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In vitro cytotoxicity evaluation of free curcumin and curcumin-loaded chitosan capped
mesoporous silica nanocarriers against MCF-7 breast cancer cells
N. Ahmadi Nasab a*, M. Keshavarz a, A. Ahmadi Nasabb, A. kolahi a
aDepartment of Nanotechnology and Advanced Materials, Materials and Energy Research Center (MERC), Tehran,
P.O. Box 31787-316, Iran
bDepartment of Industrial Engineering, K.N. Toosi University of Technology, Tehran, P.O. Box 15875-4416, Iran
*na.84ir@gmail.com
Abstract: Breast cancer is the most common cancer among women worldwide and second most common cancer overall.
Natural products and extracts have shown great potential as anticancer agents for the treatment of tumors. Curcumin is a
polyphenol derived from the rhizome of the Curcuma longa plant which has anticancer properties, but it has poor
bioavailability and it is a major obstacle to use of it for treatment. So, in this study mesoporous silica capped by chitosan
natural polymer have been employed to improve the bioavailability of curcumin. The efficiency of pure curcumin and
curcumin loaded nanocarriers against MCF-7 breast cancer cells was investigated. In vitro cytotoxicity evaluation of free
curcumin and curcumin loaded Nanocarriers were determined by using the MTT colorimetric assay. Our study confirmed
that curcumin-loaded chitosan capped mesoporous silica nanocarriers has more cytotoxicity than free curcumin against
the MCF-7 breast cancer cells. According to this study, it is proposed to practical use of curcumin loaded chitosan capped
mesoporous silica nanocarriers for developing novel drug delivery system against human breast cancer.
Keywords: curcumin, cytotoxicity, mesoporous silica nanocarriers, breast cancer
Introduction
Curcumin is one of the natural compounds and shows
promising anticancer activity due to inhibition of multiple
cell signaling pathways such as proliferation, blocking
cell, transformation, invasion and induction of apoptosis.
However the major obstacle to use of curcumin for clinical
treatment is the poor bioavailability of curcumin.
Curcumin has low solubility in aqueous solutions, fast
metabolism and rapid systemic elimination in bile and
urine. So it has Poor absorption and low concentration in
plasma and tissue [1]. Several approaches have been
introduced to improve the bioavailability of curcumin,
including structural reforms, use of adjuvants, liposomes,
Niosomes, exosomes, phospholipid complexes and
nanoparticles. Using of nanoparticles as the drug carriers
is extremely promising for enhancing the bioavailability of
lipophilic compounds as well as curcumin [2]. A group of
these nanoparticles mesoporous silica nanoparticles
(MSNs), have very desirable properties such as good
biocompatibility, relative high surface area and tunable
pore, adjustable pore diameter, excellent stability and
modifiable of surface propertie. A member of this family,
MCM-41, has potential to carry drug host/delivery system.
The potential toxicity of unmodified MSNs is due to
interactions of surface silanols with cellular membranes
that improves their biocompatibility [3, 4]. Therefore, the
coating of MSNs with a polymer shell can decrease the
toxicity of silanol groups with cellular membranes [5].
Chitosan is a natural biopolymer with interesting
properties, such as biocompatibility, non-toxicity, and
biodegradability. Chitosan is water soluble from pH 1.0 to
11.0 because of owning large quantities of primary amino
groups. Consequently, Chitosan is a suitable candidate as
a pH-sensitive polymer in the fields of controlled drug
delivery systems [6, 7].
In this study, the inhibitory effect of free curcumin and
curcumin loaded CS-MCM-41 against MCF-7 breast
cancer cells has studied in an in vitro model of breast
cancer, because we want to find a more efficient and safer
approach to improve the current methods of treatment with
chemotherapy to counteract this malignancy.
Materials and Methods
2.1 Preparation of Chitosan-Capped Mesoporous silica
nanoparticles (CS-MCM-41): CS-MCM-41 was prepared
by CTAB templated, base-catalyzed condensation reaction
of TEOS, that was according to procedure reported by our
prior work [1]. 0.2g of CTAB was dissolved in 38.4 mL of
DW. After that, 0.2 mL of 2 M aqueous solution of NaOH
was introduced to the CTAB solution at 80 °C. Then, 1 mL
of TEOS was added to this mixture and stirring about 8
min. The mixture was stirred for 2 h to produce the white
Mesoporous silica nanoparticles suspension. The obtained
solid was washed with water and methanol, then refluxed
for 6 h in a methanolic solution to remove extract the
CTAB. Finally, MSNs were washed with water/ methanol
and then, were heated to 80 °C under high vacuum. CS-
MCM-41 were prepared as Hu et al method [8]. 0.5 g of
chitosan was added to 50 mL of citric acid solution 1.5 wt
% under constant stirring to obtain 1 % w/v chitosan
solution. In the next step, 50 mg of powdered MCM-41
was dispersed in 5 mL of ethanol. The sample was adjusted
to a pH=4 with acetic acid. Then, 0.05 g of GPTMS was
injected into this mixture and was stirred at RT for 3 h.
Afterwards, 10 mL of chitosan solution was added to
mixture and was stirred at RT for 12 h. The CS-MCM-41
was collected by centrifugation and was rinsed by DW and
ethanol for 3-4 times prior to freeze-dried overnight.
2.2 Drug Loading: 1 mg of curcumin was dissolved in 10
mL of ethanol. Then 10 mg of CS-MCM-41 was dispersed
in an aqueous solution of curcumin and sonicated. pH
value of this mixture was adjusted to 3 with 1 M HCl and
was stirred in darkness at 4℃ for 24 h. The pH of mixture
was adjusted to 8 by adding aqueous solution of 0.2 M
aqueous solution of NaOH and it was stirred at 4℃ in dark
for 1 h. Then, sample was centrifuged at 10000 rpm for 20
min and obtained precipitate was collected.
2.3 Cell culture conditions: The human breast
adenocarcinoma cells (MCF-7) were obtained from the
Pasteur Institute (Tehran, Iran) and grown in a complete
Dubecco’s modified eagle’s medium supplemented with
10% FBS 100 mg/ml, penicillin (100 UmL-1) and
streptomycin (0.1 mgmL-1). The cells were maintained at
37 °C under humidified atmosphere with 5% CO2, the
culture medium was changed every 3 days. The cells were
passaged every 3 days.
2.4 In vitro cell viability assay: The MTT colorimetric
assay was used to investigate the toxicity effect of free
curcumin and curcumin loaded Nanocarriers against MCF-
7 breast cancer cells. Specifically, the cells were seeded in
96 well plates with a density of 1×104 cells in each well
and incubated at 37°C in a humidified atmosphere
including 5% CO2. After 24 h medium was carefully
removed and new media with several concentrations (0–50
μg/mL) of free curcumin and curcumin loaded
Nanocarriers were added to each well. Cells were treated
with these media for 24 h. After incubation periods of time,
Supernatants were gently removed and 100 µl medium
containing MTT 0.5 mg/ml was added to each well and
incubated for 4h at 37°C in dark. Then, media were
eliminated and 100 µl dimethyl sulfoxide was added to
each well to dissolve the formazan crystals. After thorough
dissolution the spectophotometrical absorbance of the
supernatant solution was instantly measured at 570 nm by
an Elx 808 absorbance microplate reader (Bio Tek
Instruments, Winooski, VT). The wells containing untreated
were used as control cells. Data were demonstrated
according to cell viability and IC50. For achieving proper
data, all experiments were done in triplicate.
Results and Discussion
The SEM image of CS-MCM-41 is shown in Figure 1 that
indicates the nanoparticles are monodispersed and have
uniform nanospheres.
Fig. 1: SEM image of CS-MCM-41.
The encapsulation efficiency and drug-loading content
were measured and the release of curcumin from the CS-
MCM-41 was carried out in PBS buffer (data not shown).
Results show that drug release was slow and sustained at
low pH compared to the environment pH in 96 h. Also in
this Research, to evaluate the cytotoxic effect of free
curcumin and curcumin-loaded CS-MCM-41, cells of the
MCF-7 breast cancer cells were treated with diverse
concentration (0–50 μg/mL) of free curcumin and curcumin
loaded CS-MCM-41 for 24 h. IC50 values after 24 h treatment
with free curcumin and curcumin loaded CS-MCM-41 was
35.01±3.48 and 15.17±1.47 μg/mL respectively (Figure 2).
CS-MCM-41 (50 μg/mL) showed an absorbance value
equivalent to 97% of control. So suggests that this
nanocarrier have extremely low cytotoxic effect on the
cells.
In this study, curcumin molecules were loaded into CS-
MCM-41 for drug delivery against MCF-7 breast cancer
cells. This results demonstrated that encapsulation of cur-
cumin in CS-MCM-41 improves the cytotoxic effect of
curcumin and reduces IC50 value, which may be due to
enhanced amont of curcumin molecules in an environment
of cancer cells.
Fig. 2: In vitro cell viability assay of different concentrations of
free curcumin and curcumin loaded CS-MCM-41 in the MCF-7
breast cancer cells for 24 h.
Conclusions
The present study disclosed that curcumin loaded chitosan
capped mesoporous silica nanocarriers as well as good
killing potency against MCF-7 breast cancer cells and have
more cytotoxicity than pure curcumin. This toxicity effect
is dose and time dependent and chitosan capped
mesoporous silica nanocarriers release curcumin dose and
time dependently. Eventually, these data illustrate that
curcumin loaded chitosan capped mesoporous silica
nanocarriers are can be used for cancer therapy.
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