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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.
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)
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-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction
assay as follows. Cells were seeded in 96 well plates (8,000 cells/well for L929 and
MG-63 cells and 15,000 cells/well for HT-29 and HepG2 cells) and cultured for
24 h in medium. After this, the medium was changed and ferrihydrite nanoparticles
in various concentrations were added for 24 h. As negative control, untreated cells
were used. Subsequently, the cells were washed, the medium was changed and
MTT solution was added to each well to a final concentration of 0.5 mg/mL and
incubated for 4 h at 37 °C. Then, the medium was colected and the insoluble
formazan product was disolved in DMSO (dimethyl sulfoxide). The absorbance of
the samples was recorded at 570 nm using a plate reader Mithras 840 from
Berthold (Germany). The data were corrected for the background and the
percentage of viable cells was obtained with the following formula:
0% Viable cells /pA A , (1)
where: Ap is the absorbance at 570 nm for the cells treated with ferrihydrite
nanoparticles of different concentrations and A0 is the absorbance of the untreated
cells.
Measurement of hemolytic activity. The hemolytic activity of the
ferrihydrite nanoparticles was determined using an adapted protocol based on the
ASTM F 756-00 standard previously described [9]. Briefly, fresh blood was
collected on heparin from healthy volunteers and diluted with phosphate-buffered
saline (PBS) to a final concentration of hemoglobin ~10 mg/mL. 50 μL of blood
were incubated for 4 h at 37 oC with the highest concentration of the tested sample.
After 4 h, the cells were centrifuged, the supernatant was collected and transferred
into 96-well tissue culture plates and mixed with an equal amount of Drabkin
reagent (Sigma-Aldrich). After 15 min, the absorbance of the samples (at 570 nm)
was read using a plate reader. As negative and positive controls, red blood cells
(RBCs) in PBS and distilled water respectively, were used. The experimental
values were corrected for background, dilution factors and used to calculate the
percentage of hemolysis (i.e., hemolytic index), according to the equation:
%Hemolysis / 100 %ASB ATB (2)
Article no. 701 C.G. Chilom et al. 4
where: ASB is the corrected absorbance of the hemoglobin released in supernatant
after treatment with ferrihydrite nanoparticles and ATB is the corrected absorbance
of the total hemoglobin released.
Morphological evaluation of cells stained with acridine orange (AO).
L929, MG-63 (32,000 cells/cover glass), HT-29 and HepG2 (50,000 cells/cover
glass) cells were grown on cover slips and treated for 24 h with ferrihydrite
nanoparticles in two different concentrations of (0.10 and 3.13 mg/mL). After the
exposure period, the cells were washed with PBS, fixed for 15 min with 3.7 %
formaldehyde, dissolved in PBS, and washed again with PBS buffer. Cells were
then stained with 20 μg/mL AO solution for 15 min and immediately washed with
PBS. The examination of the samples was done with an epifluorescence
microscope (Olympus BX51) using an appropriate filter cube (excitation filter
543/22 nm; dichroic filter 562 nm and emission filter 593/40 nm).
3. RESULTS AND DISCUSSIONS
3.1. THE STRUCTURAL ANALYSIS OF FERRIHYDRITE NANOPARTICLES
The nanoparticles produced by K. oxytoca bacteria are coated with a thin
layer of polysaccharides, which induces an impediment to their characterization by
infrared spectroscopy due of the multitude of signals that should be addressed and
which would only give a general indication of the sample content. Therefore, in
order to structurally characterize the biogenic nanoparticles produced by bacteria
K. oxytoca, the Transmission Electron Microscopy (TEM) technique was used.
Experiments of different amplification degrees, performed in an amorphous
solution, have been conducted because TEM images, have a low resolution, as
shown in Fig. 1. After the analyze of TEM images, several areas have been
identified for biogenic nanoparticles. For each of these areas, the nanoparticle
diameters and the lengthes were determined (Fig. 1, Table). The average diameter
of the ferrihydrite nanoparticles areas determined by TEM, was 7.63 nm.
To confirm that this sample are, indeed, ferrihydrite nanoparticles, the
electron diffraction was used. The position of the pattern obtained in TEM
experiment was compared with literature data for amorphous Fe2O3. It has been
found that the positions of the diffraction electron bands associated with TEM
images are in agreement with the values of the interplanar distances of the specific
amorphous Fe2O3. These results confirm that our sample contains ferrihydrite
nanoparticles in an amorphous state.
5 Biomedical application of biogenic ferrihydrite nanoparticles Article no. 701
Fig. 1 – TEM images of an amorphous solution of the biogenic nanoparticles produced by bacteria
Klebsiella oxytoca. Table: the diameter and the length of biogenic nanoparticles areas.
3.2. SPECTROPHOTOMETRIC CHARACTERIZATION OF BIOGENIC FERRIHYDRITE
NANOPARTICLES
Initial aqueous sample containing ferrihydrite nanoparticles in concentration
of 12.5 g/L was sonicated for 2 min by 1210 Branson sonicator. After that, two sets
of samples (in distilled water and in HEPES buffer) were obtained by dilution
(1:20, 1:30, and 1:45). The two sets of samples contained ferrihydrite nanoparticles
in the following concentrations: 0.625 g/L, 0.412 g/L, and 0.277 g/L.
UV-Vis absorption spectra of the samples were recorded in the wavelength
range, 250–800 nm, at room temperature. The absorption spectra of the ferrihydrite
nanoparticles in aqueous solution are presented (Fig. 2) as functions of
concentration. One can observe a very large absorption in 250–400 nm spectral
range. These spectra are in accord with some preliminary studies of ferrihydrite
Article no. 701 C.G. Chilom et al. 6
nanoparticles produced by K. oxytoca [10]. The same absorption can be observed
in a buffer solution, in the same dilutions.
One can notice a very strong absorption in the spectral range, 250–400 nm,
which may be caused by polysaccharides (many polysaccharides have their
absorption around the 230 nm) and eventually, by traces of proteins (which have
specific absorption at 280 nm).
Fig. 2 – The UV-Vis absorption spectra of ferrihydrite nanoparticles in aqueous solutions (A) and in
HEPES buffer solutions (pH = 7.4) (B), as functions of concentration: 0.625 g/L (―);
0.416 g/L (----); 0.277 g/L (-·-·-).
For checking if some proteins are presented in the structure of the analysed
complexes, their fluorescence emission spectra were recorded. The preliminary
results do not attest the presence of proteins containing fluorescent amino acids
(e.g., the fluorescence emission, at specific wavelengths of aromatic amino acids, is
not present).
Absorption study was completed with a Fourier Transform Infrared (FT-IR)
analysis with the objective to obtain information on the organic compounds bound
to the ferrihydrite nanoparticles. In order to perform this analysis, an aqueous
solution of ferrihydrite nanoparticles was dropped on silica plate and left in normal
atmosphere at room temperature, for water evaporation. The infrared absorption
spectrum (4 cm-1
resolution) is presented in Fig. 3, at high concentration of the
sample.
FT-IR spectrum of the ferrihydrite nanoparticles shows five main peaks at
1,007 cm-1
, 1,103 cm-1
, 1,400 cm-1
, 1,541 cm-1
, 1,653 cm-1
, and a large band
centered on 3,335 cm-1
. The absorption peaks at 1,653 cm-1
(due to asymmetrical
COO- stretching vibration) and 1,400 cm
-1 (due to symmetrical COO
- stretching
vibration) can be related to carboxyl groups of polysaccharides [11]. At the same
time, the peaks at 1,653 cm-1
and 1,541 cm-1
can be related to Amide I and Amide
II, respectively. The Amide bands are characteristic to proteins indicating the
7 Biomedical application of biogenic ferrihydrite nanoparticles Article no. 701
presence of peptide bonds. The Amide I band is due to C = O stretching vibration,
while the Amide II is due to N-H bending vibration [12]. The peak at 3,335 cm-1
can be attributed to the Amide A band (N-H stretching vibration). The important
absorption band centered on 3,335 cm-1
can cover absorptions around 2,929.5–
2,926.8 cm-1
(CH vibration of C1) and around the 3,255–3,216.2 cm-1
(bending
vibration of O-H). These absorptions together with 1,400 cm-1
peak indicate the
glucose presence in the ferrihydrite nanoparticles [13].
Fig. 3 – The FT-IR transmission spectrum of ferrihydrite nanoparticles produced
by Klebsiella oxytoca, in high concentration of the sample on the silica plate.
In order to investigate the binding of biogenic ferrihydrite nanoparticles
coated with polysaccharides to HSA and BSA sites, the fluorescent properties of
Trp residues were used. The fluorescence emission spectra of BSA and HSA in the
presence of ferrihydrite, after excitation at 290 nm, are shown in Figure 4.
It was noted that, as the concentration of the ligand (naoparticules of biogenic
ferrihydrite) increases, the tryptophan fluorescence emission of the two proteins
decreases. This is due to the changes that occur in the immediate vicinity of
tryptophan when the sites of each protein begin to be saturated with ligand.
In order to quantitatively analyze the mechanism of ferrihydrite nanoparticle
binding to serum albumin sites, the Scatchard equation was used:
n
A0 QKFF ][ + 1 = / (3)
where KA is the affinity constant (the binding constant), n is the stoichiometry,
which represents the number of binding sites occupied with ligand, and [Q] is the
concentration of ligand.
Article no. 701 C.G. Chilom et al. 8
Fig. 4 – Quantitative analysis of the binding mechanism of ferrihydrite nanoparticles to the sites
of BSA and HSA using Scatchard equation. Fluorescence emission of the complex
of ferrihydrite nanoparticles with BSA and HSA, was measured in 100 mM HEPES, pH 7.4 at 25 oC
(λex = 290 nm).
The experimental data were processed with equation (1) and the values of the
binding constants were determined (Fig. 4). Considering the model with one
binding site, where one ligand molecule binds to one protein molecule (n = 1), it
resulted that KA = 2.26 × 103 M
-1 for the nanoparticles ferrihydrite-BSA complex,
and KA = 3.89 × 103 M
-1 for the nanoparticles ferrihydrite-HSA complex. These
affinity constant values give an indication of the strength of binding. One can
deduce that the ferrihydrite nanoparticles bind with a weak strength to the sites of
both serum albumins. This result is important, because once transported to the site
of their action the nanoparticles can be easily removed from the protein complex
and may be delivered to act upon the desired target as planned.
It is known that the proteins are molecules whose native structures can be
denatured under the action of physical and chemical agents, such as pH,
9 Biomedical application of biogenic ferrihydrite nanoparticles Article no. 701
temperature, radiation, detergents, or other chemicals. These agents act on the
bonds in the protein structure, causing their ruptures which destabilize their native
structure affecting thus their biological activity.
The study of protein thermal stability and of their complexes with ligands is
of great importance, due to the fact that many ligands participate in physical-
chemical processes taking place at relatively high temperatures where the tertiary
structure of proteins begins to unpack, the proteins losing their biological activity.
Most often, the thermal denaturation of proteins is reversible, but there are
situations where, after denaturing, the proteins no longer regain their native
structures. The thermal denaturation of BSA, HSA and of their complexes with
biogenic ferrihydrite nanoparticles is presented in Figure 5. In both cases, the
structures of proteins and their complexes with nanoparticles undergone major
changes at high temperatures.
Fig. 5 – The denaturation of BSA, HSA and of their complexes with biogenic ferrihydrite
nanoparticles. All samples were diluted in 100 mM HEPES, at pH = 7.4.
Article no. 701 C.G. Chilom et al. 10
In order to investigate the influence of ferrihydrite nanoparticles on the
thermal stability of the complexes, the values of the maximum emission intensities,
depending on the temperature, were represented (Fig. 5). The thermal denaturation
of proteins BSA and HSA and of their complexes with ferrihydrite nanoparticles is
a process that takes place in one step. It seems that the ferrihydrite nanoparticles
stabilize the protein structures against thermal denaturation.
3.3. CYTOTOXICITY OF FERRIHYDRITE NANOPARTICLES AGAINST HEALTHY
AND CANCER CELLS
The cell viability of a normal cell line (L929) and of three cancer cell lines
(HT-29, HepG2 and MG-63) incubated with ferrihydrite nanoparticles in various
concentrations was evaluated by assessment of the cell metabolic activity (MTT
assay).
Ferrihydrite nanoparticles are less toxic for normal cells as compared with the
cancer cells (Fig. 6). At the highest tested concentration (3.13 mg/mL) L929, the
cell viability is around 80 %, while the viability of the cancer cells decreased below
70 %. In the case of cancer cells, it seems that the ferrihydrite nanoparticles have a
fast effect at smaller concentrations reaching then a plateau. Among the cancer
cells, MG-63 proved to be the most affected by the treatment with the ferrihydrite
nanoparticles, especially at smaller concentrations. We were not able to compute
the IC50 value (half maximal inhibitory concentration), but we can notice that the
ferrihydrite nanoparticles have more specificity for cancer cells as compared with
normal ones.
Fig. 6 – Cell viability of L929, HT-29, HepG2 and MG-63 cell lines incubated for 24 h, with different
concentrations of ferrihydrite nanoparticles (NP). The data are reported as mean SD of at least two
independent experiments.
11 Biomedical application of biogenic ferrihydrite nanoparticles Article no. 701
Additionally, hemolysis assay was used to assess the biocompatibility of the
samples. Less than 5 % hemolysis is considered as non-hemolytic according to the
ASTM F 756-00 standard [ASTM]. The percentage of hemolysis induced by the
ferrihydrite nanoparticles at the highest concentration tested was 0.1 ± 0.05 %,
showing that the compound is hemocompatible.
3.4. EFFECTS OF THE FERRIHYDRITE NANOPARTICLES
ON CELLULAR MORPHOLOGY
The effects of the ferrihydrite nanoparticles on cells morphology were
studied for two different concentrations (0.10 and 3.13 mg/mL). Untreated L929
cells (i.e., the control) exhibited an elongated morphology (Fig. 7 a).
Fig. 7 – Morphological changes induced by ferrihydrite nanoparticles on L929, HT-29, HepG2
and MG-63 cells. Magnification of all images was 40×.
Article no. 701 C.G. Chilom et al. 12
Cells treated with the smallest concentration of nanoparticles (Fig. 7 b) do
not show any morphological changes, while at the highest concentration (Fig. 7 c)
the cells shrunk, round up, and show condensation of the nuclei. Similar behaviour
was observed for the HT-29 cells (Fig. 7 d–f) and HepG2 cells (Fig. 7 g–i). On the
contrary, MG-63 cells treated with ferrihydrite nanoparticles (Fig. 7 l) exhibit
membrane deterioration. For all cells, at the highest tested concentration, the
number of cells decreased substantially as compared with the control and the lower
concentration.
4. CONCLUSIONS
The size of the ferrihydrite nanoparticles is of the order of 10 nm.
Spectroscopic studies (absorption, fluorescence emission, and FT-IR) confirm the
presence of polysaccharides but also of some traces of proteins. Although
ferrihydrite nanoparticles coated with polysaccharides have a poor interaction with
serum albumins, their presence increases the serum albumin thermal stability.
The ferrihydrite particles are hemocompatible even at the highest tested
concentrations and also show a higher specificity for the cancer cells as compared
with the normal ones.
Further studies are necessary to better understand the interactions between
the ferrihydrite nanoparticles and the biological components. However, based on
our results, the ferrihydrite nanoparticles could become promising candidates for
biomedical applications.
Acknowledgements. The work was accomplished with the financial support of the IUNC
Structural and spectrophotometric characterization of biogenic systems, Grants 4-1069-2009/2014
and 04-4-1121-2015/2017. Biological studies were accomplished with the financial support of
Romanian National Authority for Scientific Research, CNDI-UEFISCDI, Project number: PN 16 42
02 03. The authors are very much indebted to Virginia Dincă and Gabriel Prodan for TEM images
and to Gabriel Socol for FT-IR spectra.
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