BIOMEDICAL APPLICATION OF BIOGENIC FERRIHYDRITE NANOPARTICLES C.G. CHILOM 1 , D.M. GĂZDARU 1 , M. BĂLĂSOIU 2,3 , M. BACALUM 4 , S.V. STOLYAR 5,6 , A.I. POPESCU *1 1 Department of Electricity, Physics of Solid and Biophysics, Faculty of Physics, University of Bucharest, Romania 2 Joint Institute for Nuclear Research Dubna, Russia 3 Department of Nuclear Physics, National Research Institute for Physics and Nuclear Engineering Horia Hulubei (IFIN-HH), Bucharest, Romania 4 Department of Life and Environmental Physics, National Research Institute for Physics and Nuclear Engineering Horia Hulubei (IFIN-HH), Bucharest, Romania 5 Siberian Federal University, Krasnoyarsk, 660041, Russia 6 Kirensky Institute of Physics, SB RAS, Krasnoyarsk, 660036, Russia * Corresponding author: [email protected]Received December 21, 2016 Abstract. Spectroscopic properties of biogenic ferrihydrite nanoparticles, produced by Klebsiella oxytoca, were investigated. Their interaction with two serum albumins was moderate. A very weak stabilization of protein structure to denaturation, in the presence of nanoparticles, was put in evidence. Nanoparticle cytotoxicity and their hemolytic effect were studied on healthy and tumour cells. Key words: bacterial nanoparticles, albumins-ferrihydrite nanoparticles interaction, cell lines, cytotoxicity, hemolytic effect. 1. INTRODUCTION Studies of nanomaterials represent a promising research field due to their wide range of applications in medicine, biotechnology, energetics, optics, environment, and so on. The conventional methods used for nanoparticle synthesis involve chemical techniques known as being both material and time consuming and also engendering toxic byproducts. Moreover, the nanoparticle synthesis requires special temperature and pressure conditions. A more environmentally and economically friendly alternative to nanoparticle synthesis is encountered in living organisms (which can range from bacteria to fungi and plants) [1]. We will remind here, that magnetotactic bacteria are able to produce magnetosomes (magnetic nanoparticles) whose envelope S-layer forms calcium carbonate layers and diatoms that produce siliceous materials [2–4]. One of the most used method for biosynthesis of metal nanoparticles (i.e., of gold, silver, magnetite, iron, zinc, etc.) is based on the use of bacteria like Bacillus licheniformis, Bacillus subtilis, Klebsiella pneumoniae, Klebsiella oxytoca, etc. [1]. Romanian Journal of Physics 62, 701 (2017)
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BIOMEDICAL APPLICATION OF BIOGENIC FERRIHYDRITE
NANOPARTICLES
C.G. CHILOM1, D.M. GĂZDARU1, M. BĂLĂSOIU2,3, M. BACALUM4,
S.V. STOLYAR5,6, A.I. POPESCU*1
1Department of Electricity, Physics of Solid and Biophysics, Faculty of Physics,
University of Bucharest, Romania 2Joint Institute for Nuclear Research Dubna, Russia
3Department of Nuclear Physics, National Research Institute for Physics and Nuclear Engineering
Horia Hulubei (IFIN-HH), Bucharest, Romania 4Department of Life and Environmental Physics, National Research Institute for Physics and Nuclear
Engineering Horia Hulubei (IFIN-HH), Bucharest, Romania 5Siberian Federal University, Krasnoyarsk, 660041, Russia
6Kirensky Institute of Physics, SB RAS, Krasnoyarsk, 660036, Russia *Corresponding author: [email protected]
Received December 21, 2016
Abstract. Spectroscopic properties of biogenic ferrihydrite nanoparticles,
produced by Klebsiella oxytoca, were investigated. Their interaction with two serum
albumins was moderate. A very weak stabilization of protein structure to denaturation,
in the presence of nanoparticles, was put in evidence. Nanoparticle cytotoxicity and
their hemolytic effect were studied on healthy and tumour cells.
Studies of nanomaterials represent a promising research field due to their wide range of applications in medicine, biotechnology, energetics, optics, environment, and so on. The conventional methods used for nanoparticle synthesis involve chemical techniques known as being both material and time consuming and also engendering toxic byproducts. Moreover, the nanoparticle synthesis requires special temperature and pressure conditions. A more environmentally and economically friendly alternative to nanoparticle synthesis is encountered in living organisms (which can range from bacteria to fungi and plants) [1]. We will remind here, that magnetotactic bacteria are able to produce magnetosomes (magnetic nanoparticles) whose envelope S-layer forms calcium carbonate layers and diatoms that produce siliceous materials [2–4]. One of the most used method for biosynthesis of metal nanoparticles (i.e., of gold, silver, magnetite, iron, zinc, etc.) is based on the use of bacteria like Bacillus licheniformis, Bacillus subtilis, Klebsiella pneumoniae, Klebsiella oxytoca, etc. [1].
Romanian Journal of Physics 62, 701 (2017)
Article no. 701 C.G. Chilom et al. 2
Klebsiella oxytoca is a gram-negative bacterium with a cylindrical rod shape
of 2–5 µm which produces ferrihydrite nanoparticles by biomineralization of iron
salt solutions. Analysis of these nanoparticles showed their interesting magnetic
properties that could be useful in nanomedicine and bioengineering [5]. The
bacteria Klebsiella oxytoca can produce two types of ferrihydrite nanoparticles, as
a function of the growth conditions [6–8].
The aim of this study is to investigate the possible biomedical applications of
ferrihydrite nanoparticles. In the first step, a supplementary structural and
spectroscopic characterization of these nanoparticles was accomplished. In the
second step, the antitumour action of the ferrihydrite nanoparticles was assessed,
based on their power to preserve cell viability, using the MTT assay. Their
hemolytic activity on human red blood cells was tested, too. The ferrihydrite
nanoparticles do not induce hemolysis at the tested concentrations, but show
efficient effects against cancer cells.
2. MATERIALS AND METHODES
Samples. Aqueous samples of biogenic particles of ferrihydrite were
provided by Siberian Federal University, Krasnoyarsk, Russia. Biogenic
nanoparticle concentration in aqueous solution was 12.5 g/L (5g of biomineral
powder dissolved in 400 mL double distilled water).
Transmission electron microscopy (TEM). Structural analysis of
ferrihydrite nanoparticles was performed with the TEM technique, using a high
resolution (1.4 Ǻ) microscope, type CM 120 PHYLIPS, with 1.2 M magnification.
Spectrophotometric analysis. Spectrophotometric analysis of ferrihydrite
nanoparticles in aqueous solution and in HEPES buffer, at pH = 7.4, was made
with a Perkin-Elmer spectrophotometer. FT-IR spectra were obtained using a
Fourier transform infrared spectrophotometer (FTIR 8400S, Shimadzu, Tokyo,
Japan) in the frequency range of 1,000 to 4,000 cm-1
. We also have used a Perkin-
Elmer fluorometer (LS55) in order to detect a possible fluorescence emission of
sample, in the case that the complexes of nanoparticles would contain traces of
proteins, and to investigate thermal denaturation and the interaction of
nanoparticles with human (HSA) and bovine serum albumins (BSA).
Cell culture and reagents. Mouse fibroblast L929 cells (ATCC, USA) were
grown in Minimum Essential Medium (MEM) supplemented with 2 mM
L-Glutamine and 10 % fetal calf serum (FCS). Human colon adenocarcinoma
HT-29 cells and human hepatocellular liver carcinoma HepG2 cells (ATCC, USA)
were grown in Dulbecco’s modified medium (DMEM) supplemented with 2 mM
L-Glutamine and 10 % FCS. Human osteosarcoma MG-63 cells (ECACC, UK)
were grown in MEM supplemented with 2 mM L-Glutamine, 10 % FCS and 1 %
Non Essential Amino Acids (complete medium, Sigma). 100 units/mL of penicillin
3 Biomedical application of biogenic ferrihydrite nanoparticles Article no. 701
and 100 µg/mL of streptomycin were added in all solutions. Cells were grown at
37 °C in a humidified incubator under an atmosphere containing 5 % CO2. Cell
cultivation media and reagents were purchased from Biochrom AG (Berlin,
Germany). Acridine orange (AO) was purchased from Sigma (Seelze, Germany).
Cells viability. Cell viability was evaluated using MTT (3-(4,5-