Materials Science & Engineering C 101 (2019) 472–486

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Antiproliferative effects of new magnetic pH-responsive drug delivery T

system composed of Fe3O4, CaAl layered double hydroxide and levodopa on
melanoma cancer cells
⁎
Nahid Shahabadia,b, , Mahtab Razlansaria, Hossein Zhalehc, Kamran Mansourib
a
Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran
b
Medical Biology Research Center (MBRC), University of Medical Sciences, Kermanshah, Iran
c
Substance Abuse Prevention Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, an efficient drug delivery system composed of Fe3O4, CaAl layered double hydroxide (LDH) and L-
Cell culture Dopa has been synthesized through hydrogen bonds between L-Dopa and CaAl-LDH encapsulated Fe3O4 nano-
Drug release particles (Fe3O4@CaAl-LDH@L-Dopa). The structural features of Fe3O4@CaAl-LDH@L-Dopa were characterized
L-Dopa
using XRD, SEM, TEM, EDX, FT-IR, VSM, TGA, XPS, zeta potential analysis and BET. All of the characterization
Layered double hydroxide
techniques show the uniform high surface area core-shell structure with about 120 nm in average size. Also, the
Magnetic nano carrier
Melanoma
obtained results clearly indicate that this drug delivery system possess high potent for adsorption of L-Dopa
(52 wt%) and high drug encapsulation efficiency (71%). The amount of L-Dopa release in low pHs (53.8%) which
simulates the environment of cancer cells is greater than higher pHs. The in vitro cytotoxic and anticancer
activities of Fe3O4@CaAl-LDH@L-Dopa were investigated against Mel-Rm Cells Melanoma (NCIt: C3224) using
LDH colorimetric assay and differential staining cell death assay. The results showed Fe3O4@CaAl-LDH@L-Dopa
with a lower concentration of L-Dopa, illustrate a higher cytotoxicity and anticancer activity.

1. Introduction
Cancer is one of the world's most fatal diseases, which annually >
10 million new cases have added to its patient population. Therefore,
development of therapeutic approaches, that mainly involves re-
searches on pharmaceutical systems, is necessary.
Nowadays, 3,4-dihydroxyphenyl-L-alanine known as L-Dopa (Fig. 1)
is considered as one of the prominent treatments for many kinds of
cancers particularly prostate and breast types and reduce the compli-
cations of disease [1–5]. L-Dopa is an important precursor for the var-
ious neurotransmitters like adrenaline, noradrenaline, and especially
dopamine. These compounds exhibit a novel antitumor activity with
considerable influences in several experimental tumor systems [6].
Also, L-Dopa can help to suppression of prolactin concentration, and
play a major role in the hormonal control of breast cancer [7].
The major problem in the cancer therapy with L-Dopa is motor
complications which generate in high doses of the drug [8]. Therefore,
finding a way to use of L-Dopa and reducing its side effects seems to be
essential. Today, new generation of therapeutic systems, focuses on the
concept of targeting and slowing the release of drugs to long-term the
therapeutic efficiency and reduces the side effect which mainly origi-
nates from the high dosage of drug in the blood.
Recent advances in cancer therapeutic manners demonstrate the
beneficial effect of formulating drugs in the controlled release drug
delivery systems [9–11]. Incorporation of L-Dopa in the controlled re-
lease nanocarriers can change the biological distribution of a drug,
prolong its therapeutic effects and decrease the instantaneous free
concentration of L-Dopa in the blood that leads to minimize the side
effects [12–14].
One of the most important factors in the design of drug delivery
systems is modifying the drug release profile through the structural or
chemical design of the drug carrier. In particular, pH-responsive sys-
tems have attracted a lot of interests due to the fact that pH in the
diseased tissue particularly in cancer cells is different from blood and
normal tissues [15,16]. The use of pH-responsive systems causes reduce
undesired drug release during drug transfer in blood circulation and
improve the release efficiency of the drugs in the diseased tissue or
cells. Using these types of delivery systems, the drug is released much
faster at diseased cells than the surrounding normal tissues. These
features cause to reduce the side effects of therapeutic systems [16–18].

⁎
Corresponding author at: Faculty of Chemistry, Razi University, Kermanshah, Iran.
E-mail address: n.shahabadi@razi.ac.ir (N. Shahabadi).

https://doi.org/10.1016/j.msec.2019.04.004
Received 6 December 2018; Received in revised form 18 March 2019; Accepted 2 April 2019
Available online 04 April 2019
0928-4931/ © 2019 Elsevier B.V. All rights reserved.
N. Shahabadi, et al.
Fig. 1. Chemical structures of L-Dopa.
Nowadays, the new class of delivering systems that have attracted a
lot of attention is layered materials, which can incorporate many kinds
of organic compounds between their layers and transport to the specific
targets. Because of the controllable release of drugs in layered mate-
rials, these new delivering systems have a great potential as a carrier in
the pharmaceutical field. One of the most important types of layered
compounds is layered double hydroxides (LDHs). The use of LDHs as-
sociated with organic biologically active compounds is an efficient al-
ternative for common drug delivery systems that may be applied to
drug therapies for different diseases. A novel approach is the use of
magnetic cores like Fe3O4 nanoparticles in LDH structures which can
help with the accurate and easy transfer of drugs to target tissue using
an external magnet. This feature addressing a great deal of drug
transportation problems faced with many delivering systems.
In order to develop a new and efficient protocol based on using the
magnetic nanoparticles in drug delivery systems [19–21] and ema-
nating from the interest in the LDH-based nanocarriers [22–24], the
new core-shell structure composed of Fe3O4, CaAl-LDH and L-Dopa
(Fe3O4@CaAl-LDH@L-Dopa) was synthesized.
The structural features of Fe3O4@CaAl-LDH@L-Dopa were char-
acterized using X-ray diffraction (XRD), Fourier transform infrared
spectroscopy (FT-IR), Zeta potential analysis, scanning electron mi-
croscopy (SEM), electron dispersive X-ray spectroscopy (EDX), trans-
mission electron microscopy (TEM), thermogravimetric analysis (TGA),
X-ray photoelectron spectroscopy (XPS), vibrating sample magneto-
metry (VSM), pore volume and specific surface area determination by
Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH)
methods and inductively coupled plasma atomic emission spectroscopy
(ICP-AES). The controlled release properties of Fe3O4@CaAl-LDH@L-
Dopa as an efficient pH-responsive cancer therapy system were in-
vestigated using spectroscopic techniques such as absorption spectro-
scopy. Moreover, cell culture and the cytotoxic activity of Fe3O4, L-
Dopa, Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH@L-Dopa were de-
termined and their effects on cancer cell viability were examined on
Mel-Rm cell lines.
2. Materials and methods
2.1. General remarks
All of the material including solvents and reagents were commer-
cially available and purchased from Sigma-Aldrich, Merck and Fluka
chemical companies. X-ray diffraction (XRD) patterns were obtained on
Inel French, EQUINOX 3000 model X-ray diffractometer using CueK
radiation. Scanning electron microscopy (SEM) has been investigated
using SU3500 microscope with scanning range from 0 to 20 keV.
Electron dispersive X-ray spectroscopy (EDX) measurements were done
with an IXRF model 550i attached to SEM. Transmission electron mi-
croscopy (TEM) analyses was performed using a TEM microscope
Philips CM 120 kV Netherland. Thermogravimetric analysis (TGA) was
performed by thermal gravimetric analysis instrument (Shimadzu TA-
60WS-TGA-50/50H) with a flow rate of 30 mL min-1 and a heating rate
of 10 °C min−1 in the air. Fourier transform infrared spectroscopy (FT-
IR) spectra were recorded with KBr pellets using a Bruker ALPHA FT-IR
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spectrometer. X-ray photoelectron spectroscopy (XPS) was investigated
using a Kratos Analytical Axis Ultra, with monochromatic aluminum
and magnesium with X-ray source of 1486.6 and 1253.6 eV and
Concentric Hemispherical Analyzer (CHA). A take-off angle of 90 was
used on a spot size of 700 lm, and 350 lm. The instrument has Ultra
High Vacuum (UHV). The magnetic property of samples was measured
using a BHV-55, Riken, Japan vibrating sample magnetometer (VSM).
The elemental analysis was investigated using inductively coupled
plasma atomic emission spectroscopy (ICP-AES) on a SpectroCiros CCD
spectrometer. The pore volume and specific surface area determination
were studied by Brunauer-Emmett-Teller (BET) and Barrett-Joyner-
Halenda (BJH) methods, respectively, by using a Quantachrome-
Autosorb 1C-VP analyzer. Prior to the measurements, the samples were
degassed at 100 °C for 6 h. The dialysis bag (molecularweight cut off
(MWCO) = 10 kDa) was purchased from Sigma-Aldrich Chemicals
Company. The cytotoxicity assay kit was prepared from Roch Chemical
Company. The optical density (OD) of wells was calculated by Bio-Tek,
ELX 800, Winooski, VT microplate reader. Zeta potentials were mea-
sured with a Nano ZS90 zeta sizer analyzer (Malvern, UK) with
0.001 mol L−1 NaNO3 electrolyte.
2.2. Preparation of Fe3O4 nanoparticles
Typically, the mixture of FeCl2·4H2O (4.3 g) and FeCl3·6H2O
(11.6 g) was prepared in 350 mL of deionized water under N2 atmo-
sphere. The obtained solution was heated to 80 °C while vigorous stir-
ring. Then, 20 mL of 25% NH4OH was quickly added to the previous
solution. The resulting dark suspension was vigorously stirred for
10 min and then, the black precipitates were separated from the mix-
ture using an external magnet and washed with deionized water and
ethanol frequently, then dried under vacuum conditions.
2.3. Preparation of Fe3O4@CaAl-LDH
Firstly, the uniform suspension was prepared using ultrasonically
dispersion of Fe3O4 (0.3 g) into 150 mL solvent (Vmethanol/Vwater = 1/1)
for 20 min. In the following, 100 mL alkaline solution of Na2CO3 and
NaOH (0.32 g and 0.48 g respectively) was added dropwise into the
prepared suspension until pH ca. 10.0 and kept for 5 min. Then 100 mL
salt solution of 0.45 g of Al(NO3)3·9H2O (1.2 mmol) and 0.85 g of Ca
(NO3)2·4H2O (3.6 mmol) was added to the previous suspension and
using the alkaline solution of Na2CO3 and NaOH, the pH of above
mixture was adjusted to 9.5–10.0. The obtained slurry dispersion was
stirred for 5 min followed collecting by magnet and washing with
deionized water and ethanol three times. Finally Fe3O4@CaAl-LDH
dried at 60 °C overnight giving the desired product.
2.4. Preparation of Fe3O4@CaAl-LDH@L-Dopa
The synthesis of Fe3O4@CaAl-LDH@L-Dopa and loading of L-Dopa
on the surface and into the structure of Fe3O4@CaAl-LDH was carried
out using anion exchange method. At first 0.01 g of Fe3O4@CaAl-LDH
was added to 30.0 mL of 2.5 mM L-Dopa solution in double distilled
water under vigorous stirring for 24 h in room temperature. In this step,
the pH of the solution was adjusted to 7.4. The resulting solid was se-
parated using external magnet, washed with distilled water and dried at
room temperature and under vacuum conditions giving product
Fe3O4@CaAl-LDH@L-Dopa.
2.5. Estimation of drug loading and drug encapsulation efficiency
In order to determining the drug encapsulation efficiency, 1 mg of
Fe3O4@CaAl-LDH was added to 3 mL solution of L-Dopa (3 mM) under
stirring for 24 h. After fully separation of solid Fe3O4@CaAl-LDH@L-
Dopa by external magnet and filtration from the suspension, 1 mL of the
filtrate was used to analyze the amount of drug residue using UV/
N. Shahabadi, et al.
visible spectrophotometry at 280 nm. The drug encapsulation efficiency
was evaluated by the following equation [25]:
Drug Encapsulation Efficiency (%) = (WDrug on the nanoparticles /WTotal used drug )
× 100
Knowing the amount of drug left in the filtrate and total con-
centration of L-Dopa (3 mM), the concentration of L-Dopa loaded on/in
the surface/interlayer of Fe3O4@CaAl-LDH and the amount of Drug
loading was determined as follow [26]:
Drug Loading (%) = (WDrug on the nanoparticles
/(WNanoparticles + WDrug on the nanoparticles )) × 100
2.6. In vitro drug release investigation
In order to evaluate the release of the L-Dopa from the Fe3O4@CaAl-
LDH carrier, 1.738 mg of Fe3O4@CaAl-LDH@L-Dopa was suspended in
2 mL of phosphate buffer saline with different pHs, and resulted sus-
pension were transferred into 3500 MWCO dialysis bag. In the next
step, the dialysis bag was placed into the 50 mL of the same buffer and
agitated in the shaker at 37 °C (90 rpm). To determine the release of L-
Dopa, 2 mL of buffer was removed and replaced with 2 mL of fresh
buffer at time points of every 5 or 10 min until 120 min. The amount of
released L-Dopa was measured by study on the absorbance of the re-
sulting samples at 280 nm and using Beer-Lambert law to assess the
concentration of L-Dopa present to calculate the amount of release.
2.7. Cell culture
In this study, we applied the Mel-Rm cells Melanoma (NCIt: C3224)
which was derived from the metastatic site: Lymph node. The Mel-Rm
cells were grown as a monolayer in DMEM culture medium supple-
mented with 5% FCS in 25 cm2 flasks (Orange Scientific, Belgium). The
cell line was seeded in 75 cm2 tissue culture flasks and maintained in
Dulbecco's MEM supplemented with 10% heat-inactivated fetal bovine
serum, 100 U mL−1 penicillin and 100 μg/mL streptomycin. The
medium was renovated every two days and the cell cultures were in-
cubated at 37 °C in a humid atmosphere (95% air and 5% CO2). While
cell cultures reached 70 to 80% confluency, they were trypsinated using
trypsin-EDTA 0.25% (Sigma) and were subcultured in 24-well culture
plates at a density of 1 × 104 cells/well.
2.8. Cell treatment
24 h after plating the cells, they have been washed with PBS
(pH 7.4). There were three Groups: Group I: incubated with L-Dopa,
Group II: incubated with Fe3O4@CaAl-LDH@L-Dopa, and Group III:
incubated with Fe3O4@CaAl-LDH. There were control and six treat-
ments in every group, including; control: 0.0 μM, treatment 1: 1 μM,
treatment 2: 2 μM, treatment 3: 4 μM, treatment 4: 8 μM, treatment 5:
16 μM, and treatment 6: 32 μM of any materials in each group respec-
tively. Thereupon, the cells have been placed in the incubator at 37 °C
with 5% CO2. The cells were cultured in DMEM, culture medium con-
taining 0.2% BSA.
2.9. Cell viability (%) measurement
Trypan blue viability measurement has been done by standard
methods. Trypan blue is the necessary stain. The usual procedure of
performing trypan blue (0.4 g/100 mL in PBS) cell viability analysis
contains manual staining and applying hemocytometer for counting.
2.10. Proliferation inhibition (%) (MTT assay)
Cell proliferation inhibition (PI) was quantified by measuring the
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MTT assay. In order to perform the test, 15 × 103 cells were loaded into
a 96-well plate and 200 μL of DMEM media containing 0.2% BSA was
added. After 24 h of incubating, 200 μL of treatments media as de-
monstrated was added to the wells. The cells were separately incubated
with various treatments medium for 24 h. After incubation, the MTT
test was accomplished in which the supernatant from each well was
removed and 50 μL of MTT solution (5 mg/mL) was added to each well
and incubated for 3 h. The supernatant from each well was then re-
moved and 100 μL of dimethyl sulfoxide was added in order to dissolve
the formazan crystals at room temperature for 30 min. The optical
density of each well was measured using an enzyme-linked im-
munosorbent assay (ELISA) reader at 570 and 630 nm. The cell pro-
liferation inhibition of the cells for each concentration was calculated
using the following formula (45):
PI (%) = (A570, 630 (sample )/ A (control ) ) × 100
2.11. Cell cytotoxicity measurement
Cell cytotoxicity was quantified by measuring the release of lactate
dehydrogenase (LDH) from damaged or destroyed cells into the media.
Cytotoxicity was measured with the LDH Cytotoxicity Detection Kit
(Roche, Germany). This kit detects LDH release from dead cells.
Therefore, the increase of LDH activity in each treatment shows that the
treatment solution has further dead cells or cytotoxicity effects on cells.
Cells were plated in 24 well culture plates with 104 cells/mL densities
for 12 h. Afterward, cells have been cultured with differentiation the
medium for 24 h. The percentage of cytotoxicity was measured by the
protocol of the company; colorimetry of LDH activity measured by
calculating the absorbance of samples at 490 or 492 nm using an ELISA
Reader (EL800; USA). The references wavelength should be > 600 nm.
Whole the tests have been replicated partly at least 3 times. Within each
experiment, we replicated each condition 4 times. The viability of cells
for every concentration has been computed applying the following
formula:
Cell cytotoxicity (%) = (A490, 630 (sample )/A (control ) ) × 100
2.12. Caspase-3 assay
Mel-Rm cells were cultured in different treatment media condition.
The caspase-3 activity of lysates from the cells treated was measured
with the caspase activity colorimetric assay kit (Bio-techne) according
to the manufacturer's protocol using a plate reader. Data were obtained
from two independent experiments.
2.13. Detection of mitochondrial membrane potential
For quantitative analysis, MMP was measured using the cell
permeable cationic fluorescence probe rhodamine 123. Briefly; Mel-Rm
cells 3 × 104 cells/well were cultured and treated in different treatment
media, then they were washed with PBS and incubated by 1 μM rho-
damine 123 in the dark for 30 min at 37 °C. Then, the absorbance of
cells was measured by calculated the absorbance of samples at 488
excitation and 525 nm emission using an ELISA Reader. The reference
wavelengths should be > 630 nm. All the tests have been repeated in-
dependently at least 3 times. Within each experiment, we replicated
each condition 4 times.
2.14. Quantification of apoptosis incidence
Fixation for all cells in this study was done by 4% w/v paraf-
ormaldehyde in PBS, pH=7.4 for 10 min at room temperature. An in
situ cell death detection kit (Roche) was used to identify the apoptotic
cells by TUNEL (Terminal Uridine deoxynucleotidyl transferase dUTP
Nick End Labeling) staining, following the manufacturer's protocol.
N. Shahabadi, et al. Materials Science & Engineering C 101 (2019) 472–486

Scheme 1. Synthetic route of Fe3O4@CaAl-LDH@L-Dopa.

Briefly, all cells were fixed, permeabilized, blocked and incubated with
a mixture of fluorescently labeled nucleotides on tdt (terminal deox-
ynucleotidy transferase) catalyzed the polymerization of labeled nu-
cleotides to 3/0H terminals of DNA fragments. The cells were then
counterstained with 10 μg/mL of propidium iodide (red) at room tem-
perature for 15 min and washed with PBS. A positive apoptosis control,
cells induced into apoptosis by 5% ethanol treatment, were included in
each assay. TUNEL positive cells were counted in eight randomly se-
lected fields from each culture under a fluorescent microscope
(Olympus AX-70), and apoptotic index was calculated by dividing the
number of apoptotic cells by the total cells.

3. Results and discussion

3.1. Structural characteristic

In this study, we report the synthesis of Fe3O4@CaAl-LDH as a

magnetic pH-responsive nanocarrier of L-Dopa for the first time.
Scheme 1 shows the synthetic route for the preparation of the designed
Fe3O4@CaAl-LDH@L-Dopa as a therapeutic system for cancer therapy.
The Fe3O4@CaAl-LDH@L-Dopa was prepared using a method that
involves the separated synthesis of Fe3O4 nanoparticles, immobilization
Fig. 2. XRD patterns of a) Fe3O4, b) Fe3O4@CaAl-LDH and c) Fe3O4@CaAl-
of CaAl-LDH on the surface and intercalation of L-Dopa into the
LDH@L-Dopa.
Fe3O4@CaAl-LDH structure.

Fe3O4@CaAl-LDH core-shell structure, shows distinctive absorption

3.1.1. XRD analysis
peaks at 10.1°, 20.2°, 30.6°, 37.3°, 48.2°, 62.6°, 63.1° which are at-
The XRD patterns of Fe3O4, Fe3O4@CaAl-LDH and Fe3O4@CaAl-
tributed to the (003), (006), (009), (015), (018), (110) and (113) facets
LDH@L-Dopa are displayed in Fig. 2. In the XRD pattern of Fe3O4 (curve
of LDH structures [27,28]. The XRD pattern of Fe3O4@CaAl-LDH@L-
a), the diffraction peaks are in accordance with cubic-phase Fe3O4
Dopa nanostructure clearly shows the characteristic peaks of CaAl-LDH
crystalline structure [JCPDS-019-0629]. The (220), (311), (400), (422),
and Fe3O4 nanoparticles. The broadening and shift of (003) basal re-
(511) and (440) reflections of typical Fe3O4 in 2θ = 30.2°, 35.7°, 43.1°,
flection of CaAl-LDH represent that the L-Dopa molecules are loaded on
53.3°, 56.8° and 62.8° prove the high crystallinity of the Fe3O4 nano-
the surface and intercalated into the layers of the Fe3O4@CaAl-LDH@L-
particles. The XRD pattern of Fe3O4@CaAl-LDH clearly demonstrates
Dopa structure (curve c). The obtained d-spacing of (003) reflections
the formation of CaAl-LDH shell around Fe3O4 nanoparticles (curve b).
were 13.2 Å and larger than 11.2 Å of Fe3O4@CaAl-LDH peak. This
In addition of Fe3O4 reflections, the diffraction pattern of resulting

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Fig. 3. SEM images of a) Fe3O4, b) Fe3O4@CaAl-LDH, c, d) Fe3O4@CaAl-LDH@L-Dopa and e–i) mapping results of Fe3O4@CaAl-LDH@L-Dopa.
result indicates that the L-Dopa molecules were intercalated into the
interlayers of LDHs.
3.1.2. FE-SEM analysis
In order to investigate the morphology of Fe3O4@CaAl-LDH@L-
Dopa, the surface structure has been studied using SEM technique
(Fig. 3). Fig. 3a indicates the nearly spherical and uniform shape of the
Fe3O4 nanoparticles. Also, the spherical structure and layered surface of
Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH-L-Dopa are clearly verified
(Fig. 3b, d). The mapping analysis of Fe3O4@CaAl-LDH@L-Dopa was
shown in Fig. 3d–h. The high intensity of the elemental dispersion of O,
C, Ca and Al shows the presence of loaded L-Dopa on the surface of LDH
structures. In addition, low intensity of Fe dispersion, confirms the
presence of Fe3O4 nanoparticles in the interior of the Fe3O4@CaAl-
LDH@L-Dopa.
3.1.3. EDX analysis
Elementary analysis of the surface was further studied by EDX
analyses. The EDX pattern of Fe3O4@CaAl-LDH shows the major peaks
of Al, Ca, Fe, O, C (Fig. 4a). Also, after intercalation of L-Dopa in the
Fe3O4@CaAl-LDH structure, the strong band of N was appeared which
confirms the presence of surface loaded L-Dopa in the structure of our
synthesized Fe3O4@CaAl-LDH@L-Dopa (Fig. 4b).
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Materials Science & Engineering C 101 (2019) 472–486
3.1.4. TEM analysis
In order to study more on the morphology of Fe3O4@CaAl-LDH@L-
Dopa, TEM images were investigated. As expected, the results clearly
indicate the core-shell structure of synthesized Fe3O4@CaAl-LDH@L-
Dopa and obviously confirmed the structural information obtained from
SEM technique (Fig. 5).
Fig. 5a clearly shows the spherical and uniform shape of the Fe3O4
nanoparticles. The low magnification image of Fe3O4@CaAl-LDH@L-
Dopa in addition to the spherical Fe3O4@CaAl-LDH nanostructure
shows the presence of thin layer of L-Dopa on the surface of carrier,
which creates the structure with approximately 120 nm of dimensions
(Fig. 5b). The high magnification image indicates the layered structures
of CaAl-LDH and L-Dopa around Fe3O4 nanoparticles (Fig. 5c).
3.1.5. FT-IR spectroscopy
The FT-IR spectra of L-Dopa, Fe3O4, Fe3O4@CaAl-LDH and
Fe3O4@CaAl-LDH@L-Dopa are depicted in Fig. 6. In Fig. 6a, the ab-
sorption peaks at 1601, 1474, and 862 cm−1 are related to the amine
group and 1594, 1419 cm−1 associated with carboxylate, confirm the
presence of L-Dopa on the surface of the Fe3O4@CaAl-LDH. The FT-IR
spectrum of Fe3O4 nanoparticles (curve b) indicates the stretching vi-
bration band of -OH at 1635 cm−1 which are attributed to the existence
of surface hydroxyl groups and H2O. In the FT-IR spectra of Fe3O4,
Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH@L-Dopa, the FeeO vibrations
N. Shahabadi, et al. Materials Science & Engineering C 101 (2019) 472–486

Fig. 4. EDX images of a) Fe3O4@CaAl-LDH and b) Fe3O4@CaAl-LDH@L-Dopa were performed in 10.0 kV.

of magnetite phase absorption bands at about 575 cm−1 were observed

[29]. Also, the FT-IR spectrum of Fe3O4@CaAl-LDH, in addition to
Fe3O4 absorption bands, shows the bands at 496 and 521 cm−1 that are
related to the M-O vibration modes of CaAl-LDH structure (curve c). It
is notable that, the characteristic absorption band around 1388 cm−1 is
related to the presence of CO32– in the LDH structure. The intensity
reduction of this band in Fe3O4@CaAl-LDH@L-Dopa spectrum clearly
indicates the replacement of CO32– anions by L-Dopa molecules. Finally,
the FT-IR spectrum of Fe3O4@CaAl-LDH@L-Dopa shows the char-
acteristic peaks of Fe3O4 nanoparticles, CaAl-LDH and L-Dopa which is
in agreement with the formation of Fe3O4@CaAl-LDH@L-Dopa struc-
ture (curve d).

3.1.6. VSM analysis

One of the advantages of using magnetic structures as drug carriers
is the ability of these systems to delivering the drug through an external
magnetic field. Therefore, the evaluation of the magnetic properties of
the carriers seems to be necessary. In order to investigate the magnetic
features of Fe3O4@CaAl-LDH@L-Dopa, the VSM measurement was
carried out at 298 K. As illustrated in Fig. 7, the magnetization of Fe3O4
nanoparticles saturated up to 55 emu/g at an applied field of 9500 Oe
and indicates the superparamagnetic behaviours at room temperature.
The presence of CaAl-LDH shell and L-Dopa molecules around Fe3O4
cores reduces the magnetic strength of the Fe3O4 nanoparticles by about
20 emu/g (Fig. 7).
Fig. 6. FT-IR Spectra of a) L-Dopa, b) Fe3O4, c) Fe3O4@CaAl-LDH and d)
3.1.7. BET analysis Fe3O4@CaAl-LDH@L-Dopa.
One of the most important features of carriers in the drug delivery
systems is having an appropriate surface for interaction with drugs. The
Fe3O4@CaAl-LDH@L-Dopa show isotherm type IV which exhibits the
surface physical properties of Fe3O4@CaAl-LDH and Fe3O4@CaAl-
mesoporous structure and presence of nano-voids in these systems.
LDH@L-Dopa were studied using N2 adsorption-desorption isotherm
Fe3O4@CaAl-LDH represents the high BET surface area (136.4 m2 g−1),
and pore distribution diagram (Fig. 8). Both of Fe3O4@CaAl-LDH and

Fig. 5. TEM images of a) Fe3O4 b) 7 nm L-Dopa, 19 nm CaAl-LDH and 84 nm Fe3O4 in Fe3O4@CaAl-LDH@L-Dopa structure and c) high magnification of Fe3O4@CaAl-
LDH@L-Dopa.

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N. Shahabadi, et al.
Fig. 7. VSM magnetization curves of Fe3O4, Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH@L-Dopa.
narrow pore size distribution (centered at 14.2 nm) with the great pore
volume (0.236 cm3 g−1), which makes Fe3O4@CaAl-LDH a suitable
option for the drug delivery system. The high surface area of this
structure improves the interaction of the drug with the carrier (Fig. 8a).
Also, the BET analysis of Fe3O4@CaAl-LDH@L-Dopa demonstrates the
specific surface area is about 112.3 m2 g−1. Moreover, BJH pore size
distribution of the drug-carrier structure shows a narrow mesopore size
distribution centered at 19.5 nm. The pore volume of Fe3O4@CaAl-
LDH@L-Dopa was estimated as 0.202 cm3 g−1 by BJH analysis (Fig. 8b).
Low surface area of Fe3O4@CaAl-LDH@L-Dopa indicates that a large
portion of nanovoids are occupied by L-Dopa.
3.1.8. XPS analysis
The surface chemical features of Fe3O4@CaAl-LDH@L-Dopa were
studied using XPS analysis with a typical wide energy scan (Fig. 9). The
obtained results from XPS spectra clearly indicate that the Fe3O4, CaAl-
LDH and L-Dopa are presented in the Fe3O4@CaAl-LDH@L-Dopa
structure (Fig. 9a). The integral peak area of the drug delivery system
estimated the Ca:Al ratio as 2.57, which is in accordance with the ob-
tained results from ICP-AES elemental analysis. The ICP-AES results
confirm the presence of 27.1 wt% of Ca and 6.25 wt% of Al. According
to the obtained results from XPS and ICP-AES data and with respect to
the stoichiometric amount of Ca and Al precursors in synthesis process,
the chemical formula of CaAl-LDH was evaluated as
Fig. 8. Isotherm and pore distribution curves of a) Fe3O4@CaAl-LDH and b) Fe3O4@CaAl-LDH@L-Dopa based on the BET and BJH method, respectively.
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Materials Science & Engineering C 101 (2019) 472–486
[Ca0.67Al0.33(OH−)2][(OH−)0.11(CO32−)0.11·2.7H2O]. It should be
noted that, according to the ICP-AES results, the weight percentage of
CaAl-LDH in Fe3O4@CaAl-LDH structure is equal to 34%.
It is notable that, the existence of the peaks at 711.4 and 724.7 eV
show the presence of Fe3O4 nano particles in the Fe3O4@CaAl-LDH@L-
Dopa structure. In high resolution XPS spectrum of 2p region of Fe, the
absence of the satellite in the Fe2p3/2 peak confirms the presence of
Fe3O4 (Fig. 9d) [30,31].
3.1.9. TGA analysis
The TGA analysis clearly shows the presence of L-Dopa in the
Fe3O4@CaAl-LDH@L-Dopa structure. Also, TGA analysis confirms that
L-Dopa was successfully loaded into/on the Layers of LDHs.
Another factor which makes the carriers an ideal option for use in
drug delivery systems is their stability in the body environment. In
order to study on the structural persistence of Fe3O4@CaAl-LDH@L-
Dopa, TGA analysis has been investigated (Fig. 10). Four mass loss steps
were happened with an increase in the temperature.
The first mass loss step in the temperature range of 35 to 150 °C is
related to the removal of surface-adsorbed and interlayer water mole-
cules of Fe3O4@CaAl-LDH@L-Dopa (7.13 wt%). The second stage at
150–320 °C was occurred due to the dehydroxylation of the layers and
decomposition of L-Dopa (11.47 wt%) [32]. The third mass loss at
320–520 °C is attributed to the combustion of L-Dopa (3.64 wt%).
N. Shahabadi, et al.
Fig. 9. XPS analysis of (a) Fe3O4@CaAl-LDH@L-Dopa with wide energy-scan, high-resolution XPS spectra of (b) 2p region of Al, (c) 2p region of Ca and (d) 2p region
of Fe.
Finally, the forth mass loss step in temperature above 570 °C is probably
due to the decomposition of the mixed metal oxide into a spinel phase
[33].
Also, according to the obtained results from ICP-AES measurements,
the drug carrier being exposed to different pHs, it was determined that
the composition of Fe3O4@CaAl-LDH remains unchanged.
3.1.10. Zeta potential analysis
Zeta potentials of Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH@L-Dopa
in the
dispersion are positive and the average zeta potentials given by the
instrument are 41 and 16 mV for Fe3O4@CaAl-LDH and Fe3O4@CaAl-
LDH@L-Dopa respectively. The positive zeta potential of the LDH
structures is in principle attributed to the structural positive charge and
the electric double layer on the LDH surface. Although the interior
charges of LDHs are fully screened by the interlayer anions, the surface
structural charges are not fully balanced by the adsorbed surface anions
because the surface-adsorbed anions can desert the electric double layer
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Materials Science & Engineering C 101 (2019) 472–486
on the surface of LDHs, and thus the LDH particles have a surface po-
sitive charge. After placing a part of L-Dopa inside the layers and on the
surface of Fe3O4@CaAl-LDH, the negative charge on the surface in-
creases which cause smaller zeta potential in Fe3O4@CaAl-LDH@L-
Dopa (Fig. 11a).
In order to investigate the size of Fe3O4@CaAl-LDH@L-Dopa, size
distribution analysis was performed using dynamic light scattering
(DLS) in aqueous solution. It was found that the average size of
Fe3O4@CaAl-LDH@L-Dopa was about 150 nm. Fig. 11b shows that the
particles range in size from 70 to 400 nm and possess an average size of
150 nm. According to the obtained results from TEM images,
Fe3O4@CaAl-LDH@L-Dopa has uniform and spherical morphology and
the mean diameter of the particles are about 130 nm (Fig. 5). The main
reason of difference between particle size derived from TEM analysis
and DLS data is aggregation of Fe3O4@CaAl-LDH@L-Dopa nanoparticle.
N. Shahabadi, et al. Materials Science & Engineering C 101 (2019) 472–486

Fig. 10. TGA curve of Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH@L-Dopa.

3.2. pH-dependent L-Dopa loading and release from Fe3O4@CaAl-LDH

In order to optimize the loading of L-Dopa on the Fe3O4@CaAl-LDH

structure and evaluation of the drug solubility in different body en-
vironments (normal and cancerous cells), the synthesis of Fe3O4@CaAl-
LDH@L-Dopa was investigated in two different pHs. As it appears from
the Fig. 12, the amount of drug loading and drug encapsulation effi-
ciency in pH = 7.4 are higher than pH = 5.5.
The L-Dopa molecules interact with the LDH layers principally via
electrostatic, hydrogen bond and van der Waal's forces. Therefore,
having a higher negative charge of interlayer drug, play an important
role in the replacement of L-Dopa in the CaAl-LDH structures. Also,
increasing the pH causes enhancing the hydrogen bond strength be-
tween L-Dopa and CaAl-LDH and facilitates the anion exchange process.
Therefore, formation of hydrogen bond between L-Dopa and CaAl-LDH
cause increases the amount of drug in the Fe3O4@CaAl-LDH@L-Dopa
structure.
The increment of the loading of L-Dopa on Fe3O4@CaAl-LDH in the
Fig. 12. Drug loading (%) and drug encapsulation efficiency (%) of L-Dopa at
pH = 7.4 (52 wt%), clearly demonstrates the higher solubility and re- 7.4 and 5.5 pHs and room temperature.
lease of the drug in cancerous cells which have a lower pHs than normal
cells. It is notable that, the lower solubility of L-Dopa at pH = 7.4,
justifies the greater amount of drug encapsulation efficiency and drug Dopa. As illustrated in Table 1, the adsorption of L-Dopa on the
content (Fig. 13). Fe3O4@CaAl-LDH was much higher than Fe3O4. Strengthening the
In the next step of our investigation, the drug loading studies were hydrogen bonds of L-Dopa-carrier and increasing the surface area of the
carried out at constant pH = 7.4 by varying the concentration of L- carrier are the main reasons for the increment of drug loading and drug
encapsulation efficiency. It is noteworthy that, the amount of L-Dopa

Fig. 11. a) Zeta potential distribution of Fe3O4@CaAl-LDH and Fe3O4@CaAl-LDH@L-Dopa, b) DLS particle size analysis of Fe3O4-CaAl-LDH@L-Dopa.

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Fig. 13. Reducing the hydrogen bond strength with decreasing pH.

Table 1 Dopa's release rate was observed. The solubility of L-Dopa is mainly
Optimization of the drug loading and drug encapsulation efficiency of L-Dopa. influenced by the pH of the media. The decrease in the pH of an aqu-
Entry Carrier Drug/ Drug Drug encapsulation
eous solution leads to increasing the solubility of L-Dopa. Therefore, L-
carrier (w/ loading (%) efficiency (%) Dopa released from Fe3O4@CaAl-LDH much faster in lower pHs. Also,
w) reducing the pH of the aqueous solution causes weakening the drug/
carrier interactions due to the protonation of amino and hydroxyl
1 Fe3O4 1.5 7 7.5
2 Fe3O4@CaAl-LDH 1 36 56
groups, and therefore the amount of L-Dopa release enhanced. The rate
3 Fe3O4@CaAl-LDH 1.5 52 71 of drug release from the carrier was slow in high pHs and the con-
4 Fe3O4@CaAl-LDH 2 55 61 centration of total released L-Dopa was evaluated as 53.68%. So, it
could be resulted that Fe3O4@CaAl-LDH can act as an ideal pH-re-
sponsive carrier for L-Dopa.
loading increased from 36% to 52% with increasing the weight ratio of
L-Dopa to carrier from 1 to 1.5. Further increase in the weight ratio of L- 3.3. Therapeutic investigation
Dopa to Fe3O4@CaAl-LDH showed no enhancement in the drug loading
and drug encapsulation efficiency (Table 1, entry 4). 3.3.1. Cell viability (%)
The drug release behaviour of Fe3O4@CaAl-LDH@L-Dopa was in- Interestingly, L-Dopa and Fe3O4@CaAl-LDH@L-Dopa induced cells
vestigated in phosphate buffer (pH = 7.4) and acetate buffer death in Mel-Rm cells. To checking this possibility, cells were exposed
(pH = 5.4). As mentioned previously, the release profile of L-Dopa is to different concentrations of L-Dopa and Fe3O4@CaAl-LDH@L-Dopa,
affected by pH changes. The extracellular pH of cancerous tissues are afterward, cell viability was measured by trypan blue assay at 24 h after
often acidic [34] and release features of Fe3O4@CaAl-LDH@L-Dopa at the exposure (Fig. 15). In L-Dopa group, the results of this experiment
pH = 5.5 was investigated to simulate the behaviour of the drug de- showed that in treatments 1–6 the percentage of cell viability was de-
livery systems in the natural physiological environment of the tumor creased compared with control cells (p < 0.05). In Fe3O4@CaAl-LDH
cells [35,36]. The L-Dopa loaded Fe3O4@CaAl-LDH with a drug loading group, exposure of the cells to the different concentrations of pure
of 52% showed the release profile attributed to the pH of the media nanoparticle was decreased the cell viability in treatments 5 and 6
(Fig. 14). At higher pHs (pH = 7.4), the obvious decrease in the L- compared with control cells (p < 0.05). The percentage of cell viabi-
lity was decreased in treatments 5 and 6 compared with treatments 1–3
(p < 0.05). In Fe3O4@CaAl-LDH@L-Dopa group, exposure of the cells
to the different concentrations of nano-drug caused the decrement of
cell viability in treatments 1–6 compared with control cells (p < 0.05).
The percentage of cell viability was decreased in treatment 6 compared
with treatments 1–4 (p < 0.05). The percentage of cell viability was
decreased in each treatment of Fe3O4@CaAl-LDH@L-Dopa compared
with the same treatments in L-Dopa treatment, respectively (p < 0.05)
[1].

3.3.2. Cell cytotoxicity (%)

Fig. 14. Release investigation of Fe3O4@CaAl-LDH@L-Dopa in pH = 7.4 and
pH = 5.5 (drug loading = 52%, 37 °C).
Interestingly, L-Dopa and Fe3O4@CaAl-LDH@L-Dopa groups in-
duced cells death in Mel-Rm cells. To check the cytotoxic effect possi-
bility, cells were exposed to different concentrations of L-Dopa and
Fe3O4@CaAl-LDH@L-Dopa groups, then, cell cytotoxicity was mea-
sured by LDH colorimetric assay at 24 h after the exposure (Fig. 16). In
L-Dopa group, the results of this experiment showed that in treatments
1–6 the percentage of cell cytotoxicity was increased compared with
control cells (p < 0.05). In Fe3O4@CaAl-LDH group, exposure of the

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Fig. 15. The cell viability of Mel-Rm cells in different

treatment media.
There were three Groups: Group I: incubated with L-Dopa,
Group II: incubated with Fe3O4@CaAl-LDH@L-Dopa, and
Group III: incubated with Fe3O4@CaAl-LDH. There were
control and six treatments in each group, including;
control: 0.0 μM, treatment 1:1 μM, treatment 2:2 μM,
treatment 3:4 μM, treatment 4:8 μM, treatment 5:16 μM,
and treatment 6:32 μM of material in each groups, re-
spectively. All data represented by mean ± S.E.M
(p < 0.05). Significant difference between treatments
and control treatment in each group; *p < 0.05; one way
ANOVA for repeated measures.

cells to the different concentrations of pure nano-particle was increased

the cell cytotoxicity in treatments 5 and 6 compared with control cells
(p < 0.05). The percentage of cell cytotoxicity was increased in
treatments 5 and 6 compared with treatments 1–3 (p < 0.05). In
Fe3O4@CaAl-LDH@L-Dopa group, exposure of the cells to the different
concentrations of nano-drug was increased the cell cytotoxicity in
treatments 1–6 compared with control cells (p < 0.05). The percen-
tage of cell cytotoxicity was increased in treatment 6 compared with
treatments 1–4 (p < 0.05). The percentage of cell cytotoxicity was
increased in each treatment of Fe3O4@CaAl-LDH@L-Dopa compared
with the same treatments in L-Dopa treatment, respectively (p < 0.05).

3.3.3. Cell proliferation inhibition and IC50 values

The inhibitory effect of the agents on the proliferation of the Mel-
Rm cell lines was evaluated and the dose-response curves are depicted
in Fig. 17. After 24 h, Cell proliferation in all treatments was decreased
as dose depended compared with control cells (p < 0.05). In L-Dopa
Fig. 17. Proliferation inhibition of Mel-Rm cell lines by L-Dopa, Fe3O4@CaAl-
group IC50 value was evaluated to be 5.48 μM. In Fe3O4@CaAl-LDH@L-
LDH, and Fe3O4@CaAl-LDH@L-Dopa after 24 h. The cells were counted by the
Dopa group, the percentage of cell proliferation was about 100%. The MTT.
cell proliferation in all treatments was decreased compared with control There were three Groups: Group I: incubated with L-Dopa, Group II: incubated
cells (p < 0.05). In treatments 1–6, cell viability was decreased and with Fe3O4@CaAl-LDH@L-Dopa, and Group III: incubated with Fe3O4@CaAl-
cell cytotoxicity was increased compared with intra-control treatment LDH. There were control and six treatments in each group, including; control:
and parallel treatments in L-Dopa group, respectively (p < 0.05). In 0.0 μM, treatment 1: 1 μM, treatment 2: 2 μM, treatment 3: 4 μM, treatment 4:
Fe3O4@CaAl-LDH@L-Dopa group, IC50 was 3.87 μM. Another important 8 μM, treatment 5: 16 μM, and treatment 6: 32 μM of material in each groups,
effect becomes apparent when analyzing Fig. 17, which summarized in respectively. All data represented by mean ± S.E.M (p < 0.05).
Table 3; L-Dopa-loaded Fe3O4@CaAl-LDH nanoparticles displays a more
pronounced anticancer activity than L-Dopa alone. It means that the
nanoencapsulation of L-Dopa in Fe3O4@CaAl-LDH nanoparticles leads
to a significant enhancement of the biological effect of L-Dopa. This

Fig. 16. The cell cytotoxicity of Mel-Rm cells in different

treatment media.
There were three Groups: Group I: incubated with L-
Dopa, Group II: incubated with Fe3O4@CaAl-LDH@L-
Dopa, and Group III: incubated with Fe3O4@CaAl-LDH.
There were control and six treatments in each group,
including; control: 0.0 μM, treatment 1: 1 μM, treatment
2: 2 μM, treatment 3: 4 μM, treatment 4: 8 μM, treatment
5: 16 μM, and treatment 6: 32 μM of material in each
groups, respectively. All data represented by
mean ± S.E.M (p < 0.05). Significant difference be-
tween treatments and control treatment in each group;
*p < 0.05; one way ANOVA for repeated measures.

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N. Shahabadi, et al.
synergistic antitumor activity reveals the great potential for future ap-
plications of this new L-Dopa release system in cancer chemotherapy.
Further investigation on this topic is required.
3.3.4. Cell death index
In the L-Dopa group (Fig. 18), the results of this experiment showed
that, in treatments 1–6, the percentage of cell death was increased
compared with control cells (p < 0.05). In Fe3O4@CaAl-LDH@L-Dopa
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Materials Science & Engineering C 101 (2019) 472–486
Fig. 18. The effects of different treatments on the cell
death on Mel-Rm cells in different treatment media.
There were three Groups: Group I: incubated with L-
Dopa, Group II: incubated with Fe3O4@CaAl-LDH@L-
Dopa, and Group III: incubated with Fe3O4@CaAl-LDH.
There were control and six treatments in each group,
including; control: 0.0 μM, treatment 1:1 μM, treatment
2:2 μM, treatment 3:4 μM, treatment 4:8 μM, treatment
5:16 μM, and treatment 6:32 μM of material in each
groups, respectively. All data represented by
mean ± S.E.M (p < 0.05). Significant difference be-
tween treatments and control treatment in each group;
*P < 0.05; one way ANOVA for repeated measurements.
Fig. 19. The effects of different treatments on the cas-
pase-3 activity on Mel-Rm cells.
There were three Groups: Group I: incubated with L-
Dopa, Group II: incubated with Fe3O4@CaAl-LDH@L-
Dopa, and Group III: incubated with Fe3O4@CaAl-LDH.
There were control and six treatments in each group,
including; control: 0.0 μM, treatment 1: 1 μM, treatment
2: 2 μM, treatment 3: 4 μM, treatment 4: 8 μM, treatment
5: 16 μM, and treatment 6: 32 μM of material in each
groups, respectively. All data represented by
mean ± S.E.M (p < 0.05). Significant difference for
treatments and control treatment in each group;
*P < 0.05; one way ANOVA for repeated measurements.
Fig. 20. The effects of different treatments on the
Mitochondrial membrane potential (Rhodamine-123 ab-
sorbance) in Mel-Rm cells.
There were three Groups: Group I: incubated with L-
Dopa, Group II: incubated with Fe3O4@CaAl-LDH@L-
Dopa, and Group III: incubated with Fe3O4@CaAl-LDH.
There were control and six treatments in each group,
including; control: 0.0 μM, treatment 1: 1 μM, treatment
2: 2 μM, treatment 3: 4 μM, treatment 4: 8 μM, treatment
5: 16 μM, and treatment 6: 32 μM of material in each
groups, respectively. All data represented by
mean ± S.E.M (p < 0.05). Significant difference be-
tween treatments and control treatment in each group;
*P < 0.05; one way ANOVA for repeated measurements.
group, exposure of the cells to the different concentrations of nano-
particle was increased the cell death in treatments 1–6 compared with
control cells (p < 0.05). The percentage of cell death was increased in
treatments 5 and 6 compared with treatments 1–3 (p < 0.05). In
Fe3O4@CaAl-LDH@L-Dopa group, exposure of the cells to the different
concentrations of nano-drug was increased the cell death in treatments
1–6 compared with intra-control cells and L-Dopa group treatments,
respectively (p < 0.05).
N. Shahabadi, et al. Materials Science & Engineering C 101 (2019) 472–486

Fig. 21. The effects of different concentrations of L-Dopa induced apoptosis in Mel-Rm cells as identified by DNA fragmentation (TUNEL staining) (400×).
Viable cell: white arrow and apoptotic cell: yellow arrow. A:control: 0.0 μM, B:treatment 1: 1 μM, C:treatment 2: 2 μM, D:treatment 3: 4 μM, E:treatment 4: 8 μM,
F:treatment 5: 16 μM, and G:treatment 6: 32 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

3.3.5. Caspase-3 assay activation in treatments 5 and 6 were highest compared with all
In most cases, L-Dopa and Fe3O4@CaAl-LDH@L-Dopa groups, treatments of L-Dopa and Fe3O4@CaAl-LDH and other intragroup
eventually mediate a common apoptotic pathway through the result treatments, respectively (p < 0.05).
was obtained in case of caspase-3 activation (Fig. 19). Furthermore, in
Fe3O4@CaAl-LDH group, Caspase3 activation in all treatments after
3.3.6. Mitochondrial membrane potential (Rhodamine-123 absorbance)
24 h was similar to control treatment. In the L-Dopa group: the activa-
In most cases, in L-Dopa and Fe3O4@CaAl-LDH@L-Dopa groups
tion of caspase-3 in control cells was less than other treatments
eventually mediate a common apoptotic pathway through the result
(treatments 1–6) (p < 0.05). Caspase-3 activation in treatments 1–6
was obtained in case of caspase-3 activation after changing the
were higher compared with Fe3O4@CaAl treatment, respectively
Mitochondrial membrane potential (Δφm). To check the change of
(p < 0.05). Caspase-3 activation in treatment 1 was less in comparison
Δφm in the treated cells, they were exposed to different concentrations
with treatments 2–6, respectively (p < 0.05).
of L-Dopa and Fe3O4@CaAl-LDH@L-Dopa, then, Δφm was measured by
In Fe3O4@CaAl-LDH@L-Dopa group: the caspase-3 activation in
Rhodamine-123 staining and colorimetric assay at 24 h after the ex-
control cells was less than other treatments (treatments 1–6)
posure. Furthermore, in Fe3O4@CaAl-LDH group, RH-123 absorption in
(p < 0.05). Caspase-3 activation in treatments 1–6 were higher com-
all treatments after 24 h was similar to control treatment. In L-Dopa
pared with L-Dopa treatments, respectively (p < 0.05). Caspase-3
group: the RH-123 absorption in control cells was higher than other

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Fig. 22. The effects of different concentrations of Fe3O4@CaAl-LDH@L-Dopa induced apoptosis in Mel-Rm cells as identified by DNA fragmentation (TUNEL staining)
(400×).
Viable cell: white arrow and apoptotic cell: yellow arrow. A:control: 0.0 μM, B:treatment 1: 1 μM, C:treatment 2: 2 μM, D:treatment 3: 4 μM, E:treatment 4: 8 μM,
F:treatment 5: 16 μM, and G:treatment 6: 32 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

Table 2 Table 3
The Effect of L-Dopa, Fe3O4@CaAl-LDH@L-Dopa and Fe3O4@CaAl-LDH with The IC50 values of Fe3O4@CaAl-LDH@L-Dopa and L-Dopa in Mel-Rm cell
different concentrations (nanomollar) on the cell viability of cells. line.
Control 100 nM 200 nM 500 nM 750 nM 900 nM Cell line Mel-Rm cell line

L-Dopa 99% 98% 98% 96% 97% 94% IC50 of Fe3O4@CaAl-LDH@L-Dopa 3.87 μM
Fe3O4@CaAl-LDH@L- 99% 98% 98% 95% 96% 95% IC50 of L-Dopa 5.48 μM
Dopa
Fe3O4@CaAl-LDH 99% 99% 98% 98% 99% 97%
respectively (p < 0.05). RH-123 absorption in treatments 5 and 6 were
the lowest, compared with all treatments of L-Dopa and Fe3O4@CaAl-
treatments (treatments 1–6) (p < 0.05). RH-123 absorption in treat- LDH@L-Dopa and other intragroup treatments, respectively (p < 0.05)
ments 1–6 were lesser compared with Fe3O4@CaAl-LDH treatment, (Figs. 20–22).
respectively (p < 0.05). RH-123 absorption in treatment 1 was higher It is notable that, the obtained results from culturing and incubating
compared with treatments 2–6, respectively (p < 0.05). In of the cells in nanomolar concentration of L-Dopa, Fe3O4@CaAl-LDH@
Fe3O4@CaAl-LDH@L-Dopa group: the RH-123 absorption in control L-Dopa, and Fe3O4@CaAl-LDH showed that L-Dopa and Fe3O4@CaAl-
cells was higher than other treatments (treatments 1–6) (p < 0.05). LDH@L-Dopa were not induced cell death and cell cytotoxicity in
RH-123 absorption in treatments 1–6 were less than L-Dopa treatments,

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melanoma cell lines (Table 2).
Also, the values of IC50 for L-Dopa and Fe3O4@CaAl-LDH@L-Dopa
(50% proliferation growth inhibition) examined in these cancer cells
were also different after 24 h of treatment (Table 3). In comparison with
a free drug (Fig. 17 and Table 3), the amount of IC50 for Fe3O4@CaAl-
LDH@L-Dopa was lower than the corresponding values for free L-Dopa.
Based on the obtained results, the loading of L-Dopa on the surface and
in the layers of Fe3O4@CaAl-LDH nanoparticles, cause increase the
anticancer activity of free L-Dopa.
4. Conclusions
In the present study, we have developed a novel and efficient tar-
geted anticancer drug delivery system based on the use of a magnetic
core-shell structure of Fe3O4@CaAl-LDH nanospheres as a carrier for L-
Dopa in the treatment of Melanoma. The structural properties of
Fe3O4@CaAl-LDH@L-Dopa were characterizes using various techniques
like XRD, SEM, TEM, EDX, FT-IR, VSM, TGA, XPS, BET, and Zeta
Potential analysis. According to the obtained results from the men-
tioned analysis, Fe3O4@CaAl-LDH@L-Dopa has a uniform core-shell
structure with about 120 nm in average size. It is noteworthy that the
use of magnetic Fe3O4@CaAl-LDH can help to the accurate and easy
transfer of L-Dopa to target tissue using an external magnet. This hybrid
nanocarrier has a high drug loading (52 wt%) and drug encapsulation
efficiency (71%). Also, in this drug delivery system, the drug release
was sensitive to pH changes. The amount of L-Dopa release from
Fe3O4@CaAl-LDH nanoparticles was much faster in lower pHs (98.5%)
which indicate Fe3O4@CaAl-LDH@L-Dopa can be used efficiently in the
cancerous cells. Therefore, this system can decrease the cytotoxic ef-
fects of L-Dopa by selective L-Dopa delivery. Furthermore, the in vitro
cytotoxic activity of this drug delivery system was investigated against
Mel-Rm cell lines. The results of cytotoxicity studies showed that the
loading of L-Dopa on the surface and in the layers of Fe3O4@CaAl-LDH
nanoparticles, cause improve the anticancer activity of free L-Dopa.
Acknowledgments
The authors thank the Razi University Research Council for support
of this work.
Disclosure statement
The authors declare that they have no competing interests.
References
[1] R. Dickey, J. Minton, Levodopa relief of bone pain from breast cancer, N. Engl. J.
Med. 15 (1972) 843.
[2] J.P. Minton, The response of breast cancer patients with bone pain to L-dopa,
Cancer 2 (1974) 358–363.
[3] W.B. Malarkey, L.S. Jacobs, W.H. Daughaday, Levodopa suppression of prolactin in
nonpuerperal galactorrhea, N. Engl. J. Med. 21 (1971) 1160–1163.
[4] B. Stoll, Brain catecholamines and breast cancer: a hypothesis, Lancet (7747)
(1972) 431.
[5] G.H. Sasaki, B.S. Leung, W.S. Fletcher, LevoDopa test and estrogen receptor assay in
prognosticating responses of patients with advanced cancer of the breast to endo-
crine therapy, Ann. Surg. 4 (1976) 392.
[6] M.M. Wick, L-Dopa methyl ester as a new antitumour agent, Nature 5628 (1977)
512–513.
[7] R.P. Dickey, J.P. Minton, L-Dopa effect on prolactin, follicle-stimulating hormone,
and luteinizing hormone in women with advanced breast cancer: a preliminary
report, Am. J. Obstet. Gynecol. 2 (1972) 267–269.
486
Materials Science & Engineering C 101 (2019) 472–486
[8] R.L. Doty, D.A. Deems, S. Stellar, Olfactory dysfunction in parkinsonism A general
deficit unrelated to neurologic signs, disease stage, or disease duration, Neurology 8
(1988) 1237–1244.
[9] F. Li, T. Li, W. Cao, et al. Near-infrared light stimuli-responsive synergistic therapy
nanoplatforms based on the coordination of tellurium-containing block polymer
and cisplatin for cancer treatment. Biomaterials. 133 (2017) 208–218.
[10] D. Yang, W. Luo, J. Wang, et al. A novel controlled release formulation of the Pin1
inhibitor ATRA to improve liver cancer therapy by simultaneously blocking mul-
tiple cancer pathways. J. Control. Release 269 (2018) 405–422.
[11] J. Shi, PW. Kantoff, R. Wooster, et al. Cancer nanomedicine: progress, challenges
and opportunities. Nat. Rev. Cancer 1 (2017) 20.
[12] AJ. Espay, FL. Pagan, BL. Walter, et al. Optimizing extended-release carbidopa/
levodopa in Parkinson disease Consensus on conversion from standard therapy.
Neurol.Clin. Pract. 1 (2017) 86–93.
[13] A. Hsu, J. Kou, L. Alani, Controlled release formulations of levodopa and uses
thereof. Google Patents; 2017.
[14] J. Margolesky, C. Singer, Extended-release oral capsule of carbidopa–levodopa in
Parkinson disease, Ther. Adv. Neurol. Disord. 11 (2018) 1756285617737728.
[15] T. Etrych, M. Jelı́nková, B. Řı́hová, et al. New HPMA copolymers containing dox-
orubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in
vivo biological properties. J. Control. Release 1 (2001) 89–102.
[16] T. Etrych, T. Mrkvan, P. Chytil, et al. N-(2-hydroxypropyl) methacrylamide-based
polymer conjugates with pH-controlled activation of doxorubicin. I. New synthesis,
physicochemical characterization and preliminary biological evaluation. J. Appl.
Polym. Sci. 5 (2008) 3050–3061.
[17] T. Mrkvan, M. Sirova, T. Etrych, et al. Chemotherapy based on HPMA copolymer
conjugates with pH-controlled release of doxorubicin triggers anti-tumor immunity.
J. Control. Release 1 (2005) 119–129.
[18] K. Ulbrich, Vr. Šubr, Polymeric anticancer drugs with pH-controlled activation.
Adv. Drug Deliv. Rev. 7 (2004) 1023–1050.
[19] J. Dobson, Magnetic nanoparticles for drug delivery, Drug Dev. Res. (1) (2006)
55–60.
[20] O. Veiseh, J.W. Gunn, M. Zhang, Design and fabrication of magnetic nanoparticles
for targeted drug delivery and imaging, Drug Deliv. Rev. 3 (2010) 284–304.
[21] SC. McBain, HH. Yiu, J. Dobson, Magnetic nanoparticles for gene and drug delivery.
Int. J. Nanomedicine 2 (2008) 169.
[22] J-H. Choy, S-J. Choi, J-M. Oh, et al. Clay minerals and layered double hydroxides
for novel biological applications. Appl. Clay Sci. 36 (2007) 122–132.
[23] S.-J. Choi, J.-H. Choy, Layered double hydroxide nanoparticles as target-specific
delivery carriers: uptake mechanism and toxicity, Nanomedicine 6 (2011) 803–814.
[24] J-H. Choy, J-S. Jung, J-M. Oh, et al. Layered double hydroxide as an efficient drug
reservoir for folate derivatives. Biomaterials. 25 (2004) 3059–3064.
[25] CP. Dora, SK. Singh, S. Kumar, et al. Development and characterization of nano-
particles of glibenclamide by solvent displacement method. Acta Pol. Pharm. 3
(2010) 283–90.
[26] B. Chu, L. Zhang, Y. Qu, et al. Synthesis, characterization and drug loading property
of monomethoxy-poly(ethylene glycol)-poly(ε-caprolactone)-poly(D,L-lactide)
(MPEG-PCLA) copolymers. Sci. Rep. 2016.
[27] F.P. de Sá, B.N. Cunha, L.M. Nunes, Effect of pH on the adsorption of sunset yellow
FCF food dye into a layered double hydroxide (CaAl-LDH-NO3), Chem. Eng. J. 215
(2013) 122–127.
[28] P. Zhang, G. Qian, ZP. Xu, et al. Effective adsorption of sodium dodecylsulfate (SDS)
by hydrocalumite (CaAl-LDH-Cl) induced by self-dissolution and re-precipitation
mechanism. J. Colloid Interface Sci. 367 (2012) 264–271.
[29] M. Yamaura, R. Camilo, L. Sampaio, et al. Preparation and characterization of (3-
aminopropyl) triethoxysilane-coated magnetite nanoparticles. J. Magn. Magn.
Mater. 279 (2004) 210–217.
[30] M. Muhler, R. Schlögl, G. Ertl, The nature of the iron oxide-based catalyst for de-
hydrogenation of ethylbenzene to styrene 2. Surface chemistry of the active phase,
J. Catal. 138 (1992) 413–444.
[31] D.D. Hawn, B.M. DeKoven, Deconvolution as a correction for photoelectron in-
elastic energy losses in the core level XPS spectra of iron oxides, Surf. Interface
Anal. 10 (1987) 63–74.
[32] AU. Kura, SHH. Al Ali, MZ. Hussein, et al. Development of a controlled-release anti-
parkinsonian nanodelivery system using levodopa as the active agent. Int. J.
Nanomedicine 8 (2013) 1103.
[33] F.L. Theiss, G.A. Ayoko, R.L. Frost, Thermogravimetric analysis of selected layered
double hydroxides, J. Therm. Anal. Calorim. 112 (2013) 649–657.
[34] Y. Kato, S. Ozawa, C. Miyamoto, et al. Acidic extracellular microenvironment and
cancer. Cancer Cell Int. 13 (2013) 89.
[35] N. Shahabadi, M. Falsafi, K. Mansouri, Improving antiproliferative effect of the
anticancer drug cytarabine on human promyelocytic leukemia cells by coating on
Fe3O4@SiO2 nanoparticles, Colloids Surf. B Biointerfaces 141 (2016) 213–222.
[36] N. Shahabadi, M. Jamshidbeigi, M. Falsafi, Functionalization of Fe3O4@SiO2
magnetic nanoparticles with nicotinamide and in vitro DNA interaction, J. Mol. Liq.
224 (2016) 227–233.