Comet Assay in Assessing the Safety of Metal Nanoparticles for Biotechnology and Medicine

Dybkova S. M.

NANOTECHNOLOGY

Scentific article

Genotoxicity is property of chemical, physical and biological factors exert adverse effects on the genetic structure of a living organism. The study of genetic damage as a result of certain substances is a key element determining the risk of new pharmaceutical agents based nanomaterials biotech and medical devices, as the primary DNA damage can trigger malignant degeneration of cells. In recent years, the new techniques that allow you to record DNA damage are developed. However, these methods do not have sufficient sensitivity and specificity, which is so necessary to monitor a wide range of primary DNA damage. The main advantage of the Comet assay is the ability to determine the level of damage in isolated eukaryotic cells of different origins, as well as an integrated assessment of the integrity of the genome. The method is extremely sensitive and fast. The aim of the study was to study the possibility of using the method of alkaline gel electrophoresis of isolated cells (Comet assay under alkaline conditions) to assess the safety of metal nanoparticles for biotechnology and medicine. The Comet assay is to register the differences in electrophoretic mobility in a constant electric field native DNA and fragments of DNA of lysed cells. It results in relaxation of DNA macromolecules and formation of fragments. When using the method of alkaline gel electrophoresis of isolated cells to assess safety of nanoparticles of metals alkaline treatment drugs lysed cells is destruction of duplex of DNA and allows individual threads regardless migrate in an electric field. In this case, the DNA migrates to the anode and forms electrophoretic trace that resembles “comet tail”, whose properties depend on the experimental DNA damage. We first demonstrated the possibility of applying the Comet assay under alkaline conditions to assess the safety of metal nanoparticles for biotechnology and medi- cine. Evaluation of metal nanoparticles genotoxicity in vitro by Comet assay under alkaline conditions was carried out with the involvement of cells culture lines: U937, CHO-K1, HEp-2, L929, PTP and sperm of bull. Evaluation of metal nanoparticles genotoxicity in vivo by Comet assay under alkaline conditions was performed involving of laboratory animals – mices and rats. Identified genotoxic properties of gold nanoparticles with size 7, 10, 17 and 20 nm, iron oxide nanoparticles with size 14, 18 and 23 nm, cobalt nanoparticles with size 5 nm, nanoparticles of zinc with size 20 nm and bismuth cubic nanoparticles with size 20 nm. Were not genotoxic gold nanoparticles with size 30, 40 and 57 nm, silver nanoparticles with size 30 and 50 nm, iron nanoparticles with size 40, 77 and 100 nm, nanoparticles with size copper 20, 40 and 70 nm. Analyzing the results of genotoxicity testing metal nanoparticles in vitro and in vivo can be concluded about the high level it’s correlation: not genotoxic gold nanoparticles with size of 30-57 nm, silver nanoparticles with size 30 nm and iron nanoparticles (Fe0) with size 40 nm. These nanoparticles can be recommended for biotechnological and medical applications. Realized testing the safety of metal nanopar- ticles of in terms of genotoxicity in vitro and in vivo made it possible to recommend Comet assay under alkaline con- ditions to assess the safety of metal nanoparticles. This method is included in the system of methods Methodical recommendations “Safety assessment of medical nanopreparations” of the State Expert Center of the Ministry of Health of Ukraine. Comet assay under alkaline conditions in vitro and in vivo is adequate for evaluation of safety of metal nanoparticles for biotechnology and medicine.

genotoxicity, metal nanoparticles, Comet assay under alkaline conditions

• Дибкова С. М. Визначення ушкоджень ДНК наночастинками металів, перспективних для біотехнології / С. М. Дибкова, М. Є. Романько, Т. Г. Грузіна [та ін.] // Біотехнологія . – 2009. – Т. 2, № 3. – С. 80-85.
• Загальні етичні принципи експериментів на тваринах // Ендокринологія. – 2003. – № 8, Т. 1. – С. 142–145.
• Методические разработки к практикуму по коллоидной химии / Под ред. А. В. Перцова. – М.: Изд-во МГУ, 1976. – 132 с.
• Методичні рекомендації «Оцінка безпеки лікарських нанопрепаратів». – Київ, 2013. – 108 с.
• Методичні рекомендації «Оцінка біобезпеки наноматеріалів органічної та неорганічної природи методом визначення генотоксичності лужним гель-електрофорезом ізольованих еукаріотичних клітин». – Київ, 2010. – 24 с.
• Патент України на корисну модель МПК (2009.01) G01N33/00 G01N33/48. Спосіб оцінки генотоксичних властивостей наноматеріалів / С. М. Дибкова, О. В. Годовський, М. Є. Романько [та ін.] // Заявл. 10. 09. 2009; Опубл. 25.03.2010; Бюл. № 6. – 10 с.
• Чекман І. С. Наногенотоксикологія: вплив наночастинок на клітину / І. С. Чекман, М. О. Говоруха, А. М. Дорошенко // Український медичний часопис. – 2011. – №1 (81). – с. 30-35.
• Didenko V. Methods in Molecular Biology. In Situ Detection of DNA Damage. Methods and protocols / V. Didenko. – Humana Press, 2002. – 279 р.

«Bulletin of problems biology and medicine» Issue 3 part 3 (112), 2014 year, 279-283 pages, index UDK 577. 151:579. 864. 1