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: prof.aurel.popescu@gmail.com

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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