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Preparation and cytotoxic properties of goethite-based nanoparticles covered with decyldimethyl(dimethylaminoethoxy) silane methiodide.

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Full Paper
Received: 27 July 2009
Revised: 18 September 2009
Accepted: 18 September 2009
Published online in Wiley Interscience: 6 November 2009
(www.interscience.com) DOI 10.1002/aoc.1573
Preparation and cytotoxic properties
of goethite-based nanoparticles covered
with decyldimethyl(dimethylaminoethoxy)
silane methiodide
Izolda Segala∗ , Alla Zablotskayaa, Edmunds Lukevicsa , Mikhail Maiorovb,
Dmitry Zablotskyb , Elmars Blumsb , Anatoly Mishneva ,
Radostina Georgievac, Irina Shestakovaa and Anita Gulbea
The present work describes the synthesis, physico-chemical and biological properties of the first water-soluble goethite
nanoparticles covered with biologically active components: oleic acid and cytotoxic decyldimethyl(dimethylaminoethoxy)silane
methiodide. The structure of initial goethite nanoparticles synthesized was proved by XRD analysis and the rough estimation
of nanoparticles core size gave the value of 8 nm. The size of colloidal water-soluble nanoparticles, determined by dynamic
light scattering, was within 19–35 nm. Magnetic properties and cytotoxicity (against HT-1080 and MG-22A tumor cell lines) of
c 2009 John Wiley & Sons, Ltd.
the nanoparticles obtained were investigated. Copyright Keywords: iron oxide nanoparticles; organosilicon compounds; choline analogs; drug delivery; cytotoxicity
Introduction
Appl. Organometal. Chem. 2010, 24, 193–197
∗
Correspondence to: Izolda Segal, Latvian Institute of Organic Synthesis,
Aizkraukles 21, Riga, LV-1006, Latvia. E-mail: seg@osi.lv
a Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga, LV-1006, Latvia
b Institute of Physics, University of Latvia, 32 Miera, Salaspils LV-2169, Latvia
c Charité – Universitätsmedizin, Berlin, Germany
c 2009 John Wiley & Sons, Ltd.
Copyright 193
During recent years magnetic nanoparticles have had a large
impact in a number of fields of biomedicine.[1] Creation of stable
small magnetic nanoparticles for biomedical applications is very
desirable among numerous existing large ones. Small biologically
active molecules used as coating materials with sufficiently
hydrophilic surfaces can evade the reticuloendothelial system.[2]
Coating of colloidal particles with a layer of a different material in
the nanometer scale is an interesting route for modifying surface
properties and fabricating composite features.
Some years ago we started investigations in this field with the
aim of obtaining magnetite-based water-soluble nanoparticles
on the basis of silyl choline and colamine analogs as model
compounds. A number of water-based magnetic solutions
containing iron oxide nanoparticles bearing mixed oleic acid and
trialkyl(β-dimethylaminoethoxy)silane methiodide cover were
synthesized and investigated for their physico-chemical properties
and in vitro antitumor effect. It was found that they possess low or
moderate cytotoxicity concerning tumor cell lines.[3,4]
To extend the research, it would be interesting to use other
iron-containing carriers for preparation of analogous water-based
colloidal systems of biological interest. Fe3+ ions are the most
desirable in the formation of intracellular and macromolecular
biologically active or MR contrast materials because of these
ions’ low toxicity and existence in nature in many tissues.[5] In
this paper we present an investigation concerning the usage
of goethite (α-FeOOH, iron oxyhydroxide)-based nanoparticles
as carriers of biologically active compounds. This is reasonable
because of its chemical structure allowing greater possibility for
surface modification of the nanoparticles due to the presence of
hydroxy groups and at the same time being stable due to the
three-valency state of iron.
The magnetic properties of goethite remain difficult to interpret.
It has been reported that they are strongly affected by both the
particle size and the degree of crystallinity.[6] Mineral goethite is
weakly magnetic and belongs to the group of antiferromagnetic
materials which need high magnetic field in order to reach
magnetic saturation.[7,8] Negligible isothermal remanence up to 4
T in synthetic goethites was reported.[9] The data adduced in the
literature for Neel point at which synthetic goethite transforms
from the antiferromagnetic state to the paramagnetic state are
different: 392 K, which is in agreement with the natural samples,[8]
and 252 K for goethite nanoparticles, which is significantly lower
than the corresponding bulk values.[10] Goethite has been found
to show superparamagnetism depending on its particle size
and temperature. Superparamagnetic properties are attributed
to the small particle size.[11,12] At low temperature (<300 K), both
remanent and induced magnetization of primary nanoparticles
are about an order of magnitude larger than for micron-sized
goethite and can act as magnetic signatures of nanophase
in controlled environments.[13] The normal superparamagnetic
behavior of synthetic goethite, precipitated from SiO2 -containing
Fe(III) solution, has been determined.[14] It has been shown
I. Segal et al.
G
O
E
T
H
I
T
E
1
OA
Toluene
solution
2
1. A
2. sedimentation
3. drying
Powder
4
1. H2O/NH3
2. shaking
3. centrifugation
1. sedimentation
2. drying
Powder
3
Water solution
5
Powder
6
drying
OA: oleic acid
A: [DcMe2SiOCH2CH2NMe3]+I-
Scheme 1. Synthesis of goethite based samples 1–6.
that various conditions of the goethite synthesis influenced
the properties of the final product. The conditions of synthesis
of goethite with potential superparamagnetic properties have
been investigated and some fine powders have been obtained
which can be used for the preparation of contrast liquids in
magnetic resonance imaging (MRI).[15] Thanks to the interesting
magnetic properties of aqueous suspensions of goethite displayed
at low magnetic fields, it also can be used for preparation of
ferrofluid.[6,16 – 18]
Experimental
Figure 1. X-Ray diffraction pattern of goethite nanoparticles 1.
Chemical Materials and Instrumentation
Solvents and reagents were purchased from Acros and Aldrich. All
solvents used were freshly dried using standard techniques and all
glassware was oven-dried. The dependence of magnetization
on external magnetic field for the samples was determined
using a magnetometer with vibrating sample (VSM), Lake Shore
Cryotronics Inc., model 7404. To measure the size of particles with a
solvate shell using dynamic light scattering technique, a ZEN1690
(Malvern Instruments Ltd) was applied.
Synthesis of Nanoparticles
194
Decyldimethyl(β-dimethylaminoethoxy)silane methiodide was
synthesized and characterized according to the literature
procedure.[19] An outline is given in Scheme 1. Nanoparticles
of goethite (α-FeOOH) 1 were obtained by precipitation from
an aqueous solution of Fe(II) sulfate and Fe(III) chloride with excess amount of sodium hydroxide under intensive stirring of the
mixture bubbled with compressed air.
Toluene-based fluid 2 was synthesized by reaction of the
goethite nanoparticles 1 with oleic acid (OA) following the same
procedure as described for magnetite nanoparticles.[3] Powder 3
with molar ratio FeOOH : OA = 2.2 : 1 was prepared from 2 by its
treatment with acetone after toluene evaporation.
Powder 4 was prepared by shaking of 2 with ndecyldimethyl(β-dimethylaminoethoxy)silane methiodide in molar ratio alkanolamine : OA = 0.3 : 1 and consecutive treatment
with acetone after toluene evaporation. Powder 4 was treated
with water in the presence of ammonium hydroxide to produce
2% water-based fluid 5 for biological experiments. Water-soluble
powder 6 was obtained by solvent evaporation from 5. The yield
of water-soluble nanoparticles 6 relative to the initial amount of
goethite 1 and to the powder 4 was 14% and 73% correspondingly.
A scheme of synthesis of goethite-based samples is presented in
Fig. 1.
www.interscience.wiley.com/journal/aoc
X-ray Analysis
The structure of the initial goethite nanoparticles synthesized was
identified by X-ray diffraction analysis using the database ICDD
PDF-4 and HighScore software. XRD pattern was measured on
an X’PertPro MPD (PANalytical B.V.) diffractometer with linear
detector PIXcel, in flat Bragg-Brentano geometry, Soller slits
0.04 rad, filtered Cu Kα radiation, generator settings 30 mA, 40 kV,
continuous scan type, 2θ interval 10–90◦ , step size 0.026 2θ , scan
step time 50 s.
Magnetic Measurements
The method of magnetogranulometry has been used for determination of the diameter of iron oxide magnetic core and magnetic
properties of nanoparticles prepared. Magnetic properties of magnetic fluids were studied in solutions without separating the carrier
fluid. The special glass container with MF was placed in the measuring system of the magnetometer. Magnetization curves were
recorded at different stages of the material treatment for the
monitoring of magnetite concentration and its condition as well.
As a rule, measurements were made at room temperature for
fields up to 10 KOe. The magnetization curves were used for the
magnetogranulometry analysis[20] to find magnetization of superparamagnetic saturation and magnetic moment of magnetite
particles in the sample from which the magnetite concentration
and the particle sizes in the sample were calculated. To analyze
the weak magnetic samples, the matrix (water or toluene) in the
same holder was measured separately. The matrix magnetization
was subtracted from full magnetization values.
Dynamic Light Scattering Measurements
Dynamic light scattering (DLS), sometimes referred to as Photon
correlation spectroscopy or quasi-elastic light scattering, is a
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2010, 24, 193–197
Preparation and cytotoxic properties of goethite-based nanoparticles
(a) 0.0008
(b)
0.0007
522-4
0.020
Arb. concenration
σ, emu/g
0.0006
0.0005
0.0004
0.0003
0.0002
0.0001
0.015
0.010
0.005
0.000
0
0
2000
4000
6000
8000
10000
4.7
5.5
6.5
7.6
8.9
10.4
12.2
d, nm
H, Oe
Figure 2. Magnetization curve (a, magnetization of water is subtracted) and size distribution (b, obtained by the method of magnetogranulometry) of
goethite particles for sample 5.
technique for measuring the size of particles typically in the
sub micron region. In DLS the speed at which the particles are
diffusing due to Brownian motion is measured by a laser. DLS gives
the colloidal particles size distribution based on its translational
diffusion coefficients in carrier liquid (the particles are modeled as
spheres). Measurements were taken at 20 ◦ C.
Cytotoxicity Assays
Monolayer tumor cell lines MG-22A (mouse hepatoma), HT-1080
(human fibrosarcoma) and normal mouse fibroblasts (NIH 3T3)
were cultivated for 72 h in DMEM (Dulbecco’s modified Eagle’s
medium) standard medium (Sigma) without an indicator and
antibiotics.[21] Tumor cell lines were taken from the European
Collection of Cell Culture. After the ampoule was thawed not more
than four passages were performed. The control cells and cells with
tested substances in the range of 2–5×104 cells/mL concentration
(depending on line nature) were placed on a separate 96-well
plates. The volume of each plate was 200 µl. MFs were diluted with
water and added to wells. The sample before addition was kept
in water bath at 80 ◦ C, 1 h. Control cells were treated in the same
manner but in the absence of test compounds. The plates were
incubated for 72 h at 37 ◦ C with 5% CO2 . The number of surviving
cells was determined using tri(4-dimethylaminophenyl)methyl
chloride (crystal violet, CV) or 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT) coloration, which was
assayed using a multiscan spectrophotometer. The quantity of cells
alive on the control plate was taken in calculations as 100%.[21,22]
The LC50 was calculated using Graph Pad Prism 3.0 program,
r < 0.05.
Results and Discussion
Appl. Organometal. Chem. 2010, 24, 193–197
c 2009 John Wiley & Sons, Ltd.
Copyright www.interscience.wiley.com/journal/aoc
195
We have prepared 2% aqueous solution 5 (Scheme 1) of
goethite-based nanoparticles covered with biologically active components: oleic acid and cytotoxic n-decyldimethyl(βdimethylaminoethoxy)silane methiodide[19] and investigated its
physico-chemical and cytotoxic properties.
The physico-chemical properties of the intermediate nanoparticles of goethite (α-FeOOH) 1, powder 3 with molar ratio FeOOH : OA = 2.2 : 1, powder 4 bearing ndecyldimethyl(β-dimethylaminoethoxy)silane methiodide in molar ratio alkanolamine : OA = 0.3 : 1 were also studied.
The X-ray diffraction of synthesized goethite nanoparticles 1
is presented in Fig. 1. The rough estimation of nanoparticles
average size calculated according to the Scherrer formula
[D = 0.9 λ/(Bstruct. cos θ )] gives the value of 80 (8 nm).
The typical field dependence of magnetization for all samples
was nonlinear. On this evidence the samples consisted not only
of antiferromagnetic goethite. For further magnetization curve
analysis, we assumed that some quantity of superparamagnetic
particles was present in our samples. For this reason the magnetization curves were used for magnetogranulometry analysis
to find superparamagnetic saturation and magnetic moment of
the superparamagnetic particles in the sample.[20] For the next
step of the estimation we assumed that superparamagnetic particles consisted of magnetite and calculated the magnetite weight
concentration and the particle sizes in the samples. The magnetogranulometry gave the particle size distribution. The most
expected particle size at the distribution was used as a particle
diameter. For distribution width estimation we directly used dmin
and dmax at level 0.5 from the distribution density maximum. The
magnetization curve (a) and size distribution (b) of nanoparticles
in aqueous solution 5 are presented in Fig. 2.
The results of the magnetization properties of samples studied
are presented in Table 1. The magnitudes of magnetization
at 10 kOe and superparamagnetic saturation of water-soluble
powder 6 and concentration of magnetic constituent were
calculated on the basis of the same parameters obtained
for aqueous solution 5: magnetization σ10 kOe × 10−3 = 33.8
emu/g, superparamagnetic saturation σs × 10−3 = 8.6 emu/g,
C × 10−3 = 9.0%.
The investigated samples 1 and 3–5 display superparamagnetism, which is equivalent to superparamagnetism of magnetite
nanoparticles with corresponding concentration and corresponding size determined for each sample (Table 1). Aqueous solution 5
was investigated using dynamic light scattering technique, which
gives the colloidal particle size distribution. It was determined
that dmin and dmax at level 0.5 from distribution density maximum
were 19 and 35 nm, correspondingly. These are different from
the sizes obtained by means of magnetogranulometry because of
molecular or ion shells on ferrite particles in colloidal solution and,
probably, association of some ferrite particles on one colloidal particle takes place. The particle size distribution for aqueous solution
5 is presented in Fig. 3.
The efficacy as cytotoxic agents of the nanoparticles obtained was studied. Aqueous solution of double-coated goethite
nanoparticles 5 was screened for in vitro cytotoxicity on monolayer
tumor cell lines HT-1080 (human fibrosarcoma), MG-22A (mouse
hepatoma) and normal mouse fibroblasts (NIH 3T3).
The results of experimental evaluation of cytotoxic properties
are presented in Table 2. The analogous data for magnetite-based
mixed coated MFs bearing the same surfactants and possessing
the highest (7) and the lowest cytotoxicities (8) are also included
for comparison.
I. Segal et al.
Table 1. Physico-chemical properties of samples 1, 3–5
No.
Physical
state
Magnetization
σ10 kOe × 10−3
(emu/g)
Superparamagnetic
saturation σs × 10−3
(emu/g)
Magnetite
concentration
C × 10−3 (%)
d
dmin a
dmax a
1
3
4
5
Powder
Powder
Powder
Solution
930
470
280
0.75
15.2
36.6
106
0.19
17
40
120
0.2
9.9
10.7
5.1
7.2
8.0
8.2
4.1
6.2
11.8
15.1
5.85
8.5
a
Magnetite core size (nm)
At level 0.5 from distribution density maximum.
Conclusion
Figure 3. Size distribution of colloidal nanoparticles for sample 5, obtained
by dynamic light scattering.
Table 2. In vitro cell cytotoxicity and intracellular NO generation
caused by goethite-based sample 5
HT-1080
MG-22A
NIH 3T3
No.
LC50 a
CV
LC50 b
MTT
NOc
CV
LC50 a
CV
LC50 b
MTT
NOc
CV
LC50 d
NR
5
7e[3]
8e[3]
620
159
378
580
98
389
150
1100
400
600
88
16
400
73
285
150
1200
400
960
1115
600
a
Concentration of nanoparticles (µg/ml) providing 50% cell killing
effect (CV: coloration).
b Concentration of nanoparticles (µg/ml) providing 50% cell killing
effect (MTT: coloration).
c NO generation (CV: coloration), determined according to Fast et al.[22]
d
Concentration of nanoparticles (µg/ml) providing 50% cell killing
effect (NR: coloration) r < 0.05.
e Magnetite (Fe O )-based nanoparticles containing oleic acid and
3 4
n-decyldimethyl(β-dimethylaminoethoxy)silane methiodide.
196
It was found that the water-based solution of goethite
nanoparticles 5 exhibits cytotoxicity against human fibrosarcoma
HT-1080 and mouse hepatoma MG-22A, which approximates to
the less active magnetite-based sample, and possesses moderate
NO induction ability. The magnetic fluid therapeutic index (TIMF )
of 5, which is calculated as the ratio of LC50 determined for normal
cells to LC50 for tumor cells,[4] ranges from 1.6 to 2.4 depending
on the kind of tumor and the test.
www.interscience.wiley.com/journal/aoc
Water colloidal solution of firstly synthesized mixed covered
goethite nanoparticles with weak magnetic properties was obtained for cytotoxicity study. It has been revealed that it is possible
to apply the method used for magnetite-based water-soluble
nanoparticles for preparation of goethite-based water-soluble
nanoparticles. The size of colloidal water-soluble nanoparticles
ranged within 19–35 nm. The aqueous solution of goethite
nanoparticles synthesized exhibited cytotoxic properties concerning tumor cell lines. The concentration of nanoparticles
providing a 50% tumor cell killing effect was by up to 2.4
times less (mouse hepatoma, MTT coloration test) than the
concentration for normal cells. Unlike magnetite-based fluids,
goethite-based fluids did not strongly influence the cell morphology. It is reasonable to continue the investigation on goethite
nanoparticles concerning the investigation of magnetic properties and improve their biological properties by variation of the
cover nature and the amount of cytotoxic agent. In perspective the goethite nanoparticles with biologically active cover
can be used as therapeutic agents possessing low magnetic
properties.
Acknowledgments
We acknowledge support from Latvian Council of Sciences in the
form of grant funding (project no. 09.1321 and joint project no.
06.0032.6).
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base, preparation, decyldimethyl, properties, dimethylaminoethyl, silane, covered, cytotoxic, nanoparticles, methiodide, goethite
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