Deferasirox loaded on fumed silica nanoparticles used in cancer treatment

Article abstract of DOI:10.1039/c5nj02790j

The present study has demonstrated a paradigm of successful in vitro drug delivery systems using Fumed Silica Nanoparticles (FSNPs) as a scaffold. The surface of FSNPs was first coated with (3-aminopropyl)trimethoxysilane (APTMS) by a silanization reaction and then was linked with deferasirox via the reaction between -NH₂ and -COOH to form well-dispersed surface functionalized biocompatible FSNPs. The obtained nanoparticles were thoroughly characterized by various spectroscopic and microscopic methods such as Fourier Transform Infrared Spectroscopy (FT-IR), Thermo Gravimetric Analysis (TGA) and Brunauer, Emmett and Teller (BET) surface area analysis. The morphology of these structures was investigated by Scanning Electron Microscopy (SEM). The cytotoxicity of these compounds were screened for antitumor activity against MCF-7, MDA-MB-231, HeLa, HT-29, Neuro-2a, L929 cell lines and cisplatin was used as a comparative standard by a MTT assay. Our results presented herein provide experimental evidence that fumed silica nanoparticles loaded with deferasirox induce apoptosis in cancer cell lines. Our flow cytometry results confirm that the investigated compound showed a high population of apoptotic cells (55.20%) and 1.2-fold higher than cisplatin (45.15%) at the same concentration and could induce apoptosis of human breast cancer cell lines (MDA-MB-231).

Full text of DOI:10.1039/c5nj02790j

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Deferasirox loaded on fumed silica nanoparticles
used in cancer treatment
Cite this:DOI: 10.1039/c5nj02790j
Faezeh Taghavi, Mostafa Gholizadeh and Amir Sh. Saljooghi*
The present study has demonstrated a paradigm of successful in vitro drug delivery systems using
Fumed Silica Nanoparticles (FSNPs) as a scaffold. The surface of FSNPs was first coated with (3-amino-
propyl)trimethoxysilane (APTMS) by a silanization reaction and then was linked with deferasirox via the
reaction between –NH2 and –COOH to form well-dispersed surface functionalized biocompatible
FSNPs. The obtained nanoparticles were thoroughly characterized by various spectroscopic and micro-
scopic methods such as Fourier Transform Infrared Spectroscopy (FT-IR), Thermo Gravimetric Analysis
(TGA) and Brunauer, Emmett and Teller (BET) surface area analysis. The morphology of these structures
was investigated by Scanning Electron Microscopy (SEM). The cytotoxicity of these compounds were
screened for antitumor activity against MCF-7, MDA-MB-231, HeLa, HT-29, Neuro-2a, L929 cell lines
and cisplatin was used as a comparative standard by a MTT assay. Our results presented herein provide
experimental evidence that fumed silica nanoparticles loaded with deferasirox induce apoptosis in cancer
cell lines. Our flow cytometry results confirm that the investigated compound showed a high population
of apoptotic cells (55.20%) and 1.2-fold higher than cisplatin (45.15%) at the same concentration and could
induce apoptosis of human breast cancer cell lines (MDA-MB-231).
Received (in Montpellier, France)
10th October 2015,
Accepted 18th January 2016
DOI: 10.1039/c5nj02790j
www.rsc.org/njc
of bacteriophage viruses,35 and synthesis of silica polypeptide
composite particles.36,37
1. Introduction
In recent years, silica nanoparticles have been confirmed to be
Silanes are linked through the formation of a Si–O–Si bond
a valuable material for biomedical research and they have between the surface and the silanol groups. Furthermore, this
attracted worldwide attention due to their easy preparation covalent attachment, a broad spectrum of available chemical
and their wide uses in various industrial applications, such as functionalities at the other functional end of the silane molecules
catalysis,1 pigments,2 pharmacy,3 electronics4 and thin film allow flexible adaptation of the surface for various applications.
substrates,5 electronics and thermal insulators,6 and humidity In particular, 3-(aminopropyl)trimethoxysilane (APTMS) has been
sensors.7 There are five types of silica-based materials used as drug broadly used because of its amino terminal group,38–41 which
delivery systems: (1) fumed silica nanoparticles,8 (2) silica based makes APTMS especially attractive for biological purposes such
xerogels,9–13 (3) ordered mesoporous silica based materials,14–18 (4) as drug delivery. A terminal amino group is capable of a position
mesoporous silica spheres,19–25 and (5) natural silica materials e.g. for reacting with the functional end of drugs or other molecules.
diatoms.26–28 Because of the high surface area that provides good This article describes modified silica with 4-[3,5-bis(2-hydroxy-
solubilisation properties in aqueous environment, fumed silica phenyl)-1,2,4-triazol-1-yl]benzoic acid (deferasirox) in anticancer
nanoparticles have been investigated as vehicles for accelerating studies. The deferasirox was covalently bound to 3-(aminopro-
the speed and the amount of dissolution of drugs that are poor to pyl)trimethoxysilane (APTMS) assembled on the particle surface
be solved in water.29,30 Fumed silica is widely used for therapeutic through amidation between the carboxylic acid end groups on
applications, and their silanol containing surface can be function- deferasirox and the pendant amine groups on the capping linker.
alized, allowing better control over the drug release which depends At last, some investigations of other nanoparticles confirmed
on the chemical nature of the functional group attached to the that drug cleaved from the nanoparticles inside cells.42 The
surface.31
synthesis of deferasirox (4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-
Coating the silica nanoparticles with amino groups leads to 1-yl]-benzoic acid, DFX or ICL670) was first reported in 1999.43 In
a large number of new applications, such as drug delivery,32 2005, deferasirox became the first FDA approved oral alternative
gene delivery,33 plasmid DNA transport,34 capture and release for treatment of iron overload and was subsequently approved in
the EU in 2006.44 It is a tridentate chelator with high selectivity
for Fe3+, and its NO2 donation arises from one triazole nitrogen
Ferdowsi University of Mashhad, Azadi Square, Mashhad, 91735-654, Iran.
E-mail: saljooghi@um.ac.ir
and two phenolate oxygen donors.45
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This iron chelator is used for treatment of iron overload in temperature, and water was added until the first sign of pre-
certain types of anemia such as b-thalassemia and treatment of cipitation was observed. The mixture was concentrated to a total
other toxic metal overload.46,47 In recent years, the potential for volume of 50% under reduced pressure and aqueous 6 M HCl
iron chelators in the treatment of cancer has emerged. ‘‘This (40 mL) was added. The resulting solid was filtered, washed with
reflects the fact that cancer cells typically require more iron water and dried for 24 h in vacuo (3.11 g, yield = 80%).
than normal cells to mediate their generally rapid DNA synthesis
and growth’’.48 The aim of the present study was to show the (KBr) n: 3317, 2540, 1680 (CQO), 1607, 1517, 1495, 1431, 1351,
potential efficiency of antitumor activity of deferasirox loaded on 1221, 988, 752 cmꢀ1 1H NMR (C3H6O-d6 400 MHz): d 7.00
Yellow powder; (3.11 g, yield = 80%); m.p. 264–266 1C IR
;
fumed silica via an APTMS as a spacer.
(s, 1H), 7.01–7.04 (m, 3H), 7.39 (m, 2H), 7.48 (d, 1H), 7.53
(d, 2H), 8.15 (d, 2H), 8.19 (d, 1H) 10.00 (s, OH), 10.78 (s, OH)
ppm; 13C NMR (C3H6O-d6, 75 MHz): d 113.7, 113.9, 116.6 (CH),
117.0 (CH), 119.5 (CH), 119.8 (CH), 124.0 (2CH), 126.9 (CH),
130.4 (2CH), 130.5, 130.7 (CH), 131.4 (CH), 132.6, 141.9 (CH),
152.1, 155.6, 156.4, 160.4, 165.7 (CQO) ppm; M.S. (70 eV) m/z
(%): 374 (M+, 100); Anal. calc. for C21H15N3O4: C, 67.56; H, 4.05;
N, 11.25. Found C, 67, 76; H, 3.85; N, 11.14.
2. Experimental
2.1. Chemicals
All solvents were purchased from Merck. 4-Hydrazino-benzoic
acid, triethyl amine (NEt3) and 2-(2-hydroxyphenyl)-4H-3, 1-benz-
oxazin-4-one without further purification, 1-ethyl-3-[3-dimethyl-
aminopropyl] carbodiimide hydrochloride (EDC) and 7-Hydroxy
benztriazole hydrate (HOBt) were purchased from Fluka (Germany).
RPMI-1640 medium, Dulbecco’s modified Eagle’s medium
(DMEM) and fetal bovine serum (FBS) were purchased from
GIBCO (Gaithersburg, USA). Penicillin and streptomycin were
purchased from Biochrom AG (Berlin, Germany). MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow
tetrazole) was purchased from Sigma Co., Ltd. Cisplatin was
purchased from Sigma Aldrich.
2.4. Loading of deferasirox on fumed silica nanoparticles
In order to bind the silica nanoparticles (NPs) to deferasirox,
at first, the nanoparticles are treated with 3-(aminopropyl)-
trimethoxysilane (APTMS). In a subsequent reaction, the silanol
groups condensate with the hydroxyl groups on the NPs’ surface
to form Si–O–Si covalent bonds. This method introduces the NP
surface of the amino group that is essential to create an amide
bond with the activated carboxylic group of deferasirox.
Fumed silica (particle size: 200 nm) was chemically modified
as follows: first, it was activated at 150 1C under vacuum (0.1 Pa)
for 5 h. 3-(Aminopropyl)trimethoxysilane (32 mL) was dissolved
in 100 mL toluene and it was added to activate silica (31.9 g).
This mixture was refluxed with stirring for 8 h; the product was
filtered and washed with toluene (soxhlet extraction for 24 h)
and finally it was dried at 40–50 1C for 6 h. Then, deferasirox
(4.2 mmol) was dissolved in 100 mL of toluene and then was
added to 1.1 g of the previous sample in the presence of EDC
(50 mg)/HOBt (40 mg) compounds to activate the carboxylate
groups of deferasirox for amide bond formation. EDC/HOBt
(also NHS) cross linking leads to amide bond formation
between activated carboxyl groups and amine groups. This
mixture was refluxed under stirring for 24 h and was filtered
and washed with 100 mL of dry toluene (soxhlet extraction) for
24 h. The solid product was dried at room temperature under
vacuum and abbreviated as FS-APTMS-DFX. The synthesis of
fumed silica nanoparticles loaded with deferasirox was carried
out according to the steps showed in Scheme 1.
2.2. Apparatus
NMR spectra were recorded on an Avance Brucker-400 MHz
spectrometer. All Chemical shifts in NMR experiments are
reported as ppm and were referenced to residual solvent.
Chemical shifts are reported in parts per million and the
signals are quoted as s (singlet), br (broad), d (doublet) and
m (multiplet). FT-IR spectra were recorded on an AVATAR-370-
FTIR Thermo Nicolet. All Mass spectra were scanned on a
Varian Mat CH-7 at 70 eV. The reaction was monitored by
TLC using silica gel plates and the products were identified
by comparison of their spectra and physical data with those of the
authentic samples. Melting points were measured on an Electro-
thermal 9100 apparatus. The Scanning Electron Microscopy (SEM,
EDS) images were taken on a Zeiss LEO 1450 VP/35Kv, Germany.
Thermo Gravimetric Analysis (TGA) was carried out using a
thermogravimetric analyzer TGA-50 (Shimadzu Japan) instru-
ment. Elemental analysis was carried out using a CHNS (O)
Analyzer Model FLASH EA 1112 series made by Thermo Finnigan.
2.5. Characterization of silica nanoparticles loaded with
2.3. Synthesis of 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]- deferasirox
benzoic acid (deferasirox)
The composition and pore structures were thoroughly studied
2-(2-Hydroxyphenyl)-4H-3,1-benzoxazin-4-one was prepared with various spectroscopic and microscopic methods such as
according to the previous reported procedure with little mod- FT-IR, TGA, SEM-EDS and BET techniques.
ifications.49,50 4-Hydrazino-benzoic acid (11.5 mmol, 1.75 g)
Fig. 1 shows the FT-IR spectra of Fumed Silica (FS), FS-APTMS,
and Et3N (11.5 mmol, 1.16 g) were dissolved in boiling EtOH and FS-APTMS-DFX. Fig. 1(a) demonstrate the peak at 1113 cmꢀ1
(80 mL). Then, 2-(2-hydroxyphenyl)-4H-3,1-benzoxazin-4-one corresponding to the Si–O–Si stretching modes of silica gel and
(10.45 mmol, 2.50 g) was added to the clear solution, and the a broad adsorption peak at 3400 cmꢀ1 belonging to Si–OH
reaction mixture was refluxed for an additional 2 h. After the groups. Fig. 1(b) indicates the FT-IR spectrum of FS-APTMS;
completion of the reaction, the solution was cooled to room the peaks at 1034–1123, 1388 and 1560 cmꢀ1 are allocated to
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Scheme 1 Genaral synthesis of FS-APTMS-DFX.
Fig. 2 TGA of: (a) silica modified with 3-(aminopropyl)trimethoxysilane
(FS-APTMS) and (b) defrasirox anchored on FS-APTMS (FS-APTMS-DFX).
The energy dispersive spectrum (EDS) indicated the presence of Si,
N, C and O elements (Fig. 4). This analysis confirms that fumed
silica nanoparticles were loaded by organic structures.
The particle size of silica nanoparticles were found by DLS
analysis with a polydispersity index (PDI) o0.2, indicating a
narrow size distribution.
Table 1 gives the specific surface area and the mean pore
diameter data of the fumed silica (FS), silica modified with
3-(aminopropyl)trimethoxysilane (FS-APTMS) and deferasirox
anchored on FS-APTMS (FS-APTMS-DFX). From the nitrogen
Fig. 1 (a) Infrared spectra of Fumed silica (FS), (b) silica modified with
3-(aminopropyl)trimethoxysilane (FS-APTMS) and (c) defrasirox anchored
on FS-APTMS (FS-APTMS-DFX).
Si–O–Si (asymmetric stretching), C–N (stretching vibration) and
N–H (bending), respectively. Furthermore, several bands with
medium intensity of 2872–2929 and 3436 cmꢀ1 are attributed to
the C–H stretching of the propyl group and N–H stretching mode.
In the FT-IR spectrum of FS-APTMS-DFX, new bands are observed
at 1466, 1565 and 1644 cmꢀ1 due to the aromatic CQC stretch
and CQN stretch of deferasirox. CQO stretch of the amide group
appears at 1711 cmꢀ1. Moreover, the presence of broad absorp-
tion peaks in 3039–3299 regions demonstrates the O–H groups of
deferasirox and the N–H stretching band of amide (Fig. 1(c)).
Observed bands confirmed that immobilization of organic mole-
cules to the silica surface has been accomplished successfully.
TGA of FS-APTMS shows weight loss below 150 1C is due
to the removal of physisorbed water or the water that was
obtained from the condensation of hydroxyl groups. Moreover,
the main weight change (about 14%) in the temperature range
of 150–600 1C was observed attributed to the decomposition
of the amino propyl group on the surface. In the case of
FS-APTMS-DFX, increase of weight loss of about 31% was found
in the temperature range between 200–600 1C. Thus, these
results confirmed the successful immobilization of the organic
structure on the silica (Fig. 2).
Scanning Electron Microscopy (SEM) was used to observe
the morphology of fumed silica nanoparticles before and after
deferasirox loading. Using scanning electron microscopy, it is
possible to obtain information about the surface properties of
the nanoadsorbents (Fig. 3).
In accordance with the scanning electron microscopy it can be
postulated that the nanoparticles were in uniform spherical shape.
Fig. 3 SEM of: (a) silica modified with 3-(aminopropyl)trimethoxysilane
(FS-APTMS) and (b) deferasirox anchored on FS-APTMS (FS-APTMS-DFX).
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comparative standard by a MTT assay. Cell viability was eval-
uated by using a colorimetric method based on the tetrazolium
salt MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]), which is reduced by living cells to yield purple
formazan crystals. Cells were seeded in 96-well plates at a
density of 2–5 ꢂ 104 cells of MCF-7, HeLa, HT-29, MDA-MD-231,
Neuro-2a and L929 per well in 200 mL of culture medium and left
to incubate overnight for optimal adherence. After careful removal
of the medium, 200 mL of a dilution series of FS-APTMS-DFX
in fresh medium were added and incubation was performed at
37 1C/5% CO2 for 72 h. FS-APTMS-DFX was first solubilized in
DMSO, diluted in medium and added to the cells in final
concentrations between 20 nM and 200 mM. The percentage of
DMSO in cell culture medium did not exceed 0.3%.51 Cisplatin
was first solubilized in saline and then was added at the same
concentrations used for the other compounds. At the end of the
incubation period, the investigated compound was removed and
the cells were incubated with 200 mL of MTT solution (500 mg mLꢀ1).
After 3–4 h at 37 1C/5% CO2, the medium was removed and the
purple formazan crystals were dissolved in 200 mL of DMSO by
shaking. The cell viability was evaluated by measurement of the
absorbance at 570 nm by using a STAT FAX-2100 microplate
reader (Awareness Technology, Palm City, FL, USA). The cell
viability calculated the division of the absorbance of each well by
that of the control wells (cells treated with medium containing
0.3% DMSO). Each experiment was repeated at least three times
and each point was determined in at least three replicates.
The amounts of EC50 were obtained using deferasirox concen-
tration in nanoparticles as described by Buchman et al.52 In this
method, the total nitrogen in each stage was obtained by
Fig. 4 The EDS spectrum of deferasirox anchored on FS-APTMS
(FS-APTMS-DFX).
Table 1 Specific surface area and the mean pore diameter data of the FS,
FS-APTMS and FS-APTMS-DFX
Mean pore
diameter (nm)
Sample
SBET (m2 gꢀ1
)
FS
170 ꢁ 5.1
73.9 ꢁ 3.2
53.4 ꢁ 1.7
11.15 ꢁ 0.37
20.23 ꢁ 0.71
22.46 ꢁ 0.67
FS-APTMS
FS-APTMS-DFX
adsorption–desorption isotherms and BET analysis the specific
surface area of FS, FS-APTMS and FS-APTMS-DFX were calcu-
lated as 170 ꢁ 5.1, 73.9 ꢁ 3.2 and 53.4 ꢁ 1.7 m2 gꢀ1 respectively.
3. Biological studies
3.1. Cell culture methods
Human breast cancer cells MCF-7 (ATCC HTB-22), MDA-MD-231 elemental analysis. In the following, we used Ninhydrin quanti-
(ATCC HTB-26), human cervix epithelial carcinoma HeLa (ATCC tative test for the determination of primary amines before and
CCL-2), human colon cancer cell line HT-29 (ATCC HTB-38), after drug (deferasirox) loading on silica nanoparticles.
mouse neuroblastoma cell line Neuro-2a (ATCC CCL-131), and
3.3. Apoptosis assay for FS-APTMS-DFX by flow cytometry
mouse fibroblast L929 cell line (ATCC CCL-1) were obtained from
the American Type Culture Collection (ATCC; Manassas, VA, USA) Evaluation of apoptosis by flow cytometry is generally accom-
and cultured at 37 1C in a humidified atmosphere of 5% CO2 plished by methods that use annexin V-FITC as a vital dye, which
in air. HeLa cells were cultured in Dulbecco’s Modified Eagle’s access phosphatidyl serine exposed to the external membrane at
Medium (DMEM) with 0.1 mM nonessential amino acids, 2 mM- the beginning of this process. The differentiation between
glutamine, 1.0 mM sodium pyruvate and 5% fetal bovine serum, apoptotic and necrotic cells can be performed by simultaneous
at 37 1C in an atmosphere of 5% CO2. Cells were plated in 96-well staining with Propidium Iodide (PI). Therefore, Annexin V-FITC
sterile plates at a density of 1 ꢂ 104 cells per well in 100 mL was used as a marker of phosphatidyl serine exposure and PI as a
of medium and incubated for 24 h. Also, MCF-7, MDA-MD-231, marker for dead cells. This combination allows differentiation
HT-29 and cells Neuro-2a were cultured in Dulbecco’s Modified among early apoptotic cells (annexin V-positive, PI-negative), late
Eagle’s Medium (DMEM) containing 10% fetal bovine serum, apoptotic/necrotic cells (annexin V-positive, PI-positive), and
100 units per mL of penicillin and 100 mg mLꢀ1 of strepto- viable cells (annexin V-negative, PI-negative).
mycin. L929 cells were cultured in RPMI-1640 containing 10% fetal
Cells lines (5 ꢂ 105) were seeded and treated with the
bovine serum, 100 units per mL of penicillin and 100 mg mLꢀ1 of investigated compound FS-APTMS-DFX and incubated for
streptomycin.
24 h at a concentration close to the EC50 at 37 1C. Following
treatment, the cells were harvested by trypsination and centri-
fugation at 1000 r.p.m. for 5 min. The supernatant was removed,
3.2. MTT assay in human cancer cell lines
FS-APTMS-DFX was screened for antitumor activity against and the cell pellet was washed in PBS followed by two washes
human breast cancer cells (MCF-7 and MDA-MD-231), human in binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl,
cervix epithelial carcinoma (HeLa), human colon cancer cell 1.8 mM CaCl2, 1 mM MgCl2). The cells were incubated with an
line (HT-29), and mouse neuroblastoma cell line (Neuro-2a), Annexin V/FITC antibody (5 mL in 100 mL binding buffer) and
mouse fibroblast L929 cell line and cisplatin were used as a incubated at 4 1C for 15 min in the dark. Samples were washed
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in binding buffer, and the supernatant was discarded. The emphasis on their role in cancer therapy was synthesized and used
pellet re-suspension 490 mL binding buffer and 10 mL propidium in a biological model.
iodide (10 mg mLꢀ1 in PBS) were added to the samples before
In this study, the potential of interesting iron chelator,
analysis by flow cytometry. Flow cytometry was done using a Partec deferasirox, that loaded on fumed silica nanoparticles as effec-
PAS flow cytometer (PartecGmbH, Germany) on FS-APTMS-DFX tive anticancer compound has been investigated in vitro. In
and cisplatin as a reference.
order to gain this aim, the in vitro cytotoxicity of FS-APTMS-DFX,
Also apoptosis was detected using an in situ cell death FS-APTMS, FS and the free drug (DFX), against MCF-7, HeLa,
detection kit (Boehringer Mannheim Corp., Indianapolis, IN) HT-29, MDA-MB-231, Neuro-2a and L929 cell lines was deter-
as described by Narla et al. and Zhu et al.53,54 Cells were mined by MTT-based assays (Table 2). For the free drug,
incubated with DFX in 0.3% DMSO or 1 : 16-diluted plasma FS-APTMS-DFX, FS-APTMS, FS and cisplatin we have measured
samples from DFX-treated mice for 48 h at 37 1C, and were fixed, the EC50 values in all cell lines. Such measurements were done
permeabilized, and incubated with the reaction mixture contain- after 72 h of incubation and using concentrations of the
ing TdT- and FITC-conjugated dUTP, and counterstained with compounds in the range 20 nM and 200 mM. The values
propidium iodide. Cells were transferred to slides and viewed determined for FS-APTMS-DFX spanned between 8.52 and
using a confocal laser scanning microscope (Bio-Rad MRC 1024) 241.3 mM, while those found for the comparative standard
mounted on a Nikon Eclipse E800 series upright microscope as ranged between 0.7 and 103 mM (Table 2). FS-APTMS-DFX
reported previously by Narla et al. and Zhu et al.
exhibits the highest selectivity against MDA-MB-231 and HT-29.
It is notable that FS-APTMS and FS is less cytotoxic than
FS-APTMS-DFX against all cancer cell lines. Also DFX is less
cytotoxic than FS-APTMS-DFX against all cancer cell lines
except HT-29. FS-APTMS-DFX in the same range is nearly three
times more cytotoxic than cisplatin against MDA-MB-231 while
it is five times more cytotoxic than DFX against this cell line.
The cytotoxicity of the free drug is the same as cisplatin and
FS-APTMS-DFX against HT-29. Also, the FS, FS-APTMS and DFX
are inactive against all cancer cell lines and L929 (except DFX
against HT-29). The effect of the synthesized compound on
mouse fibroblast cell line (L929) was evaluated as control,
simultaneously. Our results confirmed that the synthesized
3.4. Statistical analysis
EC50 values were expressed as mean ꢁ standard deviation (SD)
from at least three independent experiments. Statistical tests
including one way ANOVA, Tukey multiple comparison or unpaired
Student’s t-tests were performed using SPSS, version 17 software.
A p value of less than 0.05 was considered as significant.
4. Results and discussion
In recent years, some studies confirmed the apparent associa- compound do not have any cytotoxicity effects on L929.
tion between excess iron and cancer, there is significant interest In order to study in which way these compounds produced
in the investigation and development of iron chelating drugs as the cellular death (i.e. necrosis or apoptosis), studies of flow
anti-neoplastic agents.56,57‘‘This reflects the fact that cancer cells cytometry were performed on FS-APTMS-DFX and cisplatin as a
typically require more iron than normal cells to mediate their reference. The results are shown in Table 3 and Fig. 5. Four
generally rapid DNA synthesis and growth’’.48
areas in the diagrams stand for necrotic cells (Q1, left square on
Developments of organofunctionalized hybrid silica nano- the top), late apoptosis or necrosis cells (Q2, right square on the
particle (SN) material technology applied to its manufacturing top), live cells (Q3, left square at the bottom), and apoptosis
procedure adds a strong tool in molecular oncology to wrap up cells (Q4, right square at the bottom), respectively. As it can be
pre-existing anticancer drugs into nanoparticle formulations seen in Table 3 and Fig. 5 the investigated compound showed a
that alter the biodistribution, lower the side effects, minimize high population of apoptotic cell (55.2%) and nearly 1.2-fold
the toxic exposure to normal tissues while maximizing tumor higher than cisplatin (45.15%) at the same concentration. The
uptake and penetration of the drug. Therefore, fumed silica results demonstrated that the newly synthesized compound
nanoparticles (with oral and dermal delivery route), with particular could induce apoptosis against MDA-MB-231 cancer cell lines.
Table 2 Cytotoxic activity of FS-APTMS-DFX tested against MCF-7, HeLa, HT-29, MDA-MB-231, Neuro-2a cancer cell lines and L929 after 72 h
continuous exposure
EC50 ꢁ SDa (mM)
Compound
MCF-7
HeLa
HT-29
MDA-MB-231
Neuro-2a
L929
FS
97.8 ꢁ 8.3
85.3 ꢁ 7.9
17.4 ꢁ 3.66
53.9 ꢁ 6.1
5.94 ꢁ 1.47
147.5 ꢁ 11.2
102.1 ꢁ 10.9
35.9 ꢁ 4.98
38.2 ꢁ 3.9
134.6 ꢁ 11.3
69.4 ꢁ 8.3
105.2 ꢁ 9.5
85.7 ꢁ 6.9
8.52 ꢁ 2.24
39.9 ꢁ 4.1
24.7 ꢁ 4.71
175.4 ꢁ 12.9
122.4 ꢁ 9.8
87.4 ꢁ 9.53
100.7 ꢁ 9.4
103 ꢁ 9.8
235.2 ꢁ 17.7
135.2 ꢁ 12.1
241.3 ꢁ 17.5
102.3 ꢁ 9.5
0.7 ꢁ 0.2
FS-APTMS
FS-PTMS-DFX
DFX
17.1 ꢁ 2.68
16.5 ꢁ 3.62
19.3 ꢁ 3.46
Cisplatin
0.45 ꢁ 0.13
a
EC50 – the effective concentration at 50% of the total effect. The experiments were done in triplicate. Data were expressed as the mean of the
triplicate. EC50 4 100 mM is considered to be inactive.55
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Table 3 Percentages of the cell death pathways observed by the flow
cytometry assay
some cancer cell lines. In this study, we used fumed silica
nanoparticles (FS) as the surface so that the drug was covalently
bound to a linker (APTMS) through the formation of an amide
bond between the carboxylic acid end groups on deferasirox
and the pendant amine groups of the linker. Fumed silica
nanoparticles can donate some characteristics such as improved
bioavailability and increasing resistance time in the body. In vitro,
the nanoparticles that are loaded with the anticancer drug were
found to show a higher apoptosis-inducing effect in cancer
cell lines than free drugs. These results are in agreement with
previous reports. Deferasirox, despite its good cell permeability, is
practically insoluble in water, being classified as a Class II drug
(poorly soluble, highly permeable) according to the Biopharma-
ceutics Classification System (BCS).58 Also, due to its insolubility
in water, the method advised to patients for taking the medicine
orally is quite cumbersome.59 Due to the high surface area that
% Vital % Apoptotic % Late apoptotic/ % Necrotic
Treatment
cells
cells
necrotic cells
cells
Control
Cisplatin
FS-APTMS-DFX 41.6
98.1
50.3
0.43
8.05
25.5
0.94
37.1
29.7
0.53
4.55
3.20
Fig. 5 Flow cytometric results after the exposure of MDA-MB-231 cancer
cells to the investigated compound and cisplatin. Four areas in the provides good solubilisation properties in aqueous environment,
diagrams represent four different cell states: necrotic cells (Q1), late
fumed silica nanoparticles have been investigated as vehicles for
enhanced rate and extent of dissolution of poorly water soluble
apoptotic or necrotic cells (Q2), living cells (Q3) and apoptotic cells (Q4).
drugs. Fumed silica nanoparticle based solid dispersions have
been demonstrated to enhance the dissolution rate and extend
in vitro and in vivo bioavailability of the water insoluble drug.60,61
Furthermore, we used dense silica nanoparticles to accumulate
drug concentration at the surface of cell monolayers; manipulations
that increased complex concentration at the cell surface enhanced
the cytotoxic effect in cancer cell lines than free drugs.62 We believe
that due to the enhanced solubility and enhanced drug concen-
tration at the surface of cancer cells, due to grafting of deferasirox
(DFX) to silica nanoparticles (FS-APTMS), FS-APTMS-DFX showed
high cytotoxicity effects in comparison to DFX. The cytotoxicity of
this compound was screened for antitumor activity against some
Fig. 6 FS-APTMS-DFX induces apoptosis in breast cancer cells. MDA-
MB-231 breast cancer (A, A0) cells were incubated with 10 mM of FS-
APTMS-DFX for 24 h, fixed, permeabilized and visualized for DNA degra-
dation in a TUNEL assay using dUTP-labeling. Red fluorescence, nuclei
stained with propidium iodide. Green or yellow (i.e., superimposed red and
green) fluorescence, apoptotic nuclei containing fragmented DNA. When
compared with controls, treated with 0.3% DMSO (A), several cells incu-
bated with FS-APTMS-DFX (A0) exhibited apoptotic nuclei.
cell lines using cisplatin as a comparative standard by the MTT
assay and flow cytometry. The pharmacological results suggest that
the fumed silica nanoparticles loaded with deferasirox is a potent
anticancer agent. Besides, the cytotoxicity of this compound is
higher than free drug (DFX). The effect of synthesized compound
on mouse fibroblast cell line (L929) was evaluated as control,
simultaneously. Our results confirmed that the synthesized com-
pound do not have any cytotoxicity effects on mouse fibroblast cell
line (L929). Furthermore, the FS-APTMS-DFX has been successfully
used for bioimaging by using a confocal laser scanning microscope,
which may be favorable for biological applications.
But the proapoptotic property needs further investigation in order
to understand the precise mechanism of action of these com-
pounds much better and basic pre-clinical research is needed
before they could be recommended for human administration.
Also the FS-APTMS-DFX induced cell death was confirmed to
be apoptotic using the TUNEL of exposed 30-OH termini of DNA
with dUTP-FITC. As shown in the confocal laser scanning micro-
scopy images in Fig. 6, (FS-APTMS-DFX)-treated MDA-MB-231
breast cancer cells, examined for dUTP-FITC incorporation (green
fluorescence) and propidium iodide counterstaining (red fluores-
cence), exhibited many apoptotic yellow nuclei (superimposed
green and red fluorescence) at 24 h after treatment.
Acknowledgements
The authors are grateful to Ferdowsi University of Mashhad,
research council and Buali Research Institute, faculty of pharmacy,
Mashhad University of Medical Sciences for supporting this work.
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