Doxorubicin/Cisplatin-Loaded Superparamagnetic Nanoparticles As A Stimuli-Responsive Co-Delivery System For Chemo-Photothermal Therapy

Abstract

Introduction
To date, numerous iron-based nanostructures have been designed for cancer therapy applications. Although some of them were promising for clinical applications, few efforts have been made to maximize the therapeutic index of these carriers. Herein, PEGylated silica-coated iron oxide nanoparticles (PS-IONs) were introduced as multipurpose stimuli-responsive co-delivery nanocarriers for a combination of dual-drug chemotherapy and photothermal therapy.

Methods
Superparamagnetic iron oxide nanoparticles were synthesized via the sonochemical method and coated by a thin layer of silica. The nanostructures were then further modified with a layer of di-carboxylate polyethylene glycol (6 kDa) and carboxylate-methoxy polyethylene glycol (6 kDa) to improve their stability, biocompatibility, and drug loading capability. Doxorubicin (DOX) and cisplatin (CDDP) were loaded on the PS-IONs through the interactions between the drug molecules and polyethylene glycol.

Results
The PS-IONs demonstrated excellent cellular uptake, cytocompatibility, and hemocompatibility at the practical dosage. Furthermore, in addition to being an appropriate MRI agent, PS-IONs demonstrated superb photothermal property in 0.5 W/cm² of 808 nm laser irradiation. The release of both drugs was effectively triggered by pH and NIR irradiation. As a result of the intracellular combination chemotherapy and 10 min of safe power laser irradiation, the highest cytotoxicity for iron-based nanocarriers (97.3±0.8%) was achieved.

Conclusion
The results of this study indicate the great potential of PS-IONs as a multifunctional targeted co-delivery system for cancer theranostic application and the advantage of employing proper combination therapy for cancer eradication.

Full text

ORIGINAL RESEARCH
Doxorubicin/Cisplatin-Loaded Superparamagnetic
Nanoparticles As A Stimuli-Responsive Co-Delivery
System For Chemo-Photothermal Therapy
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Mona Khafaji
1
Masoud Zamani
2
Manouchehr Vossoughi
2,3
Azam Iraji zad
1,4
1
Institute for Nanoscience and
Nanotechnology, Sharif University of
Technology, Tehran 14588-89694, Iran;
2
Institute for Biotechnology and
Environment (IBE), Sharif University of
Technology, Tehran, Iran;
3
Department of
Chemical and Petroleum Engineering, Sharif
University of Technology, Tehran, Iran;
4
Department of Physics, Sharif University of
Technology, Tehran 14588, Iran
Introduction: To date, numerous iron-based nanostructures have been designed for cancer
therapy applications. Although some of them were promising for clinical applications, few
efforts have been made to maximize the therapeutic index of these carriers. Herein,
PEGylated silica-coated iron oxide nanoparticles (PS-IONs) were introduced as multipurpose
stimuli-responsive co-delivery nanocarriers for a combination of dual-drug chemotherapy
and photothermal therapy.
Methods: Superparamagnetic iron oxide nanoparticles were synthesized via the sonochem-
ical method and coated by a thin layer of silica. The nanostructures were then further
modified with a layer of di-carboxylate polyethylene glycol (6 kDa) and carboxylate-meth-
oxy polyethylene glycol (6 kDa) to improve their stability, biocompatibility, and drug loading
capability. Doxorubicin (DOX) and cisplatin (CDDP) were loaded on the PS-IONs through
the interactions between the drug molecules and polyethylene glycol.
Results: The PS-IONs demonstrated excellent cellular uptake, cytocompatibility, and hemo-
compatibility at the practical dosage. Furthermore, in addition to being an appropriate MRI
agent, PS-IONs demonstrated superb photothermal property in 0.5 W/cm
2
of 808 nm laser
irradiation. The release of both drugs was effectively triggered by pH and NIR irradiation. As
a result of the intracellular combination chemotherapy and 10 min of safe power laser
irradiation, the highest cytotoxicity for iron-based nanocarriers (97.3±0.8%) was achieved.
Conclusion: The results of this study indicate the great potential of PS-IONs as a multi-
functional targeted co-delivery system for cancer theranostic application and the advantage
of employing proper combination therapy for cancer eradication.
Keywords: iron oxide nanoparticles, chemo/photothermal therapy, dual-drug delivery,
control release
Introduction
Cancer is the second leading global cause of death that inflicts more people every
year as lifestyles change and breast cancer is the most prevalent cancer type among
females, with one in every eight women suffering from it in their lifetime.
1,2
The
current clinical cancer therapies including chemotherapy, radiotherapy, and surgery
lack the desirable effectiveness and are associated with severe side effects.
3–5
For
these reasons, finding an improved cancer therapy method has been the subject of
extensive research efforts worldwide.
It has been proven that a mixed use of anticancer drugs and/or treatment methods
enhances the treatment efficiency by synergistic therapeutic effect and by overcoming
Correspondence: Mona Khafaji
Institute for Nanoscience and
Nanotechnology, Sharif University of
Technology, PO Box 11365-11155, Tehran
14588-89694, Iran
Tel +98-21-66164123
Fax +98-21-66164117
Email mona.khafaji@gmail.com
Manouchehr Vossoughi
Department of Chemical and Petroleum
Engineering, Sharif University of
Technology, PO Box 11365-8639, Tehran,
Iran
Tel +98-21-66164104
Fax +98-21-66005417
Email vosoughi@sharif.edu
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drug resistance.
6,7
Today, clinics practically benefitfromthe
enhanced therapeutic effect of radiotherapy and/or dual
drug chemotherapy by two drugs of different action
mechanisms after the surgery has been performed.
Nevertheless, due to the nonspecificity and high dosage of
drugs in these methods, a combined use of them intensifies
the side effects and deteriorates the quality of life for
patients.
8,9
Over the last decade, with advancement in
science and technology, scientists have sought a combina-
tion therapy for enhancing the therapeutic index while
reducing side effects. In this regard nanotechnology has
been the most sought after science to which the major
share of research in this field is dedicated. The unique
properties that some materials represent at the nanoscale
enable them to be used for innovative applications; due to
their very small size and surface properties, some types of
nanoparticles can accumulate in specific tissues or cells and
demonstrate a great potential for application in cancer diag-
nosis and therapy.
4,6,10
The application of nanostructures as
drug carriers
11
and bioimaging
12
and thermal treatment
agents
13
has been studied for more than a decade. Some
of these nanostructures, eg gold
13-15
and iron,
16
can produce
heat as a response to external stimuli such as a magnetic
field or laser irradiation. By specific surface modification of
these nanoparticles, anticancer drugs can be loaded on them
making them good candidates for a combination chemo and
thermal therapy.
12,17
However, sometimes the high degree
of complexity of the synthesis and modification process
practically limits the applicability of nanostructures.
Among all the investigated nanostructures superpara-
magnetic iron oxide nanoparticles (SPIONs) have attracted
extensive attention in cancer therapy due to their signifi-
cant biocompatibility, biodegradability, ease of synthesis
and surface modification, magnetic targeting
18
excellent
contrast for magnetic resonance imaging (MRI)
19
and
application in thermal treatments.
19,20
SPION-based
nanostructures with various modifications have been stu-
died as simultaneous drug carriers and hyperthermia
agents.
21,22
However, since their application as photother-
mal agents in comparison to magnetic hyperthermia pro-
vides a better control over the temperature increase –it
increases the temperature in the exact target site –most
modifications on SPIONs in the recent years have been
performed with the goal of combination chemo- and
photothermal-therapy.
17,23–25
The primary goal of all the research efforts in this area is
to achieve a more effective therapy with minimized side
effects. Diverse novel nanostructures have been studied but
they could not always improve the therapeutic index.
Indeed, although these new structures have their own mer-
its, the employed design failed to improve the efficacy of
both drug delivery and photothermal therapy. Therefore, the
maximum cytotoxicity was achieved when the amount of
drug was rather significant or an unsafe laser power was
applied.
26–28
For example, incorporation of graphene into
SPION-based nanostructure significantly enhances the drug
loading content, yet a desirable photothermal cell killing
cannot be achieved unless an intense and unsafe laser power
is employed.
29,30
Not to mention, the biocompatibility of
graphene is still a challenge for the scientific community as
there are numerous contradictory reports on it.
31,32
In order
to take advantage of the surface plasmon resonance proper-
ties of gold in the photothermal therapy, gold nanoshell was
incorporated to SPION-based nanocarriers. However, due to
the weak NIR absorption of spherical gold nanostructures, it
was inevitable that a high-intensity laser power would be
used to achieve a suitable photothermal cell killing.
14,15
Furthermore, preparation of these nanostructures usually
follows a complicated and multi-step synthesis procedure
which practically limits their application. Therefore, the
need for a safe method with an easily synthesizable struc-
ture that could enhance the efficiency of combined chemo-
photothermal therapy is well felt.
Herein, for the first time, we present PEGylated silica-
coated SPIONs (PS-IONs) as dual drug carriers for
Doxorubicin (DOX) and Cisplatin (CDDP), and as photo-
thermal and MRI-contrast agents. By loading two drugs
with different action mechanisms on PS-IONs, it would be
possible to invade the cancer cells by a three-branched
shotgun design, ie targeted drug delivery, dual drug che-
motherapy, and photothermal therapy. The physicochem-
ical properties of the synthesized nanoparticles were
examined by transmission electron microscopy (TEM),
vibrating sample magnetometer (VSM), X-ray diffraction
(XRD), Fourier transform infrared spectroscopy (FT-IR),
dynamic light scattering (DLS), magnetic resonance ima-
ging (MRI), and by exposure to a 808 nm laser beam. The
cytocompatibility and cellular uptake of PS-IONs were
investigated both quantitatively and qualitatively using
fibroblast and breast cancer cells, respectively. The influ-
ence of PS-IONs on blood coagulation cascade and red
blood cells was also studied. Furthermore, the perfor-
mance of PS-IONs as a controlled release system was
measured by in vitro simulating the photothermal therapy
and acidic cancerous condition. Finally, the effectiveness
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of combined therapy on the viability of breast cancer cells
was investigated.
Materials And Methods
Materials
Iron (III) chloride hexahydrate (FeCl
3
.6H
2
O), iron (II) chlor-
ide tetrahydrate (FeCl
2
.4H
2
O), chromium trioxide (CrO
3
),
polyethylene glycol (PEG, Mw 6 kDa), methoxypolyethylene
glycol (m-PEG, Mw 6 kDa), ammonia (NH
3
, 25%), tetraethy-
lorthosilicate (TEOS), potassium chloride (KCl), disodium
hydrogen phosphate (Na
2
HPO
4
), potassium dihydrogen phos-
phate (KH
2
PO
4
), sodium chloride (NaCl), ethylenediaminete-
traacetic acid (EDTA), acetone, and isopropanol were
purchased from Merck. Cis-diammineplatinum (II) dichloride
(CDDP), (3-aminopropyl) trimethoxysilane (APTMS), N-(3-
dimethylaminopropyl)-Hydroxysuccinimide (NHS), trypsin,
penicillin-streptomycin, and 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) were obtained from
Sigma-Aldrich.
The dicarboxylate-PEG and carboxylated m-PEG were
achieved from oxidation of PEG and m-PEG using Jone’s
reagent as described by Lele.
13,33
Hydrochloric acid (HCl, 37%) and methanol were pur-
chased from Chemlab (Belgium). Doxorubicin hydrochlor-
ide (DOX) was obtained from Pfizer (Perth, Australia).
Fetal bovine serum (FBS), RPMI 1640 and high glucose
DMEM cell culture mediums were purchased from
Thermo Fisher Scientific (Gibco, USA).
All chemicals were of analytical grade and used as
received without further purifications. Deionized (DI)
water with a resistivity of 18.2 MΩand double distilled
water were used for the preparation of all chemical and
biological samples, respectively.
Synthesis Of PS-IONs
The sonochemical method was used to synthesize iron
oxide nanoparticles (IONs) in order to achieve smaller
particles with a narrow size distribution.
34
Briefly,
FeCl
2
.4H
2
O (0.002 moles) and FeCl
3
.6H
2
O (0.004
moles) were dissolved in 2 M HCl and titrated with 4%
ammonia solution at 30–32°C in the ultrasonic bath for
1 hr. The produced particles were immediately rinsed
several times with methanol to neutralize the pH and
were dispersed in 50 mL of absolute ethanol.
In the next step, IONs were coated by silica according
to Stöber’s method.
34
30 mL of the just-prepared IONs
suspension was alkalinized to pH 8–10 using diluted
ammonia solution and was put in an ultrasonic bath at
30–32°C. After 15 min, 1.8 mL of TEOS was added to
the suspension. After 1 hr, 25 µL of APTMS was added to
the reaction mixture to cease the silica coating process and
also to functionalize the surface with amine groups.
Finally, the as-prepared core-shell nanostructures were
separated from the solution by the use of an external magnetic
field and rinsed three times with ultrapure water and was
diluted to 30 mL. The structures were then further modified
with a layer of dicarboxylate PEG (6 kDa) and carboxylated-
methoxypolyethylene glycol (6 kDa) to improve their biocom-
patibility and drug loading capability. To this end, 1 mL of the
silica-coated SPIONs suspension was diluted 5 times and was
sonicated for 5 min. Then a diluted solution of an extra amount
of dicarboxylate PEG (18 mg) and m-PEG (36 mg) was added
to the silica-coated iron oxide nanoparticles and stirred with an
equivalent amount of EDC (1.7 mg)/NHS (1 mg) for 2 hrs.
The final product was rinsed three times and kept as a suspen-
sion in ethanol for future use.
Characterization Of The PS-IONs
Furier transform infrared spectra were recorded on an ABB
Bomem MB-100 spectrometer in the range of 400–4000 cm
−1
.
Magnetic properties of Fe
3
O
4
nanoparticles (IONs) and PS-
IONs were evaluated by MDK Kawir Magnetic vibrating
sample magnetometery. The applied magnetic field was in
the range of 0 to 9 kOe. The morphology of PS-IONs was
observed by transmission electron microscopy (Zeiss EM10C,
80 kV). Crystallographic information and mean particle size of
samples were obtained from X-ray diffraction. A P Analytical
X’Pert PRO MPD (The Netherlands) X-ray diffractometer
with CuKα-irradiation (λ= 1.5406 ᵒA, 10ᵒ<2θ<80ᵒ)was
used to measure the X-ray diffraction patterns.
The platinum concentration was quantified using
inductively coupled plasma optical emission spectroscopy
(ICP-OES, SPECTRO ARCOS). The fluorescence spectra
of doxorubicin were collected on a fluorescence spectro-
photometer (Varian, Cary Eclipse) at room temperature.
Magnetic resonance images were recorded using a 3 T
clinical MRI scanner (Magnetotrio, Siemens). The relax-
ivity was determined using spin-echo acquisition utilizing
a repetition time (TR) of 3000 ms and 32 echo-times (TE)
ranging from 12 to 384 ms where the field of view was
7 cm, slice thickness was 3 mm and acquisition matrix was
256×128. The transverse relaxation time (T
2
) of water
protons was obtained by fitting a logarithmic curve to the
mean of the measured MR signals. By linear least square
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fitting of 1/T
2
(s
−1
) versus iron concentration (mM) the
transverse relaxivity, r
2,
was calculated.
All the samples were irradiated by a Hi-Tech
Optoelectronic (China) optic fiber coupled continuous wave
diode laser at 808±5 nm (0.5 W/cm
2
).
In Vitro Cellular Assay
Cell Culture
Human breast cancer cells (MCF7; National Cell Bank of
Iran) and a mouse fibroblast cell line (L929; National Cell
Bank of Iran) were used as a model of cancer and normal
cells to evaluate the cytotoxicity of PS-IONs. The MCF7
and L929 cells were cultured in high glucose DMEM and
RPMI mediums contained 10% FBS and 1% penicillin/
streptomycin, respectively. The cultures were incubated at
37°C in a humidified atmosphere of 5% CO
2
.The mediums
were replaced every other day and upon 70% confluency,
the cultures were subcultured.
Cell Viability Assay
MTT assay was carried out to determine the viability of
normal fibroblast cells after one, two, and three days of
cell culture in the presence of different amounts of PS-
IONs. Briefly, 1 mL of cell suspension (50,000 cells/mL)
were plated in plasma treated 24-well plates and cultured
for 24 hrs to allow them to attach, and then exposed to a
serial concentration of PS-IONs in nine different groups
(10 to 500 µg/mL) and further incubated at 37°C in a
humidified atmosphere of 5% CO
2
. The cell viability was
measured using MTT assay standard protocol.
13
The per-
cent of cell viability was calculated as follows:
% cell viability ¼
absorbance of samples
absorbance of blank
absorbance of control
absorbance of blank
100 (1)
MTT assay was also performed in order to compare the
cytotoxicity effect of CDDP and DOX with that of PS-
IONs and drug-loaded PS-IONs on MCF7 human breast
cancer cells. The laser effect was also evaluated on each
group and results were presented as the average records of
three replications. Briefly, the one-day cultured cells (33
wells, 50,000 cells/well) were divided into eleven groups
and exposed to a defined concentration of the drugs and/or
PS-IONs, as presented in Table 1. After overnight incuba-
tion, in order to study the photothermal therapy effective-
ness of PS-IONs, samples of five groups were exposed to a
laser beam (0.5 W/cm
2
, 10 min) and further incubated
overnight. The viability of the cells in each group was
measured using MTT standard protocol at the end of the
third day.
Cellular Uptake
An elemental analysis method was used to quantitatively
examine the cellular uptake of PS-IONs. To do so,
MCF7 cells (50,000 cell/well) were seeded in 24-well
plates and cultured overnight. Then, the culture medium
was replaced with a new medium containing a series of
PS-IONs concentrations followed by incubation at 37ᵒC
for 24 hrs. After rinsing with phosphate buffer saline
(PBS) solution (1 mL×3), the cells were detached using
trypsin solution and collected in separate vials. The cells
were then disintegrated using a high concentration nitric
acid at 70°C to release the PS-IONs in the solution for
elemental measurement by ICP-OES. Non-treated cells
were used as blank and results were averaged from three
replications.
In Vitro Hemocompatibility Assay
APTT and PT assay: Prothrombin time (PT) and activated
partial thromboplastin time (APTT) were applied to evaluate
the thrombogenic activities of PS-IONs. Briefly, a speci fic
amount of 2 mg/mL PS-IONs suspension in PBS was mixed
with the human blood plasma which was drawn from healthy
adult volunteer and mixed with 3.2% sodium citrate as an
anticoagulation agent to obtain the final concentration of
200 μg/mL of PS-IONs and incubated for 30 min at 37°C.
Afterward, to 0.1 mL plasma solution, the specificreagentsof
PT and APTT test were added and left in 37°C for 3 min.
Finally, using a photo-optical clot detection instrument
(Coatron M1, TECO, Germany), the clotting times were
recorded.
Peripheral blood smear test: To obtain a suspension of
200 μg/mL of PS-IONs, a specific amount of 2 mg/mL PS-
IONs suspension in PBS was mixed with fresh blood
(EDTA-anticoagulated) and was shacked continuously for
10 mins. Then in 40 min time intervals a drop of the blood
sample was spread on a glass coverslip followed by fixa-
tion with methanol at room temperature then the blood
cells were dyed using Giemsa stain.
It should be mentioned that the use of relevant human
material was approved by Pasteur Institute of Iran ethics
committee. For all the investigations involving human
subjects (cellular and blood experiments), a written
informed consent has been obtained from the involved
participants.
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Drug Loading And Release
In order to load the drugs on the nanoparticles, 30 mg of
PS-IONs was dispersed in 30 mL of deionized water.
Different amounts of DOX and CDDP were added to the
suspension to reach the final concentration of 15–40 ppm
for each of them and the mixture was shaken for 72 h at
room temperature. Then, the drug-loaded nanoparticles
were separated using an external magnetic field and rinsed
several times with deionized water to remove the unat-
tached drugs. All supernatants containing the unloaded
DOX and/or CDDP were collected. The amount of
unloaded DOX was determined by measuring fluorescence
emission at 592 nm (excitation at 485 nm). The amount of
unloaded CDDP was calculated by measuring the platinum
content using an elemental analysis (ICP–OES) relative to
a calibration curve obtained under identical conditions.
The drug loading content (DLC) and drug loading effi-
ciency (DLE) were calculated according to equations 2
and 3, respectively.
DLC ¼WD=Wn
ðÞ100 (2)
DLE ¼WtWu
Wt

100 (3)
where the W
D
,W
n
,W
u
, and W
t
are weights of the loaded
drug, nanoparticles, unloaded drug, and initially added
drug, respectively.
To study the release profile of DOX and CDDP from
the nanocarriers, a dialysis process was performed. All
release studies were performed under sink conditions
(amount of drug <10% of the solubility). To this end, 1
mg of drug-loaded nanoparticles were dispersed in 5 mL
of PBS and dialyzed against 30 mL of PBS using a 10 kDa
dialysis bag (Sigma) in two different pH values of 7.4 and
5.5 at 37°C under constant shaking at 150 rpm. In order to
simulate the photothermal condition, a separate group of
each pH value was subjected to a 30 min temperature
increase to 42°C. At predetermined time intervals, 5 mL
of the solution outside the dialysis bag was withdrawn for
analysis and replaced with the same volume of fresh PBS
solution.
Statistical Analysis
Statistical package for social science (SPSS v. 25.0) soft-
ware was used for statistical analysis of all the experi-
ments. Multiple samples were analyzed by one-way
ANOVA. To compare the control samples with the treated
ones, the Student–Newman–Keuls post hoc test was done.
Measures are reported as means ± standard deviations and
statistically significant difference was reported at p < 0.05.
The XRD data were analyzed using X’Pert HighScore
Pluse software. All the graphs were plotted by Microsoft
Excel 2010 software.
Results And Discussion
Synthesis Of PS-IONs
Iron oxide nanoparticles (IONs) were first synthesized
through the sonochemical method and then coated with a
silica shell to improve their biocompatibility, thermal sta-
bility, and dispersibility in aqueous solutions.
34
This rela-
tively simple method also enables the functionalization of
the particles’surface for further applications. APTMS was
used to both halt the increase-in-thickness of the silica
shell on the IONs and to functionalize the surface of silica
coated-IONs with amine groups. The zeta potential of the
NH
2
modified silica-coated IONs was 27.7 mV. The pre-
sence of amine groups on the surface not only enhances
the stability of particles due to their positive charge but
Table 1 Characteristics Of Eleven Groups For Cytotoxicity Assessment By MCF-7 Breast Cancer Cells
Group No. Control DOX (4.2 µg/mL) CDDP (2.8 µg/mL) PS-IONs (200 µg/mL) Drug-Loaded PS-IONs Laser
1✓
2✓ ✓
3✓
4✓ ✓
5✓
6✓ ✓
7✓✓
8✓
9✓✓
10 ✓
11 ✓✓
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also enables the attachment of PEG molecules from their
carboxylic acid ends.
After preparation of the silica-coated IONs, their sur-
faces were modified with PEG to increase their circulation
time in the bloodstream and enhance their drug loading
ability for targeted drug delivery application. After pegy-
lation, the zeta potential of nanoparticles decreased to
−2.27 mV which confirms the presence of the COOH
groups on the surface of PS-IONs instead of NH
2
ones.
The detailed procedure for the preparation of the PS-IONs
and the drug loading on them is illustrated in Scheme 1.
Figure 1a and bshow the TEM images of the PS-IONs.
It could be observed that the nanoparticles were spherical
in shape with a narrow size distribution and an average
size of 20±3 nm. The IONs could be distinguished from
the silica through the difference in their contrasts.
Scheme 1 Illustration of the synthesis procedure of PS-IONs and drug loading mechanism.
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According to TEM images, the silica shell was thin
enough to limit the adverse effect on magnetization satura-
tion. The severe aggregations of the PS-IONs were pre-
sumably due to the hydrogen bonding of PEG molecules
among the nanoparticles.
X-ray diffraction (XRD) experiments were also per-
formed to investigate the crystalline structure of IONs and
PS-IONs (Figure 1c). The characteristic diffraction peaks in
both spectra could be assigned to the (220), (311), (400),
(422), (511) and (440) planes of the spinel structure of
magnetite (Fe
3
O
4
) according to the JCPDS 19–0629.
Furthermore, the broad peak at around 2θ= 20° indicates
the presence of amorphous silica in the synthesized
nanoparticles.
35
The Scherer equation was employed to cal-
culate the average crystallite size from the (311) peak in each
spectrum. The calculated particle size for both samples was
15 nm which is in agreement with the observations of the
TEM images indicating that the silica coating process does
not induce major unfavorable effects such as dissolution or
growth of the crystals on the Fe
3
O
4
nanocrystals.
Considering that the size of PS-IONs was less than a
magnetic domain for these components (25 nm for Fe
3
O
4
),
it was expected that they possess a superparamagnetic
behavior.
34
With this in mind, VSM analysis was performed
on PS-IONs to evaluate their magnetic behavior (Figure 1d).
The results show that the magnetization saturations of the
IONs and PS-IONs were 66.6 emug
−1
and 16.75 emug
−1
,
respectively. These data were consistent with the previous
observations which indicate that silica coating decreases the
magnetization saturation of IONs by at least four folds.
36
The
magnetic residue of both IONs and silica-coated ones were
4mT, hence both samples were classified as superparamag-
netic nanoparticles.
34
The superparamagnetic behavior in
silica-coated particles possibly results from bipolar-bipolar
interactions between the magnetic cores. Therefore, since the
bipolar-bipolar magnetic interactions had a reverse relation-
ship with the distance, the interactions were intensified as a
result of theentrapment ofmore than one ION in a silica shell
and/or the physical attachment by the hydrogen bonding of
PEG molecules.
19,34
FT-IR spectroscopy was employed to verify the forma-
tion of the silica shell surrounding the Fe
3
O
4
nanoparticles
and also to investigate the success of the PEGylation
process. Figure 1e shows the IR spectra of IONs (black),
Figure 1 (a, b) TEM images of PS-IONs (scale bars are 150 nm and 40 nm, respectively). (c) XRD patterns [the yellow, green, pink, blue, violet, light brown and orange
squares relate to (220), (311), (400), (422), (511), (440) and (530) planes of the spinel structure of magnetite, respectively], (d) hysteresis loops, (e) FT-IR spectra, and (f)
DLS particles size distribution profiles of IONs (black line) and PS-IONs (red line).
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PS-IONs (red), and PEG (blue). The presence of silica,
magnetite, and PEG in the PS-IONs was clearly shown by
the observation of the characteristic bands of all three,
magnetite (560, 1400 cm
−1
, Fe-O band vibration), silica
(1080 cm
−1
asymmetric Si-O-Si stretching), and PEG
(2925 cm
−1
CH
2
stretching vibration, 1150 cm
−1
C-O-C
stretching), in the PS-IONs’spectrum (red line). There was
also an evidence of bonds formation between silica and
Fe
3
O
4
(1190 cm
−1
, Fe-O-Si vibration) confirming the for-
mation of the desired structure. Unlike the IONs, dissol-
ving the PS-IONs even in hot hydrochloride acid (37%
w/v) was not possible except after prewashing in a hydro-
fluoric acid to remove the silica coating, endorsing the full
coverage of silica on the nanoparticles.
The hydrodynamic size of the nanoparticles in aqueous
solution was determined by DLS (Figure 1f). The results
showed a single peak with a small polydispersity index
which points to the negligible aggregation of PS-IONs in
aqueous solution. The average diameters in a hydrated state
were 58.8 (PDI=0.171) and 122 nm (PDI=0.156) for IONs
and PS-IONs, respectively. The measured size of IONs by
the DLS method was significantly higher compared to the
TEM measurements. According to previous reports, this may
be occurred due to the fast flocculation of bare magnetic
nanoparticles, the presence of water molecules on their sur-
face, and/or the magnetic dipole-dipole attraction between
magnetic nanoparticles.
37–39
Also, the measured size for PS-
IONs was approximately six times higher than that measured
by TEM. This increase may be due to the fact that more than
one ION was trapped in the same silica coat and/or irrever-
sible aggregates formed through thePEG bridges. In addition
the hydrophilic PEG chains were extended in the aqueous
solution or/and dynamic association in the liquid.
8,34
In view
of the fact that the nanocarriers’size plays an important role
in their biodistribution and accumulation in the organs and
cells, the synthesized PS-IONs are absolutely ideal as nano-
carriers for cancer therapy applications.
In Vitro Cytocompatibility
The most important characteristic of a nanocarrier in a given
drug delivery application is its negligible toxicity to normal
cells. The cytotoxic effects of PS-IONs on L929 fibroblast
cells were evaluated by conducting a MTT assay. Figure 2
shows that the presence of nanoparticles did not induce any
significant detrimental effect on the viability of normal cells at
24, 48, and 72 h time points. The viability of cells was more
than 88±5.2% even after exposure to 500 μg/mL of nanopar-
ticles for 72 h, endorsing the success of the employed surface
modification in the improvement of the nanoparticles’
biocompatibility.
40
Unlike some of the previous observations,
the increase in the cell density in comparison to the control in
some samples is attributed to their metabolic changes after
being exposed to nanoparticles rather than the dissolution of
IONs.
19
Light microscopy images of the cells exposed to
different concentrations of PS-IONs for defined times indi-
cated that the presence of the nanoparticles in the culture
medium had no adverse effect on the attachment and morphol-
ogy of the cells (Figure S1). This significant improvement in
the biocompatibility was attributed to both the silica coating
and the PEGylation which makes the prepared particles sui-
table candidates for a drug delivery application.
Cellular Uptake
It is essential for the nanoparticles to internalize into target cells
in the highest amount in order to be considered as effective
drug delivery vehicles. The cellular uptake of PS-IONs by
MCF7 breast cancer cells was studied both quantitatively and
qualitatively. According to Figure 3a, the PS-IONs could be
obviously visualized in the cytoplasm of the cells as brown
spots indicating that their accumulation in the cells did not
affect the cells’normal morphology. In addition, the cumula-
tive cellular uptake of PS-IONs was linearly increased as their
concentration in the medium increased up to 200 µg/mL. The
percentage of the uptaked PS-IONs increased linearly at low
Figure 2 Viability of L929 normalfibroblast cells after 24, 48, and 72 hrs of incubation
with different concentrations of PS-IONs (ranging from 10 to 500 µg/mL). There was
no significant difference between the control and other groups.
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Figure 3 (a) Light microscopy images of the cells (the total magnification was 400 times) exposed to 100 (left) and 200 µg/mL (right) of PS-IONs for 24 hrs. The brown dots
show the uptaken nanoparticles. (b) Concentration dependent cellular uptake percentage. The inset shows the amount of PS-IONs uptake (µg/mL) versus initial PS-IONs
concentration (µg/mL) in the culture media.
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concentrations (up to 50 µg/mL), while further increase of the
PS-IONs concentration in the culture medium caused their
uptake rate to decelerate so that for concentrations above 100
µg/mL the uptake percentage declined indicating the cells were
approaching their saturated uptake capacity (Figure 3b). This
excellent cellular uptake was related not only to the optimum
particle size and the spherical morphology of PS-IONs but also
to their surface characteristics. By altering the serum protein
adsorption on the nanoparticles surface, the coating affects the
nanoparticles-cells interactions and thus improved the cellular
uptake.
41,42
So, the presence of PEG on the particles surface
improved the cellular uptake and viability through prevention
of the attachment of proteins to their surface.
43,44
In Vitro Hemocompatibility
Another determinant factor in the clinical applicability of
nanocarriers is their means of interaction with the blood
cells. A proper drug delivery system should not induce any
adverse effect on the morphology and activity of the blood
cells.
45
Accordingly, the hemocompatibility of the as-
synthesized nanoparticles was investigated by studying
their effect on blood coagulation and the morphology of
red blood cells (RBCs).
13,46,47
The coagulation times in
the presence of PBS (control) and 200 μg/mL of PS-IONs
were investigated. The results showed that the PT and
APTT have not been affected in the presence of such a
high concentration of nanoparticles and still remained in
the normal reference range (Table 2).
Normally RBCs have a biconcave disk shape which
easily changes in interaction with foreign substances.
13,46
The investigation of RBCs morphology, when exposed to a
foreign material, is a routine method to evaluate the new
biomaterial’s hemocompatibility. Hence, the peripheral
blood smear test was performed to study the influence of
the PS-IONs on the shape of RBCs. It can be observed from
Figure 4 and Figure S2 that the RBCs had undergone
neither deformation nor aggregation after exposure to the
200 µg/mL of PS-IONs even after 4 hrs of incubation when
compared to the controls. This excellent hemocompatibility
was attributed to the negligible electrostatic interaction
between negatively charged RBCs and PEGylated nanopar-
ticles, resulting from the presence of non-reactive methoxy
groups on their surface.
In Vitro Drug Loading And Release
The ability of nanoparticles to carry and deliver a suffi-
cient amount of anticancer drug to the tumor tissues is of
great importance. Among the various anticancer drugs,
CDDP and DOX are widely used for solid cancerous
tumor treatment, therefore loading each of them on nano-
carriers has been extensively investigated.
48-50
As the var-
ious anticancer drugs have different action mechanisms, it
has been proven that cancerous cells could be killed more
effectively by the use of dual-drug chemotherapy.
8,9
Therefore, the potency of the PS-IONs was examined as
magnetically targeted dual-drug carriers by loading both
DOX and CDDP on them. The loading efficiency of DOX
in the presence of a constant amount of CDDP (15 ppm)
was investigated. As shown in Figure 5a the DLC was
raised by increasing the initial DOX concentration. At the
optimum conditions (30 ppm DOX), the obtained DLE
was 69.3±1.4% with DLC of 21±0.42 µg DOX/mg PS-
IONs. At the normal pH, DOX has a positive charge while
the carboxyl groups on the particles surface were nega-
tively charged.
51
Therefore, DOX could be efficiently
loaded into PEG chains through electrostatic interactions,
as well as the formation of amide and hydrogen bonds.
11,22
To investigate the loading of CDDP on the nanoparti-
cles, different concentrations of CDDP were added to the
solutions containing constant amounts of DOX loaded PS-
IONs (1 mg/mL) and DOX (15 ppm) (Figure 5b). The
amount of loaded CDDP obtained in the optimum condi-
tion (30 ppm CDDP) was 14±0.4 μg CDDP/mg PS-IONs
with a DLE of 46.6±1.4%, resulting in a total DLC of
3.5% w/w. In the absence of DOX, the DLC of CDDP was
increased by 100% while the DLE remained unchanged.
Hence, the DLE of CDDP is not a function of its initial
concentration. It can be concluded that the molecules of
both drugs compete against each other to seize the avail-
able carboxyl groups on the surface of the particles. So,
the higher DLC of DOX in comparison to CDDP is attrib-
uted to the higher reaction rate of hydrogen or electrostatic
bonding of DOX rather than coordination bonding of
CDDP with the carboxyl groups of the particles’surface.
After the drug loading, the zeta potential of PS-IONs was
changed positively to reach +1.25 mV. According to pre-
vious reports, attachment of CDDP and DOX slightly
Table 2 Blood Coagulation Times After Dilution With The Same
Volumes Of PBS And PS-IONs Suspension (final Concentration
Of 200 μg/mL)
Normal
Range
Diluted
With PBS
Diluted With PS-
IONs (200 µg/mL)
APTT (s) 28 to 38 39.0 38.0
PT (s) 11 to 14 13.0 13.2
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increase the zeta potential. So the zeta potential changes
confirmed the successful drug loading.
52,53
IR spectra of
the drug-loaded PS-IONs were also employed to confirm
the presence of both drugs on them. As it can be seen in
Figure S3, the characteristic peaks of both DOX (3512,
632 cm
−1
) and CDDP (3296, 1319 cm
−1
) were presented
in the spectrum of the drug-loaded PS-IONs, demonstrat-
ing the reliability of the loading method.
Having a controlled drug release in the body and tumor
tissues is an indispensable characteristic of a drug delivery
system for cancer therapy. Minimizing the drug release in
the normal tissues not only reduces the side effects of the
drugs, it also maximizes the drug dosage at the tumor site
as well, resulting in a more efficient and favorable treat-
ment. To study the in vivo release behavior of drugs
from the as-synthesized PS-IONs, the pH conditions of
normal and cancerous tissues were simulated in vitro.
Furthermore, the effect of photothermal treatment using
nanoparticles was investigated by imposing an initial tem-
perature increase to 42°C for 30 min. As shown in
Figure 5c only about 23±2.2% of DOX released from the
PS-IONs after 24 h at the physiological condition, while
the release reached to 41±2.7% and 45±2.6% by decreas-
ing the pH to 5.5 and by applying the photothermal con-
dition, respectively. This indicates that both the
temperature increase and the pH decrease stimulate the
drug release. Applying the photothermal and acidic pH
conditions simultaneously increased the release rate three
folds. This pH- and thermo-responsive drug release could
be explained according to the drug-PEG interactions. The
interaction intensity between DOX molecules (pK
a
= 8.2)
and PEG chains weakens when decreasing the pH of the
media from neutral or weak alkaline to acidic condition.
Therefore DOX could remain loaded on the nanoparticles
during the blood circulation and could escape from them
more easily within the tumor cells. Similarly, through
increasing the system’s energy level, the temperature rise
has the same effect on the interaction intensity between
DOX and PEG.
11,22
As Figure 5D shows, the CDDP release was more
temperature-dependent than pH-dependent. As mentioned
before, CDDP molecules were loaded through the coordi-
nation of interactions between the hydrolyzed drug mole-
cules and the terminated carboxyl groups of PEG. By
decreasing the pH, the terminated carboxyl groups were
protonated which attenuated the interaction between the
drug molecules and the PS-IONs. In addition, according to
the equilibrium hydrolysis reaction of CDDP (Figure S4)
in acidic conditions and in presence of excess amount of
chlorine ions the rate of mono- and dichloro-cisplatin
formation increases which results in a faster release of
drug molecules. Moreover, through supplying the coordi-
nation dissociation energy, higher temperature leads to an
increase in CDDP release rate by more than two folds.
54,55
Applying the photothermal treatment and acidic pH con-
ditions simultaneously increased the release rate by 2.7
folds.
The release rate of CDDP in all situations was more
than that of DOX (Figure S5) which indicated the differ-
ence in drug-PS-IONs interactions. Unlike the CDDP, the
DOX molecules could entrap between the PEG chains by
hydrogen bonding in addition to the terminal attachment
which resulted in the higher DLC.
11,18
This was a reason for
the slower release of DOX, because some of the DOX
molecules had to diffuse through the PEG chains in order
to enter the solution while the terminally-loaded CDDP
molecules could directly be released into the solution.
These data indicate that the designed dual stimuli-triggered
Figure 4 Light microscopy images (the total magnification was 400 times) of blood smear prepared samples from EDTA-anticoagulated blood without dilution (control) and
after addition of PS-IONs (final concentration 200 μg/mL) and PBS after 4 h. The RBCs showed neither deformation nor aggregation when compared to the controls.
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drug release system provides a favorable drug delivery
platform for combination with photothermal therapy.
PS-IONs As MRI Contrast Agent
The ability of PS-IONs to serve as MRI contrast agents
was investigated using a 3T clinical instrument. The
recorded images showed proton relaxation enhancement
compared to the deionized water control (Figure 6a). The
calculated r
2
for the PS-IONs was 127.31 mM
−1
s
−1
(Figure 6b) which is comparable with that obtained for
PEGylated or silica coated IONs.
40,56
The obtained trans-
verse relaxivity was almost twice the amount of its bare
IONs.
57,58
This enhancement could be explained according
to the structural characteristics of PS-IONs. As reported
before, the field gradients generated by magnetized nano-
particles resulted in dephasing of the magnetic moment in
Figure 5 Loading capacity of (a)DOXand(b) CDDP on PS-IONs as a function of their initial concentation in the solutions containing 15 ppmof CDDP and DOX, respectively. In
vitro release profiles of (c)DOXand(d) CDDP from PS-IONs at different temperature and pH conditions. Asterisks (*) indicate significant difference (P < 0.05).
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water’s protons. Therefore magnetic nanoparticles induced
spin-spin relaxation as the proton passes through such field
gradients. By increasing the iron concentration, the mag-
netic moment becomes stronger resulting in distortion of
spin coherence of water protons in transverse relaxation.
56
The transverse relaxivity is related to the hydrody-
namic diameter of the aggregated nanostructure and the
magnetic saturation. Although the magnetic saturation of
PS-IONs was reduced comparing to its initial value, the
significant increase in the PS-IONs hydrodynamic dia-
meter was the leading factor in the MRI contrast enhance-
ment. The PEGylated nanoparticles could make small
reversible aggregates through the hydrogen bonding.
Formation of these cluster-like structures not only
increased the effective hydrodynamic diameter of nano-
particles several times but also shortened the distance
among magnetic particles which, in turn, induced a more
inhomogeneous local magnetic field. Finally, improved
hydrophilicity of PEGylated nanoparticles facilitated the
diffusion of water molecules near the surface of magnetic
particles, immobilizes them by hydrogen bonding, and
extended the interaction between the magnetic field and
protons.
56,59
Cytotoxicity Effect Of Multiple Treatment
Method
The efficiency of the multiple-treatment-method was stu-
died on MCF7 breast cancer cells in vitro and was com-
pared with individual chemotherapy and photothermal
therapy according to Table 1 . The obtained results are
summarized in Figure 7. As was expected, the sole laser
treatment (0.5 W/cm
2
, 808nm) neither had a significant
effect on the cells’viability nor exerts an effective interac-
tion with the CDDP and DOX drugs. The viability of the
cells exposed to DOX (4.2 µg/mL) and CDDP (2.8 µg/mL)
for 48 h, were 20.5±4% and 57±5%, respectively. Although
the free drugs have significantly decreased the cell viability,
these data indicate the inefficacy of the drugs at such low
concentrations. This was the reason for the extensive side
effects of traditional cancer chemotherapy, as it requires
Figure 7 MTT viability assay of MCF7 cells after exposure to 0.5 W/cm
2
near-IR
laser irradiation for 10 min, incubation with 200 μg/mL of PS-IONs, chemotherapy
with DOX (4.2 μg/mL) or CDDP (2.8 μg/mL), dual-drug chemotherapy, photother-
mal treatment, and multiple treatment method. Values are mean±SD. Double
asterisks (**) indicate nonsignificant difference (P>0.05).
Figure 6 (a)T
2
-weighted MR images of PS-IONs in aqueous media at various concentrations and different echo times. (b)T
2
relaxation rate (R
2
) versus iron concentration
in PS-IONs.
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administration of a high dosage of drugs in the whole body
to reach a desirable cytotoxicity for tumor cells.
60
DOX, an anthracycline antibiotic, restrains the DNA
remodeling through intercalating between DNA nucleotides
and inhibiting the activity of topoisomerase II (TOP2).
61
CDDP, a powerful DNA chelating agent, induces cancer
cells apoptosis by covalently bonding with DNA purine
bases to form DNA adducts which could inhibit cellular
transcription and replication.
62
These two drugs have inde-
pendent action mechanisms, and consequently, DOX could
further impede the repairing of CDDP-damaged DNA by
TOP2. Therefore, the combination chemotherapy by DOX
and CDDP could disrupt the cellular repair mechanisms. A
mixed use of cancer drugs with different mechanisms of
action is a clinical method to overcome the cell’s resistance.
It would also reduce the side effects of treatment by
decreasing the administered dosage and improving the treat-
ment efficiency.
8,9,63
As it is shown in Figure 7,dual-drug
chemotherapy had significantly enhanced the cytotoxicity
so that the cells’viability declined to 10.65±0.9%. This
result confirmed the synergetic effect of chemotherapy
drugs on the cancer cells.
As it was qualitatively shown in Figure 3, PS-IONs had
no unfavorable effect on the cancer cells’morphology.
They also demonstrated very good biocompatibility
(Figure 2) and excellent cellular uptake. By loading the
drugs on these nanocarriers, they would escape the drug
resistance by entering the cells through endocytosis enhan-
cing the cytotoxicity.
64,65
The results show that although
almost half of each drug were remained loaded on the
nanocarriers (50±3% of CDDP and 45±2.6% of DOX,
according to in vitro simulation), they resulted in the
same cytotoxicity as the free drugs. This was most likely
due to the effective cell membrane penetration and accu-
mulation of the drug-loaded PS-IONs into MCF7 cells by
endocytosis and phagocytosis. Consequently, this phenom-
enon increased the accumulated drugs within the cells
since they would release more rapidly due to the intracel-
lular acidic condition of cancer cells.
66,67
In addition to drug delivery, the PS-IONs show remark-
able ability as photothermal agents. Free PS-IONs demon-
strated superior cytotoxicity over CDDP (2.8 µg/mL) by
reducing the cell viability to 30±4%, after an exposure to
low power laser irradiation (0.5 W/cm
2
, 10 min) which
increased the temperature from 37°C to 42°C. In contrast
to previous studies, a low power laser irradiation, close to
the maximum permissible exposure (MPE) for skin (0.33
W/cm
2
), had been used for photothermal therapy.
6,16,24
By
emitting the NIR light beam to the drug-loaded PS-IONs, a
significant enhancement in cytotoxicity had been obtained.
The multiple-treatment-method increased the cancer cell
killing efficiency to 97.3±0.8% in a single dose therapy.
This result surpasses the best of previously reported out-
comes for SPION-based nanocarriers despite the fact that
very low drug concentration and laser power intensity is
simultaneously employed in our study (Tab le 3). This excel-
lent result was partly attributed to the higher accumulated
free drugs content in the cells as a result of the photothermal
and acidic condition in comparison to the non-photothermal
condition (see Figure 5). Furthermore, besides the photo-
thermal-induced cytotoxicity, the therapeutic index of
CDDP was enhanced by the increase in temperature.
68
Therefore, as a result of cellular internalization, controlled
intracellular drug release, dual drug chemotherapy, and
photothermal therapy, PS-IONs were an outstanding candi-
dates for cancer therapy applications.
Conclusion
The superparamagnetic PS-IONs were prepared by a rela-
tively simple and efficient sonochemical method. The par-
ticles have favorable chemical stability and water
dispersibility and also demonstrated excellent bio- and
hemocompatibility. The SiO
2
and Fe
3
O
4
nanoparticles
were known as in vivo biodegradable nanomaterials with
non-toxic byproducts.
69
The PS-IONs could act as dual-
drug nanocarriers. DOX was loaded on particles by both
hydrogen bonding and electrostatic interactions
11,18,22
with
DLE and DLC of 63.9% and 2.1%, respectively, while
CDDP could efficiently conjugate with carboxyl groups
of PEG molecules through coordination interaction result-
ing in DLE and DLC of 46.6% and 1.4%, respectively.
Both drugs had a slow release profile in normal blood
conditions. Only 31% of CDDP and 22% of DOX were
released during 30 h under normal conditions which sig-
nificantly decrease the drug accumulation and toxicity in
the normal tissues. These drug carriers revealed a dual
stimuli-triggered release behavior. High release rates of
69% and 84% were obtained for DOX and CDDP, respec-
tively, during 30 h in a simulated photothermal condition
in an acidic cancerous environment. Synergistic photother-
mal therapy and DOX/CDDP combination chemotherapy
could be achieved through the use of PS-IONs nanocar-
riers. PS-IONs efficiently delivered both DOX and CDDP
into the MCF-7 cells and demonstrated potent antitumor
activity in vitro which was significantly intensified by
exposure to a low power near-IR laser irradiation. As the
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Table 3 Summary Of The Parameters And Outcomes Of Some SPION-Based Chemo-Photothermal Therapy Studies
Nanostructure Concentration
(µg/mL)
Laser [(W/cm
2
);
(λ(nm); t(min)]
Drug;
Concentration
(µg/mL)
Cell
Line
Relaxivity
(mM
−1
s
−1
)
Cyto-toxicity
(%)
Comments
This study 200 0.5; 808; 10 DOX; 4.2
CDDP; 2.8
MCF-7 127.31 97.3 ± 0.8 –
rGO-Fe
2
O
3
@Au NPs
30
50 2; 808; 5 DOX; 50 HeLa 54.99 ~96 –
GO-PEG-γ-Fe
2
O
329
Not reported 2; 808; 5 DOX; 6 HeLa 33.15 ~93 Loading factor was 1.18
Porous hollow Fe
3
O
423
150 µg Fe/mL 1.5; 808; 10 DOX; Not
reported
4T1 119.66 ~75 –
Lactobionic acid/Fe
3
O
4
@polydopamine/PEG
73
125 1; 808; 30 DOX; ~46 HepG2 Not studied 75.4 Cytotoxicity increased to 83.7% after
exposure to magnetic field
Fe
3
O
4
@mSiO
2
-PO*-folic acid
26
125 2; 808; 30 DOX; ~5.6 HeLa Not studied 81 –
Fe
3
O
4
@MnO
2
@PPy
27
600 1; 638; 10 DOX; 420 HepG2 Not studied 91.6 Samples were placed in a magnetic field
for 30 min
IONPs@AuNPs@SiNWs
15
300 1; 808; 20 DOX; 50 MCF-7/
ADR
Not studied 90 Samples were placed in a magnetic field
for 3 hrs
Cu
9
S
5
@mSiO
2
@Fe
3
O
4
-PEG
74
200 0.76; 980; 10 DOX; 25 HeLa 13.583 83 –
Fe
3
O
4
@MoS
2
/PEG/2-deoxy-D-glucose
28
200 0.5; 808; 3 DOX; ~101 MDA-
MB-
231
48.86 ~70 The cell viability decreased to 1.4% after
the second treatment
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drug-loaded nanoparticles can enter the cells via endocy-
tosis, they could overcome the P-gp drug resistance
mechanism. Moreover, only the free drugs were pumped
out by resistance mechanisms of the cells.
64,65,70,71
Also,
the Fe
3
O
4
nanoparticles may act as the P-gp inhibitor in
some drug resistance cancer cells.
72
Therefore, this deliv-
ery system can potentially be used for other combination
therapies and holds a great promise as an effective tool to
treat drug-resistance cancer tumors.
Acknowledgments
The authors gratefully thank Prof. M. Reaz Hormozi-nez-
had from the Department of Chemistry of the Sharif
University of Technology for his help and support, and
Dr. Rabeei and Mr. Shahbazi for doing the blood tests at
Vardavard Medical Laboratory.
Author Contributions
All authors contributed to data analysis, drafting and revising
the article, gave final approval of the version to be published,
and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work.
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