Evaluation of Biogenic Characteristics of Iron Nanoparticles and Its Alloys in Vitro

This research was performed to study the effect of iron nanoparticles with variable size, its oxides and alloys on living systems. Analysis of the results showed manifestation of acute toxicity over a wide concentration range for FeCo alloy nanoparticles and low toxic characteristics for iron nanoparticles and its oxides. To develop acute toxicity high concentrations of iron nanoparticles and iron oxide nanoparticles were required. Calculation of toxicity index allowed to rank studied nanoparticles samples in descending order of toxic action: FeCo →Fe → Fe3O4 →Fe = Fe3O4. Correlation was direct between particle size and the intensity of the impacton living systems. According to obtained data,the smaller the particle size the greater its biological activity. The presence of cobalt in the alloy FeCo nanoparticles contributed to the increase of biological activityand as a consequence increase toxic effects. These studies revealed biological activity of iron nanoparticle and its oxides without development of toxic effect at doses of 0.25 0.000718 M which characterizes them as biotic.


Introduction
Evaluation of nanomaterials characteristics is one of the most important trends in modern biology (Jiang et al., 2015;Liu et al., 2015).Search for nanoforms with no toxic effect both in small and large quantities is necessary considering accumulation of nanomaterials in the environment and development of preparations based on them (Ajdary et al., 2015;Gong et al., 2015;Dragavon et al., 2012;Melancon et al., 2012).
Effective and general method to study the biological activity of nanoparticles in relation to living systems is to assess value of microorganism's bioluminescence under their influence.
Preparations based on metal nanoparticles and their derivatives are widespread in biology.Therefore this study shall include assessment of biological activity not only of metal nanoparticles but also of their oxides and alloys with other metals.

Materials and Methods
The test object was genetically engineered luminescent strain Escherichia coli K12 TG1 constitutively expressing luxCDABE genes of natural marine microorganism Photobacterium leiongnathi 54D10 which was produced by the SIS "Immunotech" (Russia, Moscow) in lyophilized condition under the commercial name "Ecolum".Immediately before the test the strain Echerichia coli K12 TG1 was restored by addition of chilled distilled water.The suspension of bacteria was maintained at +2-4 °C for 30 min after which the temperature of the bacterial suspension was brought to 15-25 °C.
The inhibition of bacterial luminescence was tested by placing into the cells in 96-well plates the test substance and the suspension of luminescent bacteria in a 1:1 ratio.Subsequently, the tray was placed in the measuring unit of an Infinite PROF200 microplate analyzer (TECAN, Austria) which dynamically registered the luminescence intensity for 180 min at intervals of 5 min.The effects of the nanomaterials on the intensity of bacterial bioluminescence (I) were evaluated using the formula: = intensities of the control and experimental samples, respectively, from the 0-th and n-th minutes of measurement.Three threshold levels of toxicity are taken into account: 1. less than 20 -a sample of "non-toxic" (luminescence quenching ≤ 20%); 2. 20 to 50 -a sample of relatively toxic (luminescence quenching 50%); 3 is equal to or greater than 50 -the sample is toxic (luminescence quenching ≥ 50%).
In studies commercial samples of metal nanoparticles "Advanced Powder Technology", Russia were used (Table 1).
Samples of nanoparticles suspensions for test were prepared under 4 М -6×10 -6 М concentration and treated by ultrasonic for 30 min.All experiments were done in triplicate and processed by variation statistics using the software package Statistika V8 (StatSoft Inc., USA).

Results
Obtained results characterize the dynamics of inhibition of bacterial bioluminescence over time and demonstrate dependence on the nature of the investigated nanomaterials along with their forms and concentrations.
The contact of E. coli with increasing concentrations of Fe а nanoparticles in the range of 4 to 0.25 M lead to complete suppression of illumination in the test object and manifestation of an acute toxicity in relation to living system in the first 80-90 minutes of contact (Figure 1, a).In contrast to samples of nanoparticles, Fe-Co alloy nanoparticles demonstrated highly toxic prolonged effect.So the range of concentrations from 4 to 0.00625 M (Figure 3) produced complete suppression of luminescence of bacteria and is characterized as acute toxic.50% inhibition of bacterial bioluminescence is observed at concentration of 0.003125 M. The above results were used to construct dose-response curves (Figure 4) for each of the tested nanoparticle samples.The ЕС 50 values corresponding to the molar concentrations of iron nanoparticles and its oxides causing 50% inhibition of bacterial bioluminescence at different durations of exposure were also determined (Table 3).

Discussion
The results showed that sols of iron nanoparticles and iron oxides nanoparticles have biological activity in a wide range of concentrations and demonstrate biotic characteristics in relation to living systems both in small and relatively high doses.
Our results correspond to the results of other authors by toxicity of nanoparticles in the bioluminescence inhibition test.The order of decreasing toxicity from zinc oxide nanoparticles to iron dioxide nanoparticles (ZnO > CuO > NiO > Fe2O3) has been described in the study of Ko KS et al., (2014) and slight toxic effect of iron nanoforms comparing with nanoparticles of other metals has been noted.Low toxicity of iron nanoparticles preparation which toxicity (EU50) appears at doses above 100mM has been described in the study of Deryabin D. G. et al., (2011).Similar results confirming low toxicity of iron nanoparticles are described in conducting of cultural research.The results of these studies demonstrate the ability of iron nanoparticles to inhibition of vital functions of bacteria cells but do not lead to the development of acute bactericidal effect in small doses (Tilston et al., 2013;Ismail R. A., 2015).
At the same time FeCo alloy nanoparticles showed signs of acute toxicity in relation to the test object in small doses as opposed to iron nanoparticles and iron oxide nanoparticles.Combined use of iron and cobalt nanoparticles leads to potentiation of toxic effect.In presence of iron and cobalt nanoparticles ЕС 50 increases in dozens of times (83) (by comparison Fe b ЕС 50 -0.25 and FeCo ЕС 50 alloy -0.003-) at all stages of contact, i.e. toxicity is characterized by prolonged manifestation.Such effect of alloy nanoparticles is determined by the chemical composition of the compounds and in particular by the presence of cobalt (30% of alloy).It is known that cobalt oxide nanoparticles in small quantities lead to the formation in cell cultures of reactive oxygen species and activation of oxidases, development of oxidative stress in cells and activate apoptotic factors (Chattopadhyay et al., 2015).Presumably the presence of cobalt in the alloy leads to an increase of toxic properties of iron reducing the toxic effect of its own as described for other combinations of nanoparticles (Li et al., 2015).
Comparison of effect of iron nanoparticle and its oxides allows to make a supposition that biological activity depends not only on the chemical composition but also on the size.Composition of studied preparations includes nanoparticles of size from 80 to 95 nm including various forms of oxides and metals.Individual comparison of iron nanoparticles and iron oxide nanoparticles showed an increase of biological activity with the decrease of particles size.Changes in the structure of the electron shell of the particles occurring in contact with the biological system explain the differences in the properties of different size particles (Chihao Liow, 2012;Encai Hao and George C. Schatz., 2004).Similar results with regard to the connection between size and exerted effect has been described earlier (Yang L, 2015;Müller K., 2007;Raynal I., 2004;SS Yu, 2011;Yu SS, 2012).
Change of direct dependence between the size and biological effect observed when comparing the iron nanoparticles and their oxides may be explained by changes in the oxides structure.Based on literature data some form of oxide nanoparticles and nanoparticles derivatives may have not only toxic effect on the cell but also safety effect e.g. by inactivating the active forms of oxygen formed in the development of oxidative stress (Seung Soo Lee, 2013;Shcherbakov A. B., 2015;Yongye Liang, 2011 ).

Conclusion
Thus, iron nanoparticles and nanoparticles of its oxides in relation to E.coli were characterized as non-toxic or slightly toxic.All test variations of nanoparticles possess similar biological activity stable over time with low variability in the narrow concentration range from 0.25 to 0.05 M.These concentrations can be defined as a biologically active in relation to living systems.

Figure 4 .
Figure 4.The relative values of luminous intensity for a luminescent strain of E. coli in contact with iron nanoparticles of different size and form; the ordinate is the relative value of luminescence intensity in comparison with the control