In Vitro Analysis of SPION Conjugated With Folic Acid and Quercetin on Cancer Cell Lines Lokman Alpsoy a , Zeynep Ülker Akal b and Abdulhadi Baykal c Fatih University, Department of a Medical Biology, b Biology and c Chemistry NANOTECHNOLOGY WORKSHOP II BRASILIAN AND TURKISH II B R A S I L I A N A N D T U R K I S H NANOTECHNOLOGY WORKSHOP
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In Vitro Analysis of SPION Conjugated With Folic Acid and Quercetin on
Cancer Cell Lines
Lokman Alpsoya, Zeynep Ülker Akalb and Abdulhadi Baykalc Fatih University, Department of aMedical Biology, bBiology and cChemistry
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Content
■ A. Brief infomation about SPIONs
– Superparamagnetic iron oxide nanoparticle (SPION) in Medicine
– Modification of SPIONs
– Internalisation of SPIONs into the cells
– SPION and cancer therapy
■ B. Our project
– Synthesis and characterisation of SPION@APTES@FA-PEG@CQ
– Biological applications of SPION@APTES@FA-PEG@CQ
■ Cytotoxicity assays
■ Apoptotic and necrotic assays
– Conclusion
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SPIONs
■ Superparamagnetic iron oxide nanoparticles (SPION)
– a superparamagnetic iron core
– small synthetic γ-Fe2O3 (maghemite),
– Fe3O4 (magnetite) particles
– a core ranging from 10 nm to 100 nm in diameter.
■ Physochemical properties of SPIONs affect
– Distrribution
– Toxicity
– Residual time in blood
– Magnetic properties
– Stability
– Internalization into the target cell
Wahajuddin and Arora S. Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers, International Journal of Nanomedicine 2012:7 3445–3471
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Medical Applications of SPIONs
■ Modified SPIONs are very useful tools for numerous applications such as
– magnetic resonance imaging (MRI)
– cancer treatment
– magnetic fluid hyperthermia
– targeted drug delivery
– tissue repair and
– catalysis
– magnetic separation technologies
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Medical Imaging
Thomas R., Park I., and Jeong YY. Magnetic Iron Oxide Nanoparticles for Multimodal Imaging and Therapy of Cancer Int. J. Mol. Sci. 2013, 14, 15910-15930
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Drug Delivery
Varshosaz J. And Farzan M. Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma,W. J Gastr. 2015, 14; 21, 12022-12041
Mok H. and Zhang M. Superparamagnetic Iron Oxide Nanoparticle-Based Delivery Systems for Biotherapeutics, Expert Opin Drug Deliv. 2013; 10(1): 73–87
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SPIONs in Cancer Therapy
Kievit FM. and Zhang M. Surface engineering of iron oxide nanoparticles for targeted cancer therapy, Acc Chem Res. 2011, 18; 44(10): 853–862
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Raju GSR., Benton L., Pavitraa E. and Yu JS. Multifunctional nanoparticles: recent progress in cancer therapeutics, Chem. Commun., 2015, 51, 13248- 13248--13259
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Thomas R., Park I., and Jeong YY. Magnetic Iron Oxide Nanoparticles for Multimodal Imaging and Therapy of Cancer Int. J. Mol. Sci. 2013, 14, 15910-15930
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Hyperthermia
Lee J,Jang J., Choi J., Moon SH. Noh S., Kim J. Et all. Exchange-coupled magnetic nanoparticles for efficient heat induction. Nature Nanotech. 2011, 6, 418–422.
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Modification of SPION
■ The aim of modification of SPIONs with biocompatible polymers are;
– Solubility
– Targetting
– Drug delivery
– Imaging
– Internalisation into the cell
■ The main objective today is optimization of the properties of these magnetic particles to:
– provide an increase in magnetic nanoparticle concentration in blood vessels;
– reduce early clearance from the body;
– minimize nonspecific cell interactions, thus minimizing side effects; and
– increase their internalization efficiency within target cells, thus reducing the total dose required
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Surface Modifications for Targeting
Muller C. and Schibli R., Folic Acid Conjugates for Nuclear Imaging of Folate Receptor–Positive Cancer, J Nucl Med, 2011vol. 52 no. 1 1-4
Castro E. and Mano JF. Magnetic Force-Based Tissue Engineering and Regenerative Medicine, J. Biomedical Nanotechnology, Volume 9, Number 7, 2013, pp. 1129-1136(8)
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Effect Mechanism of Folate (Vitamin B9)
Wilson PM., Danenberg PV., Johnston PG. Et all. Standing the test of time: targeting thymidylate biosynthesis in cancer therapy NATURE R. CLINICAL ONC, 2014, 11, 282–298
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Representation of available mechanisms for SPIONs uptake and their actions inside the cells.
Cores J., Caranasos TG. and Cheng K. Magnetically Targeted Stem Cell Delivery forRegenerative Medicine, J. Funct. Biomater. 2015, 6, 526-546
Cellular uptake of SPIONs
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In this study
We aimed to synthesize SPION conjugated with folate
and quercetin and to determine its biological
activities on cancer cells. NA
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KEY Elements for our project
■ Folate (Folic acid) is B9 vitamin which is essantial for nucleotide and
DNA synthesis
– Cells can be clasified as folate receptor positive (FR+) and folate
receptor negative (FR-).
– FR+ cells expresses FR
– FR- cells have less FR on surface of the cell membrane
– Folat conjugated drugs affect FR+ cells more than FR- cells
■ Quercetin, a kind of flavonoid is produced by plants .
■ U87 (glioblastoma, brain cancer, FR+) and L929 (mouse fibroblast as a
standard cell, FR-) were used in this study.
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4000 3500 3000 2500 2000 1500 1000 500
v)
iv)
iii)
ii)
i)
Wavenumber (cm-1
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% T
ran
sm
itta
nc
e (
a.u
.)
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm-1
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i)
ii)
iii)
iv)
Figure 1. FT-IR analysis of
i) SPION,
ii) SPION@APTES,
iii) COOH-PEG-NH2,
iv) FA-PEG,
v) SPION@APTES@FA-PEG
Figure 2. FT-IR analysis of
i) Choloroacetic acid,
ii) Carboxylated Quercetin, (CQ),
iii) Quercetin
iv) SPION@APTES@FA-PEG@CQ
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-15000 -10000 -5000 0 5000 10000 15000
-20
-10
0
10
20
M(e
mu
/g)
H (Oe)
100 200 300 400 500 600 700 800
30
40
50
60
70
80
90
100
Temperature (oC)
% W
eig
ht
Lo
ss
(a)
(b)
Figure 3 and 4. TEM micrograph and particle size distribution
histogram of SPION@APTES@ FA- PEG@CQ nanocomposite Figure 5. Room temperature M-H curve of
SPION@APTES@ FA- PEG@CQ nanocomposite
Figure 6. TG curves of (a) SPION@APTES@PEG-
FA and (b) SPION@APTES@ FA- PEG@CQ
20 30 40 50 60 70
D=11+5 nm
CP
S (
a.u
.)
Two Theta (Deg)
fit
exp
31
1
22
0
40
0
42
2
51
1
44
0
Figure 7. XRD powder pattern and line profile
fitting of SPION@APTES@ FA- PEG@CQ
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Figure 8. Quercetin release from SPION@APTES@ FA-PEG@CQ at pH
4.4 and 7.4
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The release of the drug at pH 7.4 is slower compared to pH 4.4
Figure 9. Prussian blue staining micrographs of L929 and U87 cells treated with
SPION@APTES@ FA- PEG@CQ at concentartion 100 µg/ml.
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Prussian blue staining images showed that there was a dramatically increase of intracellular iron,
as visualized by blue granules in Prussian blue staining of U87
Figure 10. Cell viability profile of SPION@APTES@FA-PEG for 48 hr
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They have not any toxic effect on cell lines in the concentration range of 0-200μg/ml significantly.
Figure 11. MTT assay results of SPION@APTES@ FA-PEG@CQ on L929 and U87 cells
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All concentrations of SPION@APTES@FA-PEG@CQ influenced cell viability in folic acid positive cells (U87) more
than folic acid negative cells (L929).
*
*
*
*
*
*
*
*
Figure 12. Effect of SPION@APTES@FA-PEG@CQ on
cell index of L929 cells, measured by RTCA
Figure 13. Effect of SPION@APTES@FA-PEG@CQ on
cell index of U87 cells, measured by RTCA
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SPION@APTES@ FA- PEG@CQ inhibied cell number of U87 more than L929
Figure 14. Annexin-V Cy5 results exposed SPION@ APTES@ FA-PEG@CQ to L929 and U87 cells.
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Cell viability decreased and necrosis and apoptosis increased in U87
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Figure 15. TUNEL results exposed SPION@APTES@ FA- PEG@CQ to L929 and U87 cells
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L929
(Control)
L929 (100
µg/ml)
U87
(Control)
U87 (100
µg/ml)
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SPION@APTES@ FA-PEG@CQ caused apoptotic effect on U87
Figure 16. Caspase 3/7 activity of SPION@APTES@ FA-PEG@CQ on L929 and U87 cells
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Caspase 3/7 activity was higher in U87 than L929
Hyperthermia
■ We will apply hyperthermia on cancer cell lines by using BNC
■ Hyperthermia studies are going on still.
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Conclusion
■ In summary, we have synthesized BNC as a multifunctional bionanosytem for drug delivery
and cancer treatment.
■ The monodisperse BNC size about 13 nm have successfully synthesized.
■ The result showed that the SPION@APTES@FA-PEG have lower toxicity both U87 and L929 cell
lines. However, SPION@APTES@FA-PEG@CQ have higher cytotoxic effects on U87 cell line.
■ The xCELLigence, MTT and Prussian blue analysis showed significant association of
SPION@APTES@ FA-PEG@CQ with the FR-positive U87 cells but not with the FR-negative L929