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RESEARCH PAPER
AS1411 aptamer-functionalized chitosan-silica
nanoparticles for targeted delivery of epigallocatechin
gallate to the SKOV-3 ovarian cancer cell lines
Leila Alizadeh &Effat Alizadeh &Amir Zarebkohan &
Elham Ahmadi &Mohammad Rahmati-Yamchi &
Roya Salehi
Received: 3 May 2019 /Accepted: 13 December 2019
#Springer Nature B.V. 2020
Abstract Chemotherapy is still one of the routine
methods for cancer therapy, but due to its poor specific-
ity to the target site and a lot of side effects, it is not
considered as a harmless approach for cancer therapy.
For improving cancer-targeted drug delivery, in the
present experiment, we synthesized chitosan-coated sil-
ica (SiO2@CS) nanoparticles and attached them to epi-
gallocatechin gallate (EGCG). The functional amine
groups of chitosan in SiO2@CS-EGCG nanoparticles
(NPs) were attached to AS1411 aptamer electrostatical-
ly. Our developed nanoparticles showed spherical core-
shell morphology with a size of around 100 nm in TEM
images. Also, the internalization efficiency of
SiO2@CS-EGCG-aptamer nanoparticles (51%) was
higher than SiO2@CS-EGCG nanoparticles (29%) in
the SKOV-3 cell line, which proved the successful rec-
ognition of nucleolin by attached AS1411 aptamer. Be-
sides, DAPI staining and Annexin V analyses showed
that SiO2@CS-EGCG-aptamer nanoparticles meritori-
ously improved the cytotoxic effect of the EGCG (with
around 93% of cells showed late apoptosis). Addition-
ally, cell cycle results showed that SiO2@CS-EGCG-
aptamer nanoparticles resulted in a decrease in S and
G2/M and increase G0/G1 and arrested cells in the G1
cell cycle. Moreover, the expression level of ERK2 and
hTERT downregulated in SiO2@CS-EGCG-aptamer
nanoparticles was treated SKOV-3 cells in comparison
with the groups that were treated with free EGCG and
SiO2@CS-EGCG. These results suggested that
SiO2@CS-EGCG-aptamer had great potential for
targeted delivery of different therapeutic agents like
EGCG to the SKOV-3 cell line.
Keywords Silica nanoparticles .Chitosan .Aptamer .
Surface modification .EGCG .Ovarian cancer .Target ed
therapy.Nanomedicine
Introduction
Cancer is one of the major causes of death in the world,
and its mortality rate was increased during these decades
(Siegel et al. 2017). The most prevalent type of ovarian
cancer is epithelial tumors. Ovarian cancer is the eighth
most widespread cause of cancer death and the seventh
most common cancer in women with 5-year survival
rates below 45%. The rate of ovarian cancer incidence
has been increasing in countries with lower economical
outcome. Women under 40 years suffered ovarian
JNanopartRes (2020) 22:5
https://doi.org/10.1007/s11051-019-4735-7
L. Alizadeh :E. Alizadeh :E. Ahmadi :
M. Rahmati-Yamchi
Department of Medical Biotechnology, Faculty of Advanced
Medical Sciences, Tabriz University of Medical Sciences, Tabriz,
Iran
A. Zarebkohan :R. Salehi (*)
Drug Applied Research Center and Department of Medical
Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz
University of Medical Sciences, Tabriz, Iran
e-mail: salehiro@tbzmed.ac.ir
M. Rahmati-Yamchi (*)
Department of Clinical Biochemistry, Faculty of Medicine, Tabriz
University of Medical Sciences, Tabriz, Iran
e-mail: rahmatibio@gmail.com
cancer rarely. Age is another risk factor for the rate of
ovarian cancer incidence (Webb and Jordan 2017). Pa-
tients who are under chemotherapy suffer from some
problems and challenges including low effectiveness,
lack of selectivity, and severe side effects. Therefore,
to overcome the limitation of chemotherapy, new ther-
apeutic methods such as targeted drug delivery systems
were developed for the treatment of cancer. Overall,
nanoparticles can infiltrate to the tumor microenviron-
ment (TME) through its poor structure of the vasculature
system, which lead to the accumulation of the drug into
the tumor site and release drugs in a controlled manner
(Li and Xie 2017).
Mesoporous silica nanoparticles (MSNs) are one of
the desirable inorganic materials due to their great large
surface area, biocompatibility, safety for mammalian
cells, and capability of inner and outer surface modifi-
cations. MSNs are inherently resistant to hydrolysis and
enzymatic degradation, and it has controllable pores and
particle size, which strictly can control drug loading and
release efficacy. The forementioned characteristics had
made these NPs as a suitable choice for the drug and
gene delivery and also imaging of cancer (Slowing et al.
2008; Hanafi-Bojd et al. 2018). Chitosan is a natural
polymer which forms by deacetylation of chitin and has
good biodegradability and biocompatibility, considered
as potential candidates for drug delivery (Muddineti
et al. 2017). Besides, due to existence of amine and
hydroxyl groups in chitosan polymers structure, these
polymers are used as linkers and drug containers in a
wide range of studies. For example, the NH
2
group of
chitosan can react with acidic components of drugs.
Also, the OH group of chitosan can react with drugs
by hydrogen bond. So, chitosan coating has used for the
functionalization of nanoparticle surfaces. In this study,
we used chitosan polymer for the attachment of aptamer
and EGCG.
With respect to the passive targeting successfully in
drug delivery systems, active targeting strategies lead to
low side effect of drugs because of their potency in
reaching to the diseased sites, precisely. In the last
decade, researchers have been devised very specific
ligands for precise active targeting of receptors.
Aptamers are short DNA or RNA oligomers which have
specific, stable three-dimensional structures in the
blood, and being resistant to the serum nuclease is one
of them. These oligomers identified their target spatial
conformation and their specificity to their receptors
higher than other ligands like antibodies and peptides
(Zhou and Rossi 2017). Also, these miracle structures
have lower immunogenicity compared with the men-
tioned ligands (Cao et al. 2009). Noteworthy, another
important property of aptamers is their tiny size in
comparison with the other ligands which is a very
important parameter in targeting strategies (Li et al.
2015).
AS1411 is a specific guanine-rich DNA aptamer
which has plenty of biological effects including inhibi-
tion of proliferation, anti-apoptotic effect on different
cancer cells such as glioblastoma (Luo et al. 2017),
reduction of Bcl-2 expression, cell cycle arrest, and
induction of tumor suppressor gene expression.
AS1411 is one of the most investigated aptamers for
targeting anticancer agents in phase I/II clinical trials
(metastatic renal cell carcinoma) (Rosenberg et al.
2014). It is noteworthy that the target of this aptamer
attaches with high affinity to nucleolin as its target
protein, which overexpressed in some cancer cells like
ovarian and breast cancer in comparison with the normal
cells (Li et al. 2014). Macropinocytosis pathway evoked
after recognition of nucleolin by AS1411 (Li et al.
2015).
Epigallocatechin-3-gallate (EGCG) is one of the
major polyphenols which can be found in green tea
and widely used as a chemo-preventive agent and
anticancer drug which pushes tumors to the apopto-
sis. A wide range of studies showed that it can
prevent the growth of different ovarian cancer cell
lines, can arrest the cell cycle in G1 or G1/S phase,
and can regulate the gene expression (Lecumberri
et al. 2013;Sak2015). ERK is one of the compo-
nents of three mitogen-activated protein kinase
(MAPK) pathways which is so important for cellular
growth, survival, and upregulation. Mutation in the
ERK signaling pathway observed in one third of
cancers. Recently, scientists tried to develop a dif-
ferent method to downregulate ERK as a beneficial
approach for inhibiting cancer promotion (Uehling
and Harris 2015). Telomerase is a ribonucleoprotein
complex which is consisted of RNA and human
telomerase reverse transcriptase (hTERT); it can
synthesize repetitive telomeric DNA at the end of
cell division and maintain the stability of the chro-
mosome. Expression of hTERT increased in 80–90%
of human cancers, and it became a target for cancer
therapy. Recently, scientists proposed that hTERT
can regulate survival, self-renewal, and proliferation
of cells (Tang et al. 2016).
5 Page 2 of 14 J Nanopart Res (2020) 22:5
Herein, we have synthesized SiO2@CS nanoparti-
cles which are loaded EGCG and then functionalized by
AS1411 aptamer. This complex can be served as an
efficient anticancer nanocarrier in which transfection
efficiency was improved because of using ligand-
targeting strategy for SKOV-3 cell line. Our developed
nanocarrier demonstrated high cytotoxic effect, good
biocompatibility, excellent internalization capacity, and
anti-proliferative and pro-apoptotic effects.
Material and method
The ovarian cancer (SKOV-3) cell lines were pur-
chased from the National Cell Bank (Pasteur Insti-
tute, Iran). Glutaraldehyde (25%) as linker, chitosan
(low molecular weight), methanol (99%), ammonia
(25%), tetraethyl orthosilicate (TEOS), 3-
aminopropyl-trimethoxysilane (95% purity)
(APTES), toluene, n-hexane, acetic acid
(CH3COOH, 99.5% purity), epigallocatechin gallate
(EGCG), penicillin G, streptomycin, propidium io-
dide (PI) and rhodamine B, dimethyl solfoxide
(DMSO), DAPI, and TRIzol were purchased from
Sigma-Aldrich (Steinem, Germany). RPMI1640 me-
dium, fetal bovine serum (FBS), and EDTA trypsin
were purchased from Gibco. Ribonuclease A was
purchased from Cinagen (Tehran, Iran). AS1411,
with sequence 5′-d(GGTGGTGGTGGTTG
TGGTGGTGGTGG)-3′, was purchased from Gen
Fanavaran (Iran). Yekta-Tajhiz RevertAid First
Strand cDNA Synthesis Kit (Yekta-Tajhiz, Iran)
and Master SYBR Green (ampliqon) were used.
Synthesis of mesoporous silica nanoparticles
MSNs were prepared through an ammonia-based catal-
ysis method. Fifty milliliters of methanol, 0.5-ml am-
monia, and 1-ml deionized water were mixed and vig-
orously stirred for 5 min under argon flow, at room
temperature. Afterward, 5.5-ml TEOS was slowly
added dropwise to the solution during 10 min and
sonicated for 2 h under argon flow and finally stirred
for 72 h. Silica nanoparticles were obtained by precipi-
tation in n-hexane, centrifuged in 8000 rpm for 20 min,
and dried in a vacuum oven at 40 °C for 48 h (Salehi
et al. 2014).
Synthesis of SiO2@CS nanoparticle
MSNs (1 g) was dispersed in 20-ml toluene with the aid
of ultrasonication for 30 min; subsequently, 1500 μl
APTES was added into the dispersion and stirred for
72 h at 70 °C. Afterward, 10-ml glutaraldehyde (2.5
wt%) was added and stirred for 40 min. Chitosan (1 g)
was dissolved in 2% acetic acid and stirred. Finally, 1 ml
of chitosan solution was added into the dispersion of
mesoporous silica nanoparticle and stirred at 80 °C for
48 h. The SiO
2
@CS nanoparticles should be collected
by centrifugation and washed with ethyl alcohol and
distilled water and finally were freeze-dried (Gulfam
and Chung 2014).
Characterization of SiO2@CS NP
Size and morphology of SiO
2
@CS NPs were char-
acterized using transmission electron microscopy
(TEM) (LEO 906, Germany) and field emission
scanning electron microscope (FESEM-EDX;
S4160, Hitachi, Japan). The average diameter of
NPs was determined by random selecting of at least
60 different NPs by using an image analyzer (Image-
Pro Plus, Media Cybrernetics). Particle size and zeta
potential of NPs were analyzed by dynamic light
scattering (DLS) (Zetasizer Nano ZS90; Malvern
Instruments, Malvern, UK). Fourier transform infra-
red (FTIR) (Bruker Tensor 27 spectrometer, Germa-
ny) was used for confirming the creation of silica
and SiO2@CS NPs by the detection of their chem-
ical functional groups.
Encapsulation of EGCG to the chitosan nanoparticles
Two hundred milligrams of nanoparticles was ultra-
sonically dispersed in distilled water then 10 mg of
EGCG added to the solution and shaked at 25 °C for
48 h. Nanoparticles were gathered by the Amicon®
centrifugal filters (Ultra-15, molecular weight cutoff
of 100 kDa, Millipore, Darmstadt, Germany), spun
at 4500 rpm for 10 min, and washed three times to
remove free EGCG, and the final EGCG-loaded
SiO
2
@CS NPs were freeze-dried. After gathering
free EGCG from the solution, the amounts of un-
bound EGCG were measured at 286 nm with a UV-
visible spectrophotometer. Drug loading efficiency
(DLE) and drug encapsulation efficiency (DEE) of
EGCG are calculated as follows.
JNanopartRes (2020) 22:5 Page 3 of 14 5
Drug loading efficiency%w=w
¼mass of drug in nanocarrier
Mass of nanocarrier 100
Drug loading efficiency %w=wðÞ
¼mass of drug in nanocarrier
Mass of feed drug 100
In vitro drug release studies
In order to evaluate EGCG release from SiO
2
@CS NPs,
the predetermined amount of EGCG-loaded SiO
2
@CS
NPs was dispersed in 2 ml of PBS solution at pH 5.4 and
7.4 (stirred at 110 rpm) and incubated 37 °C. One
milliliter of supernatant was drawn at different time
intervals (1–120 h) and replaced with 1 ml of PBS
solution. The amount of EGCG released from
SiO
2
@CS NPs was calculated from a standard curve
using a UV-visible spectrophotometer at 286 nm
wavelength.
Conjugation of the AS1411 aptamer
to SiO
2
@CS-EGCG NPs
First, 6 mg of SiO
2
@CS-EGCG NPs was dispersed in 3-
ml diethyl pyrocarbonate (DEPC) water and 30 μlof
aptamer added to the solution and stirred overnight in
the dark condition. SiO
2
@CS-EGCG-aptamer nanopar-
ticles were washed in DEPC water using an Amicon
ultra centrifugal filter (10 kd) and centrifuged to remove
unconjugated aptamer. The final complex was resus-
pended in DEPC water and kept in a –70 °C.
Confirmation of aptamer conjugation
on SiO
2
@CS-EGCG
The aptamer conjugation to the nanoparticles was con-
firmed by changes in the zeta potential of different forms
of nanoparticles (SiO
2
@CS-EGCG and SiO
2
@CS-
EGCG-aptamer) and uptake of fluorescent-labeled
nanoparticles quantitatively. To address this, we used
DLS system and flow cytometry, respectively.
Cell line experiment
SKOV-3 human ovarian cancer cells were purchased
from the National Cell Bank of Iran and cultured in
T25 culture flask. The cells were cultured in Roswell
Park Memorial Institute 1640 (RPMI 1640 medium;
Gibco BRL Life Technologies which contains 10% fetal
bovine serum, 1% streptomycin (50 μg/ml), and peni-
cillin (50 IU/ml) at 37 °C in a humidified incubator
supplied with 5% CO2 SKOV-3; when the confluency
of cell population reached 70%, they were detached
with 0.25% trypsin in PBS (pH 7.4) and centrifuged at
2500 rpm for 5 min at room temperature. Afterward,
they were seeded in 96-well microplates (with a cell
density of 3 × 103 cells/well) in 200 μl RPMI 10%
FBS and incubated in a humidified atmosphere contain-
ing 5% CO2 at 37 °C for 24 h, in order to attach the cells
to the bottom of the well.
In vitro cell viability study
SKOV-3 cells were seeded in a 96-well plate (3000 per
well) and incubated for 24 h at 37 °C with 5% CO
2
.
SiO
2
@CS (200, 400, 600, 800, 1000 μg/ml), free
EGCG, and SiO
2
@CS-EGCG with EGCG concentra-
tions of 40, 50, 60, and 70 μg/ml were added to the wells
and incubated for 72 h at 37 °C with 5% CO
2
.The
medium was removed, and cells were washed two times
with PBS then treated with 50 μl of MTT (3- (4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) solution (2 mg/ml) in PBS (pH 7.4); and 150 μl
RPMI with 10% FBS was added to all wells and incu-
bated for a further 4 h at 37 °C. After the removal of the
medium, cells were treated with 25 μlSorenson’s buffer
and 200 μl DMSO. Finally, the absorbance was mea-
sured at 570 nm after shaking for 10 min, using a
microplate ELISA reader (Awareness Technology, Palm
City, FL, USA). All statistical analyses of tests were
carried out by GraphPad Prism v6.07 P< 0.05 and were
considered significant.
Cellular uptake studies
Cellular internalization of SiO
2
@CS, SiO
2
@CS-
EGCG, and SiO
2
@CS-EGCG-aptamer labeled with
rhodamine B nanoparticles was investigated by flow
cytometry technique. Briefly, a solution of 0.2 mg/ml
of rhodamine B and 20-mg nanoparticles in 9-ml PBS
was prepared and stirred overnight at room temperature.
5 Page 4 of 14 J Nanopart Res (2020) 22:5
Then, the rhodamine B-labeled NPs were collected by
Amicon® centrifugal filters and washed three times to
eliminate the unloaded rhodamine B. This protocol was
done for all three groups of SiO
2
@CS, SiO
2
@CS-
EGCG, and SiO
2
@CS-EGCG-aptamer nanoparticles.
Firstly, the SKOV-3 cells (5 × 10
5
cells/well) were seed-
ed in 6-well culture plates and incubated for 24 h. After
incubation, the attached cells were treated with 2 ml of
rhodamine B-labeled SiO
2
@CS, SiO
2
@CS-EGCG, and
SiO
2
@CS-EGCG-aptamer which diluted in RPMI me-
dium, then cells were incubated at 37 °C for 0.5, 1.5, and
3 h. Afterward, the medium was eliminated and cells
were washed with 2-ml cold PBS three times and then
were detached with 0.5-ml trypsin-EDTA. The cells
with no treatment were used as a negative control group.
Cell suspension was centrifuged and dispersed in 1-ml
PBS and their fluorescent intensity was evaluated by
flow cytometry (MACS MultiStand,Miltenyi Biotech,
Germany).
Cell cycle analyses
SKOV-3 cells (5 × 10
5
cells/well) were seeded and after
24 h of incubation, the cells were treated with free
EGCG, SiO
2
@CS-EGCG, and SiO
2
@CS-EGCG-
aptamer which diluted in RPMI medium, at their IC
50
dose. The cells without treatment were considered as the
control group. The effects of treatment groups on the
cell cycle were evaluated after 72 h incubations. The
cells were rinsed in cold PBS three times, trypsinized,
and harvested. Then, different groups of treated SKOV-
3 cells were fixed in 300-μl cold ethanol, and after 3 days
of incubation at 4 °C, stained with 10-μl propidium
iodide (PI) solution and 10-μl Rnase A and incubated
for 10 min in the dark at room temperature. Population
frequencies of cells in different cell cycle phases were
detected with FACSCalibur flow cytometry (Becton
Dickinson Immunocytometry Systems, San Jose, CA,
USA).
Cell apoptosis analyses
Cell apoptosis was evaluated using the Annexin V-FITC
staining detection method. SKOV-3 cells (5 × 10
5
cells/
well) were seeded in a 6-well plate and incubated for
24 h. Then, cells were treated with NPs, SiO
2
@CS-
EGCG, free EGCG, and SiO
2
@CS-EGCG-aptamer at
their IC
50
dose, and after 72 h, cells were washed with
cold PBS three times, trypsinized, and transferred to the
separated falcons. Finally, they were centrifuged and
dispersed in 1-ml PBS. After that, we added 200-μl
binding buffer and 2.5-μl Annexin V; finally, we added
PI to all the groups except the control group which has
FITC. The percentage of early and late apoptosis was
investigated by the FACSCalibur flow cytometry
(Becton Dickinson).
DAPI staining study
DAPI staining was done to detect nuclei fragmentation
and condensation of the cells. Nuclei afterward expo-
sure to free EGCG, SiO
2
@CS, SiO2@CS-EGCG, and
SiO
2
@CS-EGCG-aptamer at their IC
50
dose was eval-
uated. SKOV-3 cells were seeded with a cell density of
5×10
5
cells per well of 6-well plate and incubated for
24 h. After 72 h of cells incubation with different group
of nanoparticles, the cells were washed three times with
cold PBS and fixed with 3 wt% paraformaldehyde for
10 min at room temperature, then the cells were washed
three times with cold PBS and permeabilized with Tri-
ton X-100 (10% w/v) for 15 min. Afterward, the cells
were washed with cold PBS and stained with
300 ng ml
−1
of DAPI for 5 min. Finally, condensation
and fragmentation of DNA in apoptotic cells were ob-
served under a multi-mode plate reader (live cell imag-
ing and multi-mode plate reader, citation 5, Biotech,
USA).
Quantitative real-time PCR assay
SKOV-3 cells were treated with free EGCG, SiO
2
@CS-
EGCG, and SiO
2
@CS-EGCG-aptamer and incubated
for 72 h. RNAwas separated from each group of treated
cells in 1 ml of TRIzol (Invitrogen) based on the man-
ufacturer’s protocol. Then, sample RNA concentrations
were calculated spectrophotometrically at 260 nm, and
RNA purities were measured spectrophotometrically at
260 nm and 280 nm and their ratio. The stability of
extracted RNA was characterized by electrophoresis in
agarose gels. Complementary DNA (cDNA) was syn-
thesized using random hexamer primers and moloney
murine leukemia virus reverse transcriptase (M-MLV)
of Yekta-Tajhiz first strand kit. The primer sequences
were mentioned in Table 1. A hTERT forward primer 5′-
CCGCCTGAGCTGTACTTTGT-3′and hTERT reverse
primer 5′-CAGGTGAGCCACGAACTGT-3′were
used to amplify a specific part of hTERT messenger
RNA,andERK2forwardprimer5′-CTAC
JNanopartRes (2020) 22:5 Page 5 of 14 5
AGCATGTCAGCATCTC-3′and ERK2 reverse primer
5′-ACAGTGGCAGGAACAAATAC-3′were used to
amplify a specific part of ERK2 messenger RNA. The
GAPDH mRNA quantified as the internal control by
forward primer 5′-TGACCTCAACTACATGGTTTAC-
3′and reverse primer 5′- GGAAGATGGTGATG
GGATTT-3′. Real-time PCR was performed with Taq
DNA Polymerase Master Mix Ampliqon and carried out
in Real-time PCR System (Biomolecular System, MIC,
Australia).
Result and discussion
Syntheses and characterizations of SiO
2
@CS
nanoparticle
Nanoparticles synthesis confirmation by FTIR
Nanoparticles were prepared through a process de-
scribed previously. FTIR spectra of silica-APTES,
silica-APTES-glutaraldehyde, and silica-APTES-
glutaraldehyde-chitosan were confirmed the differ-
ent steps of NPs preparation and their modifications
whichshowedinFig.1.First,APTESwereusedfor
functionalization of SiO
2
surface; two absorption
peaks at 3444 and 1641 cm
−1
canbeattributedto
the N–H stretching vibration and NH
2
bending of
the free NH
2
groupinAPTESwhichattachedtothe
surface of silica. An intense peak in 1029 was the
characteristic of the Si–O–Si bond in silica nanopar-
ticles. After the interaction of glutaraldehyde with
the amine groups of APTES, the presence of a new
absorption peak at 1657 cm
−1
was related to C=N of
imine. After the interaction of chitosan to
glutaraldehyde, the intensity of peak at 3429 cm
−1
originates from the stretching vibrations of O–Hand
N–H bonds which increased obviously. Chitosan is
one of the most useful natural cationic polymers in
drug delivery systems, but it has poor solubility, so
we added silica to improve its solubility, drug load-
ing, aptamer attachment, and biocompatibility of
complex.
Size and zeta potential evaluation
DLS and zeta potential NP size distribution was evalu-
ated by dynamic light scattering (DLS) method. Obvi-
ously, because of the DLS method, exert in the liquid
phases shows a bigger mean hydrodynamic particle size
compared with SEM which is considered the diameter
of dried NPs (Rahimi et al. 2017). For this reason, the
average hydrodynamic size of SiO
2
@CS and
SiO
2
@CS-EGCG NPs was 186.6 nm and 257 nm, re-
spectively, by PDI around 1 (Fig. 2). The zeta potential
value of the SiO
2
@CS NPs was + 17.8, while
SiO2@CS-EGCG was + 7.14. This decrease in the zeta
potential of NPs after drug loading was attributed to the
hydroxyl group of EGCG and showed attachment of
SiO
2
@CS NP to the EGCG, too (Fig. 3). Adding silica
nanoparticles to the chitosan decreases its zeta potential
and leads to the decrease of the cytotoxic effect of
chitosan in high concentration.
SEM and TEM The morphology and size of nanopar-
ticles were clearly investigated by SEM technique
(Fig. 4a). For the estimation of the average size of
NPs in SEM images, about 100 NPs were chosen
and evaluated by ImageJ program. The size of
SiO
2
@CS NPs was obtained around 100 nm. The
morphology of SiO
2
@CS NPs was investigated by
TEM technique. Its result showed the spherical mor-
phology with particle size in the range of 100–
150 nm (Fig. 4b).
EGCG loading and release study
EGCG as an anticancer drug was loaded on SiO
2
@CS
NPs with 80% drug loading. The weight ratio of drug to
nanoparticles was 1 to 20. It seems that the hydrogen
bonding interaction of hydroxyl groups of drug with the
amine groups of chitosan polymer and electrostatic in-
teractions between them are the main reason of drug
loading in this complex. Released EGCG from
Tabl e 1 . Sequence of primers
Primer name Sequence
ERK2
Forward 5′-CTACAGCATGTCAGCATCTC-3′
Reverse 5′-ACAGTGGCAGGAACAAATAC-3′
hTERT
Forward 5′-CCGCCTGAGCTGTACTTTGT-3′
Reverse 5′-CAGGTGAGCCACGAACTGT-3′
GAPDH
Forward 5′-TGACCTCAACTACATGGTTTAC-3′
Reverse 5′-GGAAGATGGTGATGGGATTT-3′
5 Page 6 of 14 J Nanopart Res (2020) 22:5
SiO
2
@CS-EGCG NPs was measured at two different
pHs (5.5 and 7.4) (Fig. 5). When SiO
2
@CS-EGCG NPs
were dispersed in acidic PBS buffer (pH 5.4), the cu-
mulative release of EGCG reached 72.91% within 96 h
and also its release in neutral PBS buffer (pH 7.4)
reached 47.43% within 96 h. As mentioned before,
when SiO
2
@CS-EGCG was subjected to the acidic
environment, the amino groups of chitosan were pro-
tonated and the chains of chitosan polymer positively
charged lead to the swelling of chitosan and opening of
pores of silica, and consequently, EGCG could quickly
release from the NPs and diffused in its environment.
Conversely at pH 7.4, the polymer chains of chitosan
were deprotonated and made a shielding layer of silica
NPs and covered their porous surface which leads to the
restriction of the EGCG release from SiO
2
@CS NPs.
Characterization of SiO2@CS-EGCG-aptamer
Zeta potential
Zeta potential of SiO
2
@CS-EGCG was + 7.14 ±
5.9 mV, and after conjugation of the aptamer, its surface
charge became −11.4 ± 3.28 mV which related to the
negative charge of nucleic acid in the structure of DNA
aptamer and confirmed the conjugation of the aptamer
to the complex (Fig. 3).
Fig. 1 FTIR spectrum of silica-APTES (a), silica-APTES-glutaraldehyde (b), and chitosan- coated silica NPs (c)
JNanopartRes (2020) 22:5 Page 7 of 14 5
In vitro cellular uptake evaluation
As mentioned above, we have evaluated the cellular
uptake of our synthesized NPs quantitatively by
flow cytometry. So, SiO
2
@CS-EGCG and
SiO
2
@CS-EGCG-Ap NPs were conjugated with
rhodamine B as a fluorescent dye, to evaluate their
cellular uptake efficiency. Our results showed that
the amount of cellular uptake increased in time
dependent manner; it was 10% for SiO
2
@CS-EGCG
in 0.5 h that reached 29.6% after increasing of
exposure time of 3 h. Also, the uptake of
SiO
2
@CS-EGCG-Apin0.5hwas3%andreached
50% in 3 h (Fig. 6). This difference between the two
types of NPs profoundly related to the presence of
aptamer on the NPs which successfully recognized
its specific target (nucleolin) on the cell surface.
Following this recognition of receptor,
macropinocytosis has begun and internalized into
the cells (Fig. 6). The nucleolin expressed in the
nucleus, cytoplasm, and on the surface of some
cancer cells which improve uptake of aptamer to
the cells. Other studies showed that nucleolin also
can improve the uptake of DNA nanoparticles and
plasmids into the nucleus (Reyes-Reyes et al. 2010).
In vitro cytotoxicity assay
SiO
2
@CS NPs are a new drug delivery system; there-
fore, the evaluation of its probable toxicity is necessary.
The in vitro cytotoxicity of free SiO
2
@CS NPs,
SiO
2
@CS-EGCG NPs, and free EGCG on SKOV-3 cell
line were evaluated with MTT assay (Fig. 7). The half
maximal inhibitory concentration (IC
50
)offreeEGCG
was 49.51 μg/ml, and IC
50
of SiO
2
@CS-EGCG was
36.81 μg/ml which showed that loading EGCG to the
SiO
2
@CS NPs decreases its IC
50
. Results showed that
the encapsulation of EGCG in SiO2@CS NPs increased
the cytotoxic effect of EGCG on the SKOV-3 cell line in
comparison with the free EGCG. According to the
nanoparticles’inherent nature, this might be happened
because of the increase entrance of the EGCG into the
cells by nanoparticles. The exact mechanism of these
NPs internalization route is unknown, but with respect
to the drug release behavior in an acidic environment,
we can conclude that this process might be done in a
clathrin-mediated endocytosis manner which ended into
the lysosomes. Noteworthy, the MTT assay results
showed no noticeable cytotoxicity of SiO
2
@CS NPs
on SKOV-3 cells, the viability of SKOV-3 cells treated
with 650 μg/ml of SiO
2
@CS was about 70%.
Fig. 3 The zeta Ppotential of a)
SiO2@CS NPs (a), b) SiO2@CS-
EGCG NPs (b), and c)
SiO2@CS-EGCG-aptamer (c)
Fig. 2 Size distribution of : (a)
SiO2@CS NPs and (b)
SiO2@CS-EGCG NPs obtained
by DLS
5 Page 8 of 14 J Nanopart Res (2020) 22:5
Cell cycle analysis
The DNA content during the cell cycle of different
groups of treated SKOV-3 cells was evaluated by flow
cytometry technique. In this method, the DNA profile in
G1, S, and G2/M could be evaluated using DNA inter-
calating dye-like propidium iodide. The result of cell
cycle analysis showed that 46% of cell group treated
with free EGCG arrested in S phase, but in the cell group
treated with SiO
2
@CS-EGCG, an extreme decrease in
the percentage of S phase arrest (decreased from 46 to
8%) and very big increase in G0/G1 arrest (increase
from 10 to 69%) was observed. The percentages of
G0/G1 cells were increased again in cells treated with
SiO
2
@CS-EGCG-aptamer to 85% (Fig. 8). Therefore,
the SiO
2
@CS-EGCG and SiO
2
@CS-EGCG-aptamer
NPs arrested cells in G0/G1 phase. In a study conducted
by Pagidas et al, they found that EGCG can increase the
percentage of G1 phase arrest, decrease in S phase and
G2 phase in SKOV-3 cell lines (10 μg/ml 2-day
treatment) which was similar with the results of treat-
ment groups which received SiO
2
@CS-EGCG and
SiO
2
@CS-EGCG-aptamer in our studies, while free
EGCG showed S phase arrest (Rao and Pagidas 2010).
In another study, Chen et al. found that EGCG can arrest
SKOV-3 cell line in G1 and S phase, but when it
combined with SNF, the percentage of a different part
of cell cycle arrest can change and it retarded to the G2\
M and S phase arrest (Chen 2013).
Cell apoptosis analyses
Apoptosis analysis of the SKOV-3 cells stained by
Annexin V-FITC showed that these cells have a higher
sensitivity to the SiO
2
@CS-EGCG NPs compared with
free EGCG. Also, functionalization of the complexes
with the aptamer increased this sensitivity, inhibited cell
proliferation, and induced more apoptosis. The results
indicated that the cells treated with SiO
2
@CS NPs
showed no obvious cytotoxicity to SKOV-3 cells, and
Fig. 5 Cumulative release of
EGCG from SiO2@CS
nanoparticles during 96 h at
various pH values (pH 5.4 and
7.4) at 37 °C
Fig. 4. Image of SiO2@CS NPs. a) SEM. and b) TEM image of SiO2@CS NPs
JNanopartRes (2020) 22:5 Page 9 of 14 5
the percentage of viable cells in both untreated control
groups and SiO
2
@CS NPs groups was 93%.
The cells treated with free EGCG had around 17%
apoptotic cells (4.5% early and 12.5% late apoptosis)
which related to lower drug uptake. The amount of
apoptotic cells in the group treated with SiO
2
@CS-
EGCG NPs reached 54% which most of them were in
early-apoptosis state (53.8%). This increase in apoptosis
can be associated with more entrance of drugs into the
cells by NPs, and early apoptosis may occur because of
the slow internalization pathway, which is mentioned
above. Consequently, cells could accommodate and
compensate their situation in response to drug
concentration. The amount of apoptotic cells treated
with SiO
2
@CS-EGCG-Ap increased to 93% which all
of them were in late-apoptosis state (Fig. 9). It is note-
worthy that our targeted nanoparticles (SiO
2
@CS-
EGCG-Ap) are about 2 times more than SiO
2
@CS-
EGCG. The main reason for this behavior profoundly
is due to the recognition of the nucleolin receptors by the
attached aptamer.
AS1411 aptamer can be internalized into the cells in a
nucleolin-mediated macropinocytosis manner (Reyes-
Reyes et al. 2010). This pathway is very faster than the
passive internalization of nanoparticles into the cells and
ended to the lysosomes; finally, (Byrne et al. 2008).
Fig. 6 SKOV-3 cellular uptake of Rho B-labelled SiO2@CS nanoparticles, SiO2@CS-EGCG, and SiO2@CS-EGCG-aptamer assessed by
flow cytometry after 0.5 h, 1.5 h, and 3 h exposure time
5 Page 10 of 14 J Nanopart Res (2020) 22:5
Obviously, the pH-responsive cargoes would be re-
leased very fast from the lysosomes to the cytoplasm.
Hence, the cells would not have to resist against high-
drug concentration. Our results showed that the
aptamer-conjugated nanoparticles are able to release a
higher amount of drug and induce late apoptosis in
treated cells. Other possible cell death mechanisms
which are reported previously include cell cycle arrest,
inhibit some signaling pathways like NF- , inhibit pro-
liferation, and cell death in cancer cells by AS1411
aptamer itself (Soundararajan et al. 2008).
DAPI staining
Evaluating the morphology of chromatin through the
DAPI staining method can differentiate between
healthy and apoptotic cells. DAPI nuclear staining
shows the degree of DNA condensation which indi-
cates the viability and mortality of cells. The cells
treated with free EGCG had some nuclei fragmenta-
tion, but it increased in cells treated with SiO
2
@CS-
EGCG and also conjugation of the aptamer to the
complex (SiO
2
@CS-EGCG-Ap) increased the nuclei
fragmentation compared with the SiO
2
@CS-EGCG
NPs (Fig. 10). The nuclear of cells treated with
SiO
2
@CS NPs was normal and similar to untreated
cells, which proved that the NPs had no obvious
cytotoxicity to SKOV-3 cells.
Quantitative real-time PCR assay
The levels of ERK2 and hTERT gene expression were
evaluated by real-time PCR. Changes in ERK2 (Fig.
11a)andhTERT(Fig.11b) expression levels among
Fig. 8 Percentage of SKOV-3 cell distribution in various cell cycle phases after 72 h treatment, control ( (a),), free EGCG ((b),), SiO2@CS-
EGCG NPs ((c), and), SiO2@CS-EGCG-aptamer (d) analyzed by flow cytometry
Fig. 7 The cell viabilities of
SKOV-3 cells after 72 h treatment
of different concentrations of free
EGCG and SiO2@CS-EGCG, P
value (0.0456) evaluated by MTT
assay
JNanopartRes (2020) 22:5 Page 11 of 14 5
Fig. 10 Fluorescence
microscopy images of DAPI
staining for SKOV-3 cells to show
amount of damage to the DNA.
The cells were stained after treat-
ment for 72 h with SiO2@CS
NPs (b), free EGCG (c),
SiO2@CS-EGCG (d),
SiO2@CS-EGCG-aptamer (e),
and cells with no treatment were
used as a control (a)
Fig. 9 Cell apoptosis assay of SKOV-3 cells incubated with EGCG (b), SiO2@CS-EGCG (c), and SiO2@CS-EGCG-aptamer (d) for 72 h
and untreated cells as a control (a) evaluated by Annexin vV-FITC and determined by flow cytometry
5 Page 12 of 14 J Nanopart Res (2020) 22:5
the control and treated SKOV-3 cells were normalized to
GAPDH mRNA expression levels and then calculated
by the 2−ΔΔct method. Results showed that ERK2 and
hTERT gene expression decreased in treated cells com-
pared with the control cells. At the same concentration,
SiO
2
@CS-EGCG showed lower expression of ERK2
and hTERT in SKOV-3 cell lines in comparison with the
cells received free EGCG (Fig. 11). Also, the conjuga-
tion of aptamer to the nanodrug complex (SiO
2
@CS-
EGCG-Ap) decreased the expression of ERK2 and
hTERT in comparison with the nanodrug group
(SiO
2
@CS-EGCG) and free EGCG in SKOV-3 cell
lines (Fig. 11). Experimental researches also showed
that EGCG can downregulate the expression level of
ERK in some other cells like renal tubular epithelial
cells in a dose-dependent manner. It is probably related
to the way that EGCG can affect some intracellular
signal pathways such as the ERK and Smad pathways,
but it is not clearly identified yet (Zhao et al. 2015).
EGCG can decrease the expression level of hTERT in
some cancer cells like MCF7 through hypomethylation
and hypoacetylation of the hTERT promoter region,
increasing of binding of E2F1 to the hTERT promoter,
and regulating the interaction of chromatin with some
proteins (Chen et al. 2013).
Conclusion
In this study, we developed a new nanodrug delivery
system for targeted delivery of EGCG to SKOV-3
ovarian cancer cell lines to improve the cancer ther-
apy efficacy. The EGCG was loaded on the
SiO2@CS NPs with encapsulation efficacy of
80%. The in vitro release behavior of EGCG was
higher in mild acidic pH which is similar to the
cancerous tissue condition. AS1411 aptamer conju-
gation to the SiO2@CS-EGCG NP increased the
uptake of SiO2@CS-EGCG to the SKOV-3 cells
compared with SiO2@CS-EGCG NP because they
bonded selectively to their targets (nucleolin) on the
surface of SKOV-3 cancer cell lines and entered to
the cells through the macropinocytosis mechanism
which it improves the efficacy of internalization.
SiO2@CS-EGCG-aptamer increased the killing rate
of SKOV-3 cell lines in comparison with SiO2@CS-
EGCG, and both of them showed better anticancer
efficacy than the free confirmed by MTT. Nuclei
fragmentations were increased in SiO2@CSEGCG-
aptamer in comparison with the SiO2@CS-EGCG
which proved through an apoptosis assay. Superior
antitumor efficacy of SiO2@CS-EGCG-aptamer was
proved by Annexin V assay in which, amount of
apoptotic cells reached 93% which all of them were
in late-apoptosis state. Cell cycle results indicated
that the SiO2@CSEGCG and SiO2@CS-EGCG-
aptamer NPs arrested cells in G0/G1 phase. Real-
time PCR results showed that SiO2@CS-EGCG
downregulated the expression level of ERK2 and
hTERT in comparison with the free EGCG and
SiO2@CS-EGCG-aptamer decreased more the ex-
pression level of two above mRNA level in compar-
ison with the SiO2@CS-EGCG. Based on these
results, the complex of SiO2@CS-EGCG-aptamer
can be an effective drug delivery system for targeted
ovarian cancer therapy.
Fig. 11 Differential gene expression level of ERK2 (a) and hTERT (b) in SKOV-3 cells treated with EGCG, SiO2@CS-EGCG, and
SiO2@CS-EGCG-aptamer
JNanopartRes (2020) 22:5 Page 13 of 14 5
Funding information This study was financially supported by a
grant [NO: 95/2-4/8] from Drug Applied Research Center, Tabriz
University of Medical Sciences, Tabriz, Iran.
Compliance with ethical standards
Conflict of interest The authors declare that they have no con-
flict of interest.
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