Environmental Engineering Reference
In-Depth Information
Fe
3
O
4
nanoparticle characterization
: iron oxide nanoparticles have been char-
acterized in terms of XRD phase composition (Rigaku Ultima IV, Cu
K
ʱ
, Tokyo,
Japan), specific surface area (nitrogen adsorption-desorption, Micromeritics ASAP
2020 (Micromeritics Instrument Corporation, Norcross, USA)) and magnetic prop-
erties (VSM 880 ADE/DMS magnetometer (DMS/ADE Technologies, Massachu-
setts, USA)).
Preparation of stable colloidal suspensions
:Fe
3
O
4
nanoparticles prepared by
the two synthesis routes have been used to prepare stable colloidal suspensions. For
this purpose, the iron oxide nanoparticles prepared by combustion synthesis were
sonicated for several hours and then covered with a first layer of oleic acid (Merck,
65-88 %) [
11
,
37
]. In the case of the co-precipitation synthesized particles, the
nanoparticles were coated with a first layer of oleic acid as soon as the precipitation
reaction started. This step was followed by a washing process with warm fresh
distilled water. Subsequently, the pH of the solution containing the magnetic
nanoparticles was adjusted to 9 with NH
4
OH (Chimopar, pro analysis) and a second
layer of oleic acid was achieved by adding some more oleic acid. The oleic acid
double layer-coated nanoparticles were dispersed in phosphate buffered saline
(PBS—Sigma Aldrich), leading to stable colloidal suspensions termed sample 1F
(derived from powder 1, prepared by combustion synthesis) and sample 2F (derived
from powder 2, prepared by co-precipitation). The two colloidal suspensions
contained a similar amount of Fe
3
O
4
nanoparticles (~5
10
2
mg/mL).
Colloidal suspension characterization
: the resulted colloidal suspensions based
on PBS were characterized by dynamic laser scattering (DLS) using a ZetaSizer
NanoZS Malvern Instrument (Worcestershire, UK). A drop of diluted aqueous
suspension of magnetic nanoparticles (MNPs) was placed on the grids and allowed
to air-dry, and transmission electron microscopy (TEM) was performed for diluted
MNPs with FEI Tecnai 12 transmission electron microscope (FEI Company,
Eindhoven, NL). The in vitro toxicity of colloidal suspensions based on magnetic
iron oxide nanoparticles on tumor and normal cell lines was investigated as follows.
Cell lines
: unprocessed bone marrow (10 mL) obtained from 10 human adult
subjects free of hematological disorders was used for isolation of mesenchymal
stem cells (MSCs). Bone marrow-derived human mesenchymal stem cells (MSCs)
were isolated following 2 cellular passages based on plastic adherence, fibroblast-
like morphology, and were further used in our experiments. The MSCs culture and
expansion media contained
alpha
-minimum essential medium (MEM; Gibco BRL,
Invitrogen, Carlsbad, CA, USA), supplemented with 10 % fetal calf serum (FCS;
PromoCell, Heidelberg, Germany) and 2 % Penicillin/Streptomycin mixture
(Pen/Strep, 10,000 IU/mL; PromoCell), and cells were grown at 37
Cin5%
CO
2
atmosphere. Medium replacement was performed every third days and when
reaching 80-90 % confluence, the cells were passed using 0.25 % Trypsin-EDTA
solution (Sigma Aldrich Company, Ayrshire, UK) followed by centrifugation
(10 min, 300 g) and replated in T75 culture flasks at a density of 10,000 cells/
cm
2
. SK-BR-3 (breast cancer) cells were procured from American Type Culture
Collection (ATCC, Manassas, VA, USA) and further maintained and expanded in
McCoy
s 5A medium (Gibco) supplemented with 10 % FCS (PromoCell) and 1 %
'
