Supporting Information
Synthesis, characterization and in vitro study of biocompatible Cinnamaldehyde
functionalized magnetite nanoparticles (CPGF NPs) for hyperthermia and drug
delivery applications in breast cancer
Kirtee D. Wani, MSc1, Brijesh S. Kadu, MSc2, Prakash Mansara, MSc1, Preeti Gupta, MSc3,
Avinash V. Deore, MSc4, Rajeev C. Chikate, PhD2, Pankaj Poddar PhD3, Sanjay D. Dhole,
PhD4, Ruchika Kaul-Ghanekar, PhD*1
1
Cell and Translational Research Laboratory, Interactive Research School for Health Affairs
(IRSHA), Bharati Vidyapeeth University Medical College Campus, Dhankawadi, Pune,
Maharashtra, India.
2
Nanoscience group, Department of Chemistry, Post-graduate & Research Center, MES
Abasaheb Garware College, Pune, Maharashtra, India.
3
Physical & Material Chemistry Division, CSIR-National Chemical Laboratory, Pune,
Maharashtra, India.
4
Department of Physics, University of Pune, Pune, Maharashtra, India.
File S1. Cytotoxic studies of cinnamaldehyde and its derivatives (Table S1) as well as those
of MNPs loaded with herbal active compounds (Table S2). Physical characterization of F, G,
C, P, GF, PGF and CPGF NPs (Figures S1-S4) and cytotoxicity data of F, G and P (Figure
S5).
Table S1. Cytotoxic studies of cinnamaldehyde and its derivatives
Cancer Cell
Lines
Cinnamaldehyde
type
K562
transcinnamaldehyde
2′-benzoyloxy
cinnamaldehyde
MCF-7
MDA-MB-231
MDA-MB-435
A375
G361
LOX
HT29
HCT116
Hs27
HEK 293
HT1080
A431
SK-N-MC
MG63
Neuro2a
SiHa
L929
Raw264.7
Mouse primary
fibroblast
Mouse
Hepatocyte
MCF10A
HEK293
MDAMB231
MCF7
MDAMB231
MCF7
Cinnamaldehyde
Cinnamaldehyde
Cinnamaldehyde
CPGF NPs
containing
Cinnamaldehyde
Concentration
range used
(μM)
120 and 180
15-60
1-25
Not given
Not given
Not given
Not given
Not given
Not given
0.8-320
5-160
1.75-900 ×10-3
MIC
(μM)
IC50
(μM)
References
Not
given
15
------
Zhang et
al., 2010
Ismail et
al., 2012
5
------------------40
40
10
160
40
20
20
80
3.2
57.2
27.7
24.5
6.3
8.1
3.4
19.7
12.6
21.4
17.9
-------------------------------------
5
-----
80
80
40
80
0.014
334.8
184.4
69.81
284.7
0.363
0.112
0.368
Cabello et
al., 2009
Singh et
al., 2009
(Our
previous
work)
Present
work
submitted
to PLOS
One
References

Zhang JH, Liu LQ, He YL, Kong WJ, Huang SA (2010) Cytotoxic effect of transcinnamaldehyde on human leukemia K562 Cells. Acta Pharmacol Sin 31:861-866.

Ismail
IA, Kang
HS, Lee
HJ, Kwon
BM, Hong
SH
(2012)
2'-
Benzoyloxycinnamaldehyde-mediated DJ-1 upregulation protects MCF-7 cells from
mitochondrial damage. Biol Pharm Bull 35:895-902.

Cabello CM, Bair WB 3rd, Lamore SD, Ley S, Bause AS, et al. (2009) The
cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell
proliferation, invasiveness, and tumor growth. Free Radic Biol Med 46:220-231.

Singh R, Koppikar SJ, Paul P, Gilda S, Paradkar AR, et al. (2009) Comparative
analysis of cytotoxic effect of aqueous cinnamon extract from Cinnamomum
zeylanicum bark with commercial cinnamaldehyde on various cell lines. Phar Bio
47:1174-1179.
Table S2. Cytotoxic studies of MNPs loaded with herbal active compounds
Herbal active
compounds
loaded onto
MNPs-Fe3O4
Artesunate
Cancer Cell
Type
Concentration Minimum
IC50 References
(μM)
inhibitory
(μM)
concentration
K562
12.5-100
12.5 µM
Silibinin
T47D
20 µM
Gambogic acid
Capan-1
20-120 µM
20-120 µM
0.25-2.0 µM
Wogonin
Raji cells
12.5-150 µM
12.5 µM
Curcumin
A2780CP
5–40 μM
MDAMB231
PC3
MCF7
0.47-15 µg/mL
5 μM
5 μM
5 μM
7 µg/mL
HT29
7 µg/mL
Gallic acid
10-100 μM
Genistein
SGC-7901
Cinnamaldehyde
MDAMB231 1.75-900 nM
0.25 µM
10 μM
0.014 μM
Not
Wang et al.,
given 2011
73
Ebrahimnezhad
et al., 2013
Not
Wang et al.,
given 2011
80
Ren et al., 2012
12.1
11.9
12.8
Not
given
Not
given
Not
given
0.363
Yallapu et al.,
2011
Dorniani et al.,
2012
Si et al., 2010
Present work
(in CPGF NPs)
0.112 μM
MCF7
0.368 submitted to
PLOS One
References

Wang Y, Han Y, Yang Y, Yang J, Guo X, et al. (2011) Effect of
interaction of magnetic nanoparticles of Fe3O4 and artesunate on apoptosis of K562
cells. Int J Nanomedicine 6:1185–1192.

Ebrahimnezhad Z, Zarghami N, Keyhani M, Amirsaadat S, Akbarzadeh A, et al.
(2013) Inhibition of hTERT gene expression by silibinin-loaded PLGA-PEG-Fe3O4 in
T47D breast cancer cell line. Bioimpacts 3:67-74.

Wang
C,
Zhang
H,
Chen
B,
Yin
H,
Wang
the enhanced anticancer efficacy of gambogic
cancer cells when
mediated
via
magnetic
W
(2011)
Study of
acid on Capan-1 pancreatic
Fe3O4 nanoparticles.
Int
J
Nanomedicine 6:1929–1935.

Ren Y, Zhang H, Chen B, Cheng J, Cai X, et al. (2012) Multifunctional magnetic
Fe3O4 nanoparticles combined with chemotherapy and hyperthermia to overcome
multidrug resistance. Int J Nanomedicine 7:2261-2269.

Yallapu MM, Othman SF, Curtis ET, Bauer NA, Chauhan N, et al. (2012) Curcuminloaded magnetic nanoparticles for breast cancer therapeutics and imaging
applications. Int J Nanomedicine 7:1761–1779.

Dorniani D, Hussein MZB, Kura AU, Fakurazi S, Shaari AH, et al. (2012)
Preparation of Fe3O4 magnetic nanoparticles coated with gallic acid for drug
delivery. Int J Nanomedicine 7:5745–5756.

Si HY, Li DP, Wang TM, Zhang HL, Ren FY, et al. (2010) Improving the anti-tumor
effect of genistein with a biocompatible superparamagnetic drug delivery system. J
Nanosci Nanotechnol 10:2325–2331.
Figure S1. TEM images of F, GF and PGF NPs
TEM image showed the particle size to be around 5-10 nm for F and GF NPs and ~10-15 nm
for PGF NPs. This indicated that the size of the NPs gradually increased from ~5 nm for nonconjugated Fe3O4 to ~20 nm for PGF NPs by successive layering of G and P onto the F NPs.
F
GF
PGF
Figure S1. TEM images of F, GF and PGF NPs
Figure S2. FTIR spectra of F, G and GF
Glycine spectrum (G) showed remarkable signatures at ~3200 cm–1 for -NH2 or -OH and that
for carboxylate stretch at ~1600 cm–1. The notable difference between Fe3O4 spectrum (F)
and glycine capped Fe3O4 (GF), was the appearance of a strong new signature at 3200 cm–1,
along with 1600 cm–1. This indicated that Fe3O4 surface was efficiently capped with glycine.
Figure S2. FTIR spectra of F, G and GF
Figure S3. FTIR spectra of GF, P and PGF
The appearance of peaks at 3200 cm–1 and 1600 cm–1 (1696 cm–1 in PGF) for both GF and
PGF suggested that the hydroxyl/amine and carboxylate functions of G and P are
successively loaded onto the Fe3O4 NPs. The overall merging of peaks indicated that pluronic
possessed similar functional groups as in glycine resulting in similar spectrum of PGF to GF.
Figure S3. FTIR spectra of GF, P and PGF
Figure S4. FTIR spectra of PGF, C and CPGF Cinnamaldehyde spectrum (C) showed
prominent peaks at 1696, 1634, 1140, 960 and 764 cm–1 arising due to bonds specified by
aldehyde structure. There was an interesting shift of –C=C– and –C=O stretches from 1696
and 1634 cm–1 in PGF towards higher energy side at 1709 and 1695 cm–1 in CPGF denoting
conjugation of cinnamaldehyde with PGF. The invariable presence of other peaks in
cinnamaldehyde such as 1140, 960 and 764 cm–1 in CPGF spectrum suggested that
cinnamaldehyde was electrostatically bound to PGF.
Figure S4. FTIR spectra of PGF, C and CPGF
Figure S5. Cytotoxicity of F, G and P
The effect of F, G and P on MDAMB231 and MCF7 was analyzed by using MTT dye. The
cells were treated with 0-640 µg/ml of F, G and P. Uncoated Fe3O4 nanoparticles were nontoxic to all the cell lines which is in accordance with the previously reported data. Both the
cell lines showed ≥100% viability post-treatment with glycine and pluronic used to coat
Fe3O4 nanoparticles. Therefore, these coating materials were non-toxic and safe.
Figure S5. Cytotoxicity of F, G and P on breast cancer cell lines