose. DNS acts on the aldehyde group in glucose and oxidizes into carboxyl group. At the same time DNS reduces to 3-amino,5-nitro salicylic acid. The absorbance is checked at 520 nm. The enzyme is incubated with I ml of 2% Nanoparticle suspension for 30 minutes at 37C. It was found that all the Nanoparticles reduced the enzyme activity significantly. Especially TiO2 & ZnO.
Nanoparticle-Serum protein interaction was also found out by incubating 1:1 volume of human serum protein mixture with 1% & 2% Nanoparticle suspension, incubating @ 37C for 30 min and then subjecting it to SDS-PAGE. The Human serum proteins were extracted from human blood using ethanol precipitation method. The protein bands start disappearing as the concentration of Nanoparticles increases.
From the battery of experiments conducted, we observed the following:
The ZnO and TiO2 Nanoparticles, which are widely used in sunscreen as pigments have undesirable effects on fibroblast cell upon prolonged exposure, and it is found that the ideal safe exposure time will be less than 4 hours at a stretch. Our phytotoxicity experiments, deviated from a previously published paper, where it was found that the Nano-Zn & Nano-ZnO were phytotoxic. There was a significant reduction in the enzyme activity upon exposure to the Nanoparticles, which may be due to various contributing factors. Protein degradation, allosteric inhibition, encapsulation by MWCNT or immobilization. The disappearance of bands in the SDS-PAGE indicates the breakage of peptide bonds in the serum protein by the Nanoparticles. The bands disappear at 1% concentration of TiO2
The Nanoparticles were characterized by using TEM and it was observed that ZnO & Alumina were the smallest Nanoparticles. The core diameter of MWCNT was found to be around 10 nm, making it easy for the protein /DNA molecules to enter into it. TiO2 is found to be around 10-30 nm. TiO2 shows larger surface area which is the reason for higher reaction rate and production of ROS.
Summarizing we can say that the Nanoparticles have toxic effects, and as the use of these Nanoparticles in various fields are increasing day by day, more and more organisms are susceptible of exposure to these particles and it may pose a health hazard to both the plants as well as animals including Humans. This calls for proper handling and disposal, and also optimization of these Nanoparticles in the future to make it more useful with minimal hazard.
Now I hope this says it all... And those 2 days of conference went really well, for I enjoyed everything except for the presentations. Not to forget those good looking girls from the chemical engineering dept of that college who were always around.. But i do have one regret..Shit..!!! I should have asked her for her number, she was drop dead gorgeous..I missed the opportunity.... :'(
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