Jana Tulinska - NanoImpactNet

The Effect of Two Iron Oxide Nanoparticles
on Immune Response
Jana Tulinska
Miroslava Kuricova, Aurelia Liskova,
Eva Neubauerova, Katarina Volkovova,
Slovak Medical University, Bratislava, Slovakia
Dagmar Bilanicova, Giulio Pojana,
University Ca’ Ca Foscari of Venice, Venice, Italy
Maria Dusinska
Norwegian Institute for Air Research, Kjeller, Kjeller,
Norway

Alternative testing strategies for the
assessment of the toxicological profile of
nanoparticles used in medical diagnostics
HEALTH-2007-201335
Project leader: Maria Dusinska, MSc, PhD.
NILU, Kjeller, Norway

Aim is to develop testing strategies and
high-throughput high throughput toxicity-testing toxicity testing protocols using
in vitro and in silico methods essential for the
risk assessment of NPs used in medical
diagnostics and compare them with in vivo
http://www.nanotest-fp7.eu
Caco2 with chitosan NPs
(green) Advancell

• Adapt the most advanced
assays for high-throughput
high throughput
automated systems and
prepare for validation
by JRC (ECVAM)
WP2 In vitro screening
WP3 In vivo pre-validation
WP5 Dissemination
Specific
objectives
• Define parameters describing properties of NP and carry out particle particle
characterisation
• Study mechanism of action of NPs in cells and organs
• Develop in vitro methods which can identify the toxicity of NPs.
• Validate in vitro findings in short-term short term in vivo models
• Assess individual susceptibility in the response to NPs.
• Develop in silico models,
WP1 Characterisation
Blood
Vascular system
perform erform Structure-Activity Structure Activity &
Liver
PBPK modelling of NPs.
Lung
WP4 Modelling
Placenta
Digestive system
Central nervous system
Kidney
Assay Automation
WP6 Management

Nanoparticles for testing in
biological system
http://www.nanotest-fp7.eu
• Titanium dioxide TiO2 - reference (benchmark) 21 nm,
Aeroxide P25, manufactured by Evonik, from JRC depository
• Uncoated iron oxide NPs (9 nm core, manufactured by
PlasmaChem)
PlasmaChem)
• Oleic acid-coated acid coated iron oxide NPs (9 nm core,
manufactured by PlasmaChem)
PlasmaChem
• Polymeric materials (polylactic glycolic acid,(PLGA-PEO) –
140nm
• Fluorescence Silica – 25nm,
• Fluorescence Silica – 50nm
• Silica – 25nm, JRC depository
• Endorem – negative control, irone oxide coated with dextran
Up: TEM TiO 2
Below: TEM Fe 3 O 4
University of Venice (P11)

Phase
Crystal structure
Chemical composition
Particle concentration (%)
Shape (by TEM)
Crystallite size distribution by
TEM (nm)
Surface chemistry
Average size in original
suspension by DLS
pH
Zeta potential in milliQ water at
pH 7 (mV)
Contaminants of toxicological
concern
Magnetite with coating
Water dispersion
Spinel (octahedral)
Fe, O
26
Oblong
5-12
Oleate micelle coating
Too high concentration
Too high concentration
-31.9
Free oleate (960 ppm), Na (26.000 ppm),
Ca (1.300 ppm), K (730 ppm)
Magnetite, no coating
Water dispersion
Spinel (octahedral)
Fe, O
2.8
Oblong
5-13
Uncoated
Too high concentration
Too high concentration
-2.8
None

NP
characterization
Size distribution and stability of coated MGT in various culture media
Conc.: 0.25 mg/ml)
Medium
(Conc.: 0.25 mg/ml) Hydrodynamic diameter (nm) 1
Size stability
with time 1
DMEM Very large agglomerates, > 900 < 10 min
DMEM +10% FBS Trimodal distribution, 18, 86 and 237 ~ 2 days
DMEM-HG Very large agglomerates, > 2000 < 5 min
DMEM-HG +10% FBS Bimodal distribution, 36 and 153 ~ 3 days
RPMI Trimodal distribution, 18, 73 and 232 ~ 2 days
RPMI +10% FBS Bimodal distribution, 39 and 165 ~ 3 days
DMEM- F12-HAM Bimodal distribution, 31 and 132 ~ 3 days
1 by DLS
DMEM-F12-HAM +10% FBS Bimodal distribution, 36 and 153 ~ 3 days
1 min
30 min

Immunomodulatory effects
Functionality of the lymphocytes was as assessed ssessed by measuring
proliferative activity after stimulation of the cells with panel of
mitogens and antigens.
Phagocytic activity and respiratory burst was as used to assess
function of neutrophils and monocytes onocytes. .
Natural atural killer cell (NK) activity was as analyzed using cytotoxicity
assay. assay.

DESIGN OF EXPERIMENT
• Blood donors: 10 human volunteers (5 women, 5 men)
• Cultures were pulsed constantly with
25 ul of nanoparticles or
medium or
cyclophosphamide in concentration 40mg/ml
and depending on the test with other supplements
Tested nanoparticles were incubated with blood in three concentrations:
0.12 ug/cm 2 3 ug/cm 2 75 ug/cm 2
The nanoparticles were added into wells in four time intervals: intervals:
4 h 24 h 48h 72h

PROLIFERATIVE RESPONSE OF HUMAN
LYMPHOCYTES IN VITRO
The proliferation of lymphocytes determines
determine
whether the cells have been activated in vitro vitro.
Principle of the assay
To o culture the cells either with
�� no stimulus (spontaneous proliferation)
�� antigen - anti-CD3 anti CD3 monoclonal antibody (the the most physiologic global T-
cell stimulus T-cells T cells receive their activation signals through the TCR/CD3
complex) complex)
�� mitogen - normal ormal T-lymphocytes lymphocytes proliferate vigorously in response to
the T-cell T cell mitogens - concanavalin A and phytohemagglutinin
�� T-dependent dependent B-cell B cell mitogen - pokeweed mitogen

Heparinized blood
derived from humans
Diluted 1:15 in
RPMI medium with
10% FCS
Incubation on
microplates with 3
concentrations of
nanoparticles for 4h,
24h, 48h and 72h and
with mitogens Con A,
PHA, PWM or antigen
(antibody CD3)
Lymphocyte proliferation
at 37°C for 72 h
In vitro lymphocyte proliferative assay
1 μCi (in 25μl medium) of
[3H]-thymidine
Results expressed as
counts per minute
(cpm)/culture)
Liquid
scintillation
counting
Cell harvesting
onto glass filter
paper
Incubation for
24h at 37°C
M. Kuricová et al 2009, Laboratory of Immunotoxicology, Slovak Medical University, Bratislava, Slovak Republic

PHAGOCYTIC ACTIVITY AND RESPIRATORY BURST
OF LEUKOCYTES
The assay evaluates phagocytic activity and
respiratory burst of polymorphonuclear
neutrophils neutrophil / monocytes monocyte exposed to the test
compound in vitro.
Principle of the assay
� Heparinised whole blood is incubated with the FITC-labelled
Staphylococcus aureus bacteria and hydroxyethidine
�� Overall verall percentage of neutrophils and monocytes showing
phagocytosis in general (ingestion of one or more bacteria per cell) and
oxidative burst activity was measured in triplicates for each variable

Phagocytic acitivity and respiratory burst of leukocytes
Heparinized blood
derived from humans
Incubation on
microplates with 3
concentrations of
nanoparticles for
4h, 24h and 48h
30 µl of blood and 10 µl
hydroethidium
C-control, S-sample
15 minutes incubation
at 37°C
3 µl of SPA particles
are added to sample
tubes
15 minutes incubation
at 37°C
Process of
phagocytosis
Samples are measured
using flow cytometer
3 µl of SPA particles
are added to Control
tubes
All tubes are put on ice.
Add 700 µl of lysing
solution

CYTOTOXIC CYTOTOXIC
ACTIVITY OF NATURAL KILLER CELLS
This test allows the quantitative
determination of the cytotoxic activity of
human natural killer (NK) cells.
Principle of the assay
�� Isolated peripheral blood mononuclear cells are incubated with the
K562 target cells
�� The K562 target cells are labeled with green fluorescent membrane membrane
dye
- to discriminate effector and target cells.
�� Killed illed target cells are identified by a DNA-stain, DNA stain, which penetrates the
dead cells and specifically stains their nuclei.
�� Calculations:
Percentage ercentage of target cells killed by effectors NK cells has been
determined.

Cytotoxic activity of natural killer cells
Isolation of effector cells (EC)
Heparinized blood diluted with
PBS, layered on Lymphoprep
Centrifuge
(2 600 rpm/30min)
Cells are resuspended in
RPMI medium with 10%
FCS
Incubation of EC on microplates
with 3 concentrations of
nanoparticles at 37°C, 5% CO2
for 4h and 24h
Target cells K562 (TC) are
prepared
Mix EC and TC cells in 50:1 ratio
Cells are analysed with the flow cytometer
Tubes placed on ice until flow
cytometric analysis
Add Propidium
iodide staining
solution per tube,
vortex and
incubate 20 min at
37°C

Conclusions
Proliferation roliferation of lymphocytes in vitro might be one of the
relevant endpoints to evaluate for NPs and uncoated
magnetite is candidate for nanoparticle immunosuppressive
control for in vitro testing.
Magnetite coated with oleic acid was more toxic to
phagocytic cells, particularly monocytes, monocytes,
then uncoated
magnetite. Respiratory burst of granulocytes seems to be
affected by exposure to coated magnetite more then
phagocytic activity of these cells. cells

Challenges
Interference of method with NPs
Reference materials and standards for the evaluation of NPs
have to be identified.
Appropriate negative and positive control needs to be
defined specific for assay but also for NPs

Consortium: M Dusinska, LM Fjellsbø, Z Magdolenova, A Rinna, E Runden Pran, A
Hudecova, K Hasplova ES Heimstad, M Harju, L Tran, B Ross, L Juillerat, B Halamoda
Kenzaui, F Marano, S Boland, R Guadagnini, M Saunders, L Cartwright, S Carreira, M
Whelan, Ch Klein, A Worth, T Palosaari, E Burello, Ch Housiadas, M Pilou, K Volkovova,
J Tulinska, A. Liskova, M. Kuricova, E. Neubauerova, A Kazimirova, M Barancokova, K
Sebekova, M Hurbankova, Z Kovacikova, L Knudsen, MS Poulsen, T Mose, M Vila, L
Gombau, B Fernandez, J Castell, A Marcomini, G Pojana, D Bilanicova, D Vallotto
.