Preparation of folic acid-conjugated dendritic mesoporous silica nanoparticles for pH-controlled release and targeted delivery of a cyclometallated gold(III) complex as an antitumor agent

Article abstract of DOI:10.1016/j.molliq.2018.07.024

The purpose of this study is to develop a targeted drug delivery system containing folic acid-conjugated dendritic amino mesoporous silica nanoparticles (DFMSNs) loaded with a cyclometallated gold(III) complex, [Au(bzpy)Cl₂] (bzpy = 2-benzylpyridine), as a cytotoxic agent (Au(drug)@DFMSNs). The products, including the Au(III) complex, DFMSNs, and Au(drug)@DFMSNs, were characterized by transmission electron microscopy (TEM), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), energy-dispersive X-ray (EDX), UV–Vis, ¹H NMR, Fourier transformation infrared (FT-IR), Brunauer-Emmett-Teller (BET) surface area analysis, inductively coupled plasma-atomic emission spectroscopy (ICP-AES), zeta potential measurements, and dynamic light scattering (DLS). The in vitro release study in different pH conditions show that the release of the Au(III) complex from the folic acid functionalized amino mesoporous silica nanoparticles is sustained and pH dependent. The in vitro cytotoxicity test of the free Au(drug), free nanocarrier (DFMSNs), Au(drug) loaded folate-conjugated nanocarrier (Au(drug)@DFMSNs), and Au(drug) loaded nanocarrier without folate-conjugation (Au(drug)@DAMSNs) against two human cancer cell lines, including MCF-7 (human breast carcinoma cell lines) and A549 (human lung carcinoma cell lines) showed that the therapeutic efficacy of Au(drug)@DFMSNs is significantly greater than those in other compounds because the cancerous cells can uptake the folate-conjugated drug nanocarrier via folate receptors-mediated endocytosis. In addition, the different staining methods and fluorescence microscopic images were used to visualize the specific targeting ability, cellular uptake, and the anticancer activity of Au(drug)@DFMSNs and Au(drug)@DAMSNs toward the MCF-7 and A549 cancer cells. The results proved that the proposed system, Au(drug)@DFMSNs, can be used as a potent anticancer agent in targeted cancer therapies for breast cancer.

Full text of DOI:10.1016/j.molliq.2018.07.024

Accepted Manuscript
Preparation of folic acid-conjugated dendritic mesoporous silica
nanoparticles for pH-controlled release and targeted delivery of a
cyclometallated gold(III) complex as an antitumor agent
Samira Malekmohammadi, Hassan Hadadzadeh, Zahra
Amirghofran
PII:
S0167-7322(18)31108-5
doi:10.1016/j.molliq.2018.07.024
MOLLIQ 9345
DOI:
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
3 March 2018
4 July 2018
5 July 2018
Please cite this article as: Samira Malekmohammadi, Hassan Hadadzadeh, Zahra
Amirghofran , Preparation of folic acid-conjugated dendritic mesoporous silica
nanoparticles for pH-controlled release and targeted delivery of a cyclometallated gold(III)
complex as an antitumor agent. Molliq (2018), doi:10.1016/j.molliq.2018.07.024
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ACCEPTED MANUSCRIPT
Journal of Molecular Liquids
Preparation of folic acid-conjugated dendritic mesoporous silica nanoparticles for
pH-controlled release and targeted delivery of a cyclometallated gold(III) complex
as an antitumor agent
a
a,
b
Samira Malekmohammadi , Hassan Hadadzadeh *, Zahra Amirghofran
a
Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran
b
Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Graphical abstract
A targeted drug delivery system containing folic acid-conjugated dendritic amino mesoporous
silica nanoparticles (DFMSNs) loaded with a cyclometallated gold(III) complex was prepared
and introduced as a cytotoxic agent.
*
Corresponding author,
Hassan Hadadzadeh
Professor of Inorganic and Bioinorganic Chemistry
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Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran
E-mail address: hadad@cc.iut.ac.ir
Abstract
The purpose of this study is to develop a targeted drug delivery system containing folic
acid-conjugated dendritic amino mesoporous silica nanoparticles (DFMSNs) loaded with a
cyclometallated gold(III) complex, [Au(bzpy)Cl2] (bzpy = 2-benzylpyridine), as a cytotoxic
agent (Au(drug)@DFMSNs). The products, including the Au(III) complex, DFMSNs, and
Au(drug)@DFMSNs, were characterized by transmission electron microscopy (TEM), field-
emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), energy-dispersive
1
X-ray (EDX), UV-Vis, H NMR, Fourier transformation infrared (FT-IR), Brunauer-Emmett-
Teller (BET) surface area analysis, inductively coupled plasma-atomic emission spectroscopy
(ICP-AES), zeta potential measurements, and dynamic light scattering (DLS). The in vitro
release study in different pH conditions show that the release of the Au(III) complex from the
folic acid functionalized amino mesoporous silica nanoparticles is sustained and pH dependent.
The in vitro cytotoxicity test of the free Au(drug), free nanocarrier (DFMSNs), Au(drug) loaded
folate-conjugated nanocarrier (Au(drug)@DFMSNs), and Au(drug) loaded nanocarrier without
folate-conjugation (Au(drug)@DAMSNs) against two human cancer cell lines, including MCF-7
(human breast carcinoma cell lines) and A549 (human lung carcinoma cell lines) showed that the
therapeutic efficacy of Au(drug)@DFMSNs is significantly greater than those in other
compounds because the cancerous cells can uptake the folate-conjugated drug nanocarrier via
folate receptors-mediated endocytosis. In addition, the different staining methods and
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fluorescence microscopic images were used to visualize the specific targeting ability, cellular
uptake, and the anticancer activity of Au(drug)@DFMSNs and Au(drug)@DAMSNs toward the
MCF-7 and A549 cancer cells. The results proved that the proposed system,
Au(drug)@DFMSNs, can be used as a potent anticancer agent in targeted cancer therapies for
breast cancer.
Keywords: Drug delivery; Folic acid; Dendritic mesoporous silica nanoparticles; Targeted
delivery; Controlled release; Gold(III) complex.
1
. Introduction
Designing an efficient drug delivery system to transport the desired drug without loss or
leakage before reaching the specific cells or tissues is a crucial requirement in cancer
chemotherapy. To achieve this goal, the delivery system must be able to lead the drug molecules
to the specific organ in a controlled manner [1]. In the past decade, mesoporous silica
nanoparticles have found an extended application in the controlled drug delivery systems [2,3].
Mesoporous materials are applied as the useful inorganic carriers for drugs due to their suitable
surface area and pore volume, tunable pore diameter, same particle size, and good
biocompatibility [4-6]. In comparison to other drug carriers, mesoporous materials are more
resistant to heat, mechanical force, and hydrolysis-induced degradations [7,8]. These properties
allow one to adjust the loading of various drug molecules and to study the type of drug release
mechanism with acceptable precision [9]. Main efforts in the studies of mesoporous materials as
drug delivery matrices have been centered on targeted and controlled drug release. The
feasibility of functionalization on the interior and exterior surfaces of mesoporous nanoparticles
makes them suitable for the other fields [10-12]. Dendritic mesoporous silica nanoparticles
(DMSNs) with a non-ordered porous structure are emerging as an efficient platform for
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biomedical purposes owing to their widespread pore size and highly available surface area
compared to other mesoporous silica nanoparticles. Dendritic mesoporous silica nanoparticles
are used in various applications including catalyst, drug delivery, and selective adsorption of
biomolecules [41].
Metal-based drugs, including transition metal complexes, have been developed to treat a
variety of ailments, viz., diabetes, ulcer, rheumatoid arthritis, inflammatory, etc. [13-16].
Cisplatin is the first-generation of metal-based antitumor drugs developed to treat many forms of
cancers [17]. Since cisplatin drug is inefficient against some cancers, interest in the development
of other metal-based complexes with more antitumor activity and a lower toxicity than cisplatin
has been rapidly growing. For this purpose, several transition metal complexes, including Fe, Au,
Rh, Ru, and Ti complexes, have been prepared and systematically tested in vitro and in vivo [18-
2
0]. Among the new non-platinum drugs, many complexes including Au in +1 and +3 oxidation
states have gained more notice due to their anti-inflammatory and neoplasm cell growth-
8
inhibiting properties [21]. Since d Au(III) is isostructural and isoelectronic with Pt(II), its
complexes are appropriate candidates for anticancer agents [20]. In this regard, many Au
complexes have been studied, which target several proteins such as Se-enzyme thioredoxin
reductase (TrxR) [23-26], Zn fingers [27,28], and aquaglyceroporins (AQGPs) and inhibit the
activity of these proteins with high potency and specificity [29-34]. When the Au(III) drug are
used as a guest for functional mesoporous nanoparticles, their combination can provide a
platform with new properties which can achieve a high therapeutic efficacy. The use of folic acid
(FA) for targeting drug delivery has developed due to the difference in expression between
normal and some cancer cells. In fact, the use of folate-targeted nanocarrier has demonstrated to
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enhance the uptake by tumor cells, which possess a large number of folate-receptors (FRs)
[35,36].
Herein, we report the preparation of dendritic folic acid-modified mesoporous silica
nanoparticles that can deliver an antitumor agent, a mononuclear cyclometallated Au(III)
complex with 2-benzylpyridine ligand ([Au(bzpy)Cl2]), to the cancer cell lines. In addition, the
proposed drug delivery system allows the use lower doses of the antitumor drug (Au(III)
complex) and helps to reduce the side effects of chemotherapy through the targeted drug delivery
and controlled drug-release. The dendritic amino mesoporous silica nanoparticles(DAMSNs)
were prepared by the condensation of tetraethylorthosilicate (TEOS) with 3-
(trimethoxysilylpropyl)diethylenetriamine (Si-DETA) in the presence of cetylpyridinium
bromide (CPB) as a cationic quaternary ammonium surfactant. In the next step, folic acid was
conjugated to the mesoporous as a targeting agent (DFMSNs). Then, the Au(III) complex,
[Au(bzpy)Cl2], was loaded to the DFMSNs nanoparticles to form the Au(drug)@DFMSNs
system (Fig. 1). Finally, the in vitro cytotoxic activity of all compounds was measured against
two different cancer cell lines including A549 (human lung carcinoma cell lines) and MCF-7
(human breast adenocarcinoma cell line) using the different colorimetric assay and controlled
with fluorescent microscope imaging.
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Fig. 1. Synthetic route for the preparation of DAMSNs, DFMSNs, and Au(drug)@DFMSNs.
2
. Experimental
2
.1. Materials
Tetraethyl orthosilicate (TEOS), 3-(trimethoxysilylpropyl) diethylenetriamine (Si-
DETA), cetylpyridinium bromide (CPB), NaAuCl4·2H2O, 2-Benzylpyridine, and folic acid
≥97%) were purchased from Sigma-Aldrich Co. N,N-dicyclohexylcarbodiimide (DCC), N-
(
hydroxysuccinimide (NHS), 4′,6-diamidino-2-phenylindole (DAPI), and all the solvents were
purchased from Merck Company. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT), and RPMI 1640 and culture medium were gifts from the Shiraz University of
Medical Sciences (IRAN).
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2
.2. Characterizations
1
IR, far-IR, UV–vis, and H NMR spectra were recorded on Bruker Vector 22 FT-IR, Bruker
Vertex 70v FT-IR, JASCO-570, and Bruker 500 Avance III, respectively. XRD patterns (2θ = 5–
1
00º) were recorded on a Philips diffractometer using Ni-filtered CuK훼 radiation. The size and
morphology of the nanoparticles were determined by field-emission scanning electron
microscopy (FE-SEM) using a JEOL scanning electron microscope. Transmission electron
microscopy (TEM) images were obtained using a Zeiss-EM10C-80 kV. Just prior to imaging, the
samples were dispersed and sonicated in deionized water and then deposited on the carbon-
coated copper grids. Nitrogen adsorption-desorption isotherms at –196°C were obtained using a
Japan BELSORP-II system following the samples were vacuum dried at 150°C overnight. To
determine the evolution of the hydrodynamic diameter and surface charge of nanoparticles by
dynamic light scattering (DLS) and zeta (ζ)-potential measurements, respectively, a Zetasizer
Nano ZS (Malvern Instruments, United Kingdom) equipped with a laser (633ꢀnm) was used.
2
.3. Synthesis of the cyclometallated Au(III) complex
Au(bzpy)Cl2] was synthesized according to a literature procedure [37]. NaAuCl4·2H2O
100 mg, 0.25 mmol) was dissolved in deionized water (10 mL). 2-Benzylpyridine (0.05 mL,
.28 mmol) was dissolved in acetonitrile (10 mL) and then added to the Au(III) solution. A
[
(
0
yellow precipitate was formed immediately. The reaction mixture was then refluxed until the
yellow precipitate turned white (Scheme 1). The resulting white precipitate, [Au(bzpy)Cl2], was
1
separated, washed with acetone, and then air-dried. The H NMR and IR spectral data of the final
product, [Au(bzpy)Cl2], were reported in the Supporting Information (SI).
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Scheme 1. Synthetic route for the preparation of (C^N) cyclometallated gold(III) complex
([Au(bzpy)Cl2]).
2
.4. Synthesis of the dendritic amino mesoporous silica nanoparticles (DAMSNs)
The dendritic amino mesoporous silica nanoparticles were prepared according to
literature, but with some modifications [38]. Cetylpyridinium bromide (1.3 mmol) and urea (5.0
mmol) were dissolved in deionized water (10 mL) at room temperature. A mixture of
cyclohexane (15 mL) and n-butanol (6.0 mmol, 0.55 mL) was then added to the solution. TEOS
1
(
4.2 mmol, 0.94 mL) and N -(3-trimethoxysilylpropyl) diethylenetriamine (Si-DETA) (1.8
mmol, 0.47 mL) were added drop-wise to the reaction mixture with vigorous stirring for 30 min
at room temperature. The mixture was heated at 70°C for 24 h, centrifuged and then washed
three times with ethanol. Afterward, the collected product was dispersed in an ethanolic solution
of ammonium nitrate (NH NO ) (0.6 wt %) and kept at 60°C with vigorous stirring for 3 h to
4
3
remove the template. The resulting precipitates (DAMSNs) were dried at 80°C for 24 h.
2
.5. Synthesis of the folic acid-modified dendritic mesoporous silica nanoparticles (DFMSNs)
Folic acid (FA) was activated according to the previous report [34]. To prepare the FA
active ester, FA (1 g) in DMSO (30 mL) and triethylamine (0.5 mL) were mixed under vigorous
sonication, and then N, N-dicyclohexylcarbodiimide (DCC) (1 g) and N-hydroxysuccinimide
(NHS) (0.6 g) were added to the mixture and stirred for 24 h. The yellow supernatant was
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separated by centrifugation, dispersed to a mixture of ether and ethanol (50 mL, 1:1), and
centrifuged again to collect the supernatant. After drying the supernatant, a yellow powder of FA
active ester was obtained. DAMSNs were added to a DMSO solution (20 mL) containing the FA
activated ester (0.4 g) and the pH of the mixture was adjusted to 8-8.5 by triethylamine. The
reaction mixture was then stirred at 40°C for 24 h. The suspension was collected by
centrifugation, washed with ethanol, and then air-dried.
2
.6. Preparation of the drug-loaded mesoporous silica nanoparticles (Au(drug)@DFMSNs)
DFMSNs (300 mg) was added to a solution of [Au(bzpy)Cl2] (100 mg) in 30 mL
phosphate-buffered saline (PBS) and stirred for 12 h at 20°C. The mass ratio of [Au(bzpy)Cl2]
to DFMSNs was 1:3. The final drug-loaded mesoporous, Au(drug)@DFMSNs, was separated by
centrifugation, washed with deionized water to eliminate the unloaded drug molecules, and then
dried under vacuum at 20°C.
2
.7. The in vitro release of the drug from the nanocarrier
Au(drug)@DFMSNs (30 mg) was dispersed in 30 mL of PBS solution with different pH
values (5.7, 6.7, and 7.4) and kept at 37°C. A specific volume of the suspension (5 mL) was
collected after the centrifugation at each specified time and the same volume of a fresh PBS
solution was then added to the remaining suspension to keep its volume constant. The
concentration of the released [Au(bzpy)Cl2] in each sample was measured by UV-Vis
spectroscopy. Afterward, the amount of the drug released from the DFMSNs nanoparticles was
calculated according to equation (1):
the amount of drug released into solution
Cumulative release (%) =
× 100
(1)
the total amount of drug loaded into the DFMSNs
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2
.8. Cell culture
The tumor cell lines containing A549 (human lung carcinoma cell lines) and MCF-7
human breast adenocarcinoma cell line) were purchased from the Culture Collection of the
(
Shiraz University of Medical Sciences in Iran. All cell studies were carried out in a humidified
incubator under standard conditions at 37°C and 5% CO2 atmosphere. The cells were grown in
an RPMI 1640 medium including 10% (v/v) heat inactive fetal bovine serum (Gibco), glutamine
(2 mM), and 1% penicillin and streptomycin.
2
.9. MTT cytotoxicity assay
The in vitro cytotoxicity of the free Au(drug), free nanocarrier (DFMSNs), Au(drug)
loaded folate-conjugated nanocarrier (Au(drug)@DFMSNs), and Au(drug) loaded nanocarrier
without folate-conjugation (Au(drug)@DAMSNs) was studied using the MTT assay. To
investigate the cytotoxic effect of the compounds, the cells were placed in 96-well plates at a
3
density of 5 × 10 cells per well and grown at 37°C in a humidified 5% CO2 incubator. The cells
were then treated with different concentrations (0.1 to 200 µg/mL) of the nanocomposite in
DMSO for 24 h. The control cultures were supplemented with the same amount of solvent. After
the drug treatment, 20 µL of the MTT solution was added to each plate and incubation was
performed at 37°C for 4 h. Then, 200 µL of DMSO was added to dissolve the formazan crystals
that formed and the absorbance was read at 570 nm using an ELISA reader (Bio-Tek, Elx 808).
The cytotoxicity was expressed as the percentage of cell viability compared to the untreated
3
0
control cells at A570.
Finally, the IC50 values for each compound were determined by plotting
the percentage inhibition against the drug concentration on a logarithmic graph.
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2
.10. Morphological change and cellular uptake
The membrane integrity and cellular uptake were evaluated using acridine orange
staining to observe the following morphological changes and also the possible occurrence of
apoptosis in the cancer cells treated with Au(drug)@DFMSNs and Au(drug)@DAMSNs with
different concentration. The treated MCF-7 cells were incubated with 100 μg/mL acridine orange
for 30 min in a CO incubator. After washing three times with PBS, the cells were monitored at
2
2
00 × magnification using an IX81 Epifluorescence microscope.
2
.11. Targeted apoptosis detection by DAPI staining
The DAPI staining assay was used to distinguish the targeted apoptosis of the MCF-7 and
−1
A549 treated cells with the same concentration (200 μgmL ) of free Au(drug),
Au(drug)@DAMSNs, and Au(drug)@DFMSNs. The treated cells were fixed with 4%
−1
paraformaldehyde (PF) and a solution containing 0.1 μgmL of DAPI (4′,6-diamidino-2-
phenylindole) in PBS was then added to each fixed sample and kept in the incubator for 10 min
to stain the nuclei in the dark. After removing the residue stain, the cells were washed three times
with PBS.
2
.12. Determination of cell apoptosis and necrosis by flow cytometry
The cell apoptosis and necrosis were investigated using Annexin V and PI staining. For
5
staining, the MCF-7 cells (2 × 10 cells/well in 6 well plates) were incubated with
−1
Au(drug)@DAMSNs and Au(drug)@DFMSNs (200 μgmL ). After 12 h, the cells were washed
three times with PBS, centrifuged, and then suspended in Annexin V buffer. Afterward, the
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treated cells were incubated for 30 min at 37°C and analyzed by flow cytometer (Becton
Dickinson, San Jose, USA).
3
. Results and discussion
3
.1. Synthesis and structural characterization
The (C^N) cyclometallated Au(III) complex, [Au(bzpy)Cl2], was prepared according to
the method described by Cinellu et al. [37]. The dendritic mesoporous silica nanoparticles were
synthesized according to the literature method [36]. After removal of the surfactant to expose the
pore channels, the Au(III) complex was loaded in the mesoporous channels to form the
Au(drug)@DFMSNs system.
The morphology and particle size of the DFMSNs nanoparticles were determined by FE-
SEM. The FE-SEM image shows that the nanoparticles have a spherical shape with similar
particle sizes (Fig. 2A). In addition, TEM was used to verify the porous structure of DFMSNs
nanocarrier. The TEM images show the presence of the pores on the nanoparticle surface and
confirm a wrinkle spherical with dendritic structures for these nanoparticles (Figs. 2B and 2C).
The average particle size of the DFMSNs nanoparticles was estimated to be 82 ± 6 nm as shown
in the histogram in Fig. 2D.
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Fig. 2. (A) FE-SEM image of DFMSNs; (B) TEM images of the DFMSNs; (C) TEM image of
the Au(drug)@DFMSNs nanoparticles; and (D) Size distribution of the DFMSNs nanoparticles.
The total surface area and pore size distribution of the folic acid-functionalized
mesoporous nanoparticles were measured by BET and BJH methods. The obtained N2
adsorption-desorption isotherm is shown in Fig. 3. The successful functionalization of the
nanoparticles was confirmed by the N2 adsorption-desorption measurements. The total surface
2
area was found to be 295 m /g. In comparison to the previous reports [41-43], a significant
decrease in the surface area of the functionalized sample shows a good interior modification for
the folic acid-modified dendritic mesoporous silica nanoparticles. The BJH pore size distribution
shows the presence of a mesoporous structure with average pore diameter and pore volume of
3
3
.4 nm and 0.82 cm /g, respectively.
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Fig. 3. Nitrogen adsorption-desorption isotherms and BJH pore size distribution of DFMSNs
(inset).
The FT-IR spectra of the dendritic mesoporous silica nanoparticles(DMSNs) with and
without CPB surfactant, dendritic amino mesoporous silica nanoparticles (DAMSNs), and folic
acid-modified dendritic mesoporous silica nanoparticles (DFMSNs) are shown in Fig. 4A. The
−1
sharp bands at 1030 cm are attributed to the Si−O−Si asymmetric stretching vibration. The
−1
bands at 780 and 950 cm are assigned to the symmetric Si−O−Si stretching and Si−O defect
−1
sites, respectively. The bands at 560 cm reflect a coupling of the Si−O stretching to the
-
1
O−Si−O and Si−O−Si bending modes. The bands at 3434 and 1635 cm are assigned to the –OH
groups present on the surface of DAMSNs and DFMSNs or due to the water molecules adsorbed
−1
by these nanoparticles. The bands at 2930 and 2880 cm are due to the C−H vibrations groups
−1
[
44-46]. Broad bands at 3050 and 3250 cm are assigned to the N−H stretching modes in the
−1
pendent DETA groups. In addition, the band at 1490 cm corresponds to the symmetric N−H
bending vibration. The FA conjugation onto the surface of amino mesoporous silica is confirmed
by the disappearance of two bands related to –NH2 group and the appearance of the characteristic
-
1
band at 1606 cm corresponding to the N–H amide stretching vibration of FA. There is a good
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agreement between the results obtained from the FT-IR and zeta potential measurements. The
surface charge of mesoporous silica nanoparticles can affect their performance for internalizing
of cells, escaping endosomal entrapment and also their controlled drug release. The zeta
potentials of DMSNs, DAMSNs, and DFMSNs are shown in Fig. 4B. Before functionalization
with aminosilane groups, the zeta potential of DMSNs is -27.3 mV, which proves the presence of
-
OH functional groups on the mesoporous surface. The zeta potential of DAMSNs is +32.4 mV,
showing the NH2 groups have strongly conjugated onto the surface of DMSNs. After the FA
conjugation, the zeta potential of DAMSNs decreases from +32.4 mV to +17.3 mV, which is
attributable to the carboxyl groups of FA, demonstrating that FA is successfully modified the
DAMSNs nanoparticles. The zeta potentials of all synthesized nanoparticles in BPS, with
different pH levels from 2 to 12 were measured. The obtained results are reported in the
Supporting Information (Fig.S1).
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Fig. 4. (A) The FT-IR spectra of the dendritic mesoporous silica nanoparticles(DMSNs) with and
without CPB surfactant, dendritic amino mesoporous silica(DAMSNs), and folic acid-modified
dendritic mesoporous silica nanoparticles (DFMSNs); (B) Zeta potentials of DMSNs, DAMSNs,
and DFMSNs.
The loading content of the Au(III) complex in the DFMSNs nanocarrier was examined by
inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The average
concentration of Au(III) was found to be 15.70 mg/L. Since the total concentration of
Au(drug)@DFMSNs was 120 mg/L, the loading percent of Au(III) in the Au(drug)@DFMSNs
system was calculated to be 13.83% (wt/wt).
The EDX analysis was applied to determine the chemical composition of
Au(drug)@DFMSNs which shows peaks for Si, O, N, and C atoms. In addition, the EDX
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spectrum reveals the presence of Au and Cl atoms that confirms the presence of the Au(III)
complex in the mesoporous silica channels (Fig. 5).
Fig. 5. The EDX mapping of Si (left) and the spectrum of the Au(III) complex-loaded
mesoporous nanocarrier (Au(drug)@DFMSNs) (right).
The crystalline properties of the samples before (Fig. 6A) and after (Fig. 6B) loading of
the drug were measured by XRD analysis using a fixed-angle ranging from 5 to 100º. The XRD
pattern of DFMSNs exhibits a very broad signal at 2θ = 22.8º, which shows the prepared
mesoporous silica is amorphous. In addition, the existence of Au(III) ions in Au(drug)@
DFMSNs was investigated by XRD (Fig. 6B). It is well known that the XRD pattern of Au
nanoparticles demonstrates four intense peaks at 38.49, 42.21, 63.88, and 78.34° [47]. In Fig.
6
B, no diffraction peaks of Au nanoparticles are observed, which confirm the presence of Au(III)
ions in Au(drug)@ DFMSNs.
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Fig. 6. XRD patterns of (A) DFMSNs, and (B) Au(drug)@DFMSNs.
Dynamic light scattering (DLS) method was used to estimate the particle size and also
dispersity of the dendritic mesoporous silica (DMSNs), dendritic amino mesoporous silica
(
DAMSNs), and folic acid-modified dendritic mesoporous silica (DFMSNs) nanoparticles in
PBS (pH = 7.4). The DLS results revealed that the particle size distribution of DMSNs is narrow
Fig.7). The hydrodynamic particle size was measured to be 100 ± 14 nm, which is a little larger
(
than the particle size observed by TEM. This is explained because DLS provides the mean
diameter of nanoparticles with a solvation layer, whereas TEM shows their size in a dry state. On
the other hand, a polydispersity index (PDI) of 0.12 indicates the presence of small amount of
aggregates of DMSNs in the buffer solution. The DLS results also show a relatively narrow
particle size distribution for the DAMSNs and DFMSNs nanoparticles with a polydispersity
index (PDI) of 0.14 and 0.19, respectively. The average particle size was estimated to be 115 ±
1
5 and 125 ± 17 nm for DAMSNs and DFMSNs, respectively, which is a little larger than that of
the DMSNs nanoparticles, suggesting that the DMSNs surface was successfully functionalized
with spatial functional groups.
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Fig. 7. Particle size distribution of A) DMSNs, B) DAMSNs, and C) DFMSNs measured by
DLS analysis.
3
.2. The in vitro release of the Au(III) complex from the nanocarrier
The amount of the Au(III) complex released from the DFMSNs carrier during the time,
was measured by UV-Vis spectroscopy. Fig. 8 indicates the plots of the drug release profiles
versus time for Au(drug)@DFMSNs in the PBS solution in different pH conditions including
tumoral microenvironment pH (pH = 5.7), normal physiological pH (pH = 7.4), and the average
pH of saliva (pH =6.7). The initial burst of the Au(II) complex from the mesoporous carrier is
due to the early release of the complex adsorbed on or near the surface of the mesoporous
nanocarrier. The Au(III) complex can form weak hydrogen bonds with the functional groups
including silanol and amino groups on the pore walls of DFMSNs. In general, as shown in Fig.
8
, the Au(III) drug release from the DFMSNs nanoparticles is pH-dependent. At pH = 5.7, the
dissolution of drug-loaded materials into the release media is greater than weak acidic and
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neutral media. Under the acidic conditions, the functional groups on the surface of DFMSNs
become protonated and cause to more intense release as compared to the neutral condition. The
diffusion of the solvent molecules into the small channels and the counter-diffusion of drug out
of the mesoporous channels are relatively restricted, leading to a delayed release of the Au(III)
complex. However, the drug molecules adsorbed in the large mesoporous channel have a good
chance to escape from the mesoporous and to diffuse into the release medium. The pH-sensitive
release of drug can enhance the therapeutic effect of the Au(drug)@DFMSNs for cancer
treatment.
Fig. 8. The in vitro release profiles of Au(drug)@DFMSNs in different pH of the PBS solutions.
A similar profile release at different conditions has been reported by different drug
delivery systems based on mesoporous silica [48-52]. These results show that the synthesized
folic acid-modified dendritic mesoporous silica can be used as a suitable drug carrier for the
sustained and pH-controlled release systems. About 50% of the drug is released from the matrix
at a neutral pH after 48 h, whereas during the same time, the amount of the drug released reaches
to about 90% at pH = 5.7.
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3
3
.4. Biological activity studies
.4.1 MTT cell proliferation assay
The cytotoxic activity of the free Au(drug), free nanocarrier (DFMSNs), Au(drug) loaded
folate-conjugated nanocarrier (Au(drug)@DFMSNs), and Au(drug) loaded nanocarrier without
folate-conjugation (Au(drug)@DAMSNs) was investigated by a colorimetric microculture assay
(
MTT assay) against two human cancer cell lines including MCF-7 and A549. The cytotoxic
activity of four compounds was measured through the spectrophotometric data by equation (2)
53]:
cell cytotoxicity = [1 – Absdrug/Abscontrol] × 100
[
%
(2)
Where, Absdrug and Abscontrol are the average absorbance of the treated cancer cells with the
compounds, free Au(drug), free DFMSNs Au(drug)@DAMSNs, and Au(drug)@DFMSNs and
non-treated cancer cells served as a control, respectively.
Fig. 9 shows the plots of the percentage inhibition versus the concentration of the
compounds (0.1–200 μg/mL) for the selected cell lines. The results show that the cytotoxicity of
Au(drug)@DFMSNs against the MCF-7 and A549 cell lines is significantly greater than other
compounds. According to the MTT results MCF-7 cells showed high cellular uptake of
Au(drug)@DFMSNs and significant cell apoptosis compared to A549 cells. It is further
concluded that FA-conjugated on to the nanocarrier, promotes the entry of them into the FR
positive MCF-7 cells that overexpressed with folate receptors and demonstrate good affinity with
FA which may enhance the rate of cellular uptake of Au(drug)@DFMSNs and more cell
apoptosis. The IC50 values are shown in Table 1. The results show that the loading process of
[
Au(bzpy)Cl ] into a mesoporous nanocarrier enhances the cytotoxic activity because the
2
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nanocarrier can help to protect the optimum concentration of the Au(III) complex through a
sustained and controlled drug-release.
Fig. 9. The cytotoxic effect of A) DFMSNs, B) Au(drug), C) Au(drug)@DAMSNs, and D)
Au(drug)@DFMSNs against the MCF-7 and A549 cancer cell lines in a concentration-dependent
manner.
Table 1. IC50 values (μg/mL) for compounds, A) DFMSNs, B) Au(drug), C)
Au(drug)@DAMSNs, and D) Au(drug)@DFMSNs, against the MCF-7 and A549 cancer cell
lines.
A
B
C
D
MCF-7
A549
54 ± 3
32 ± 2
27 ± 5
18 ± 3
57 ± 4
35 ± 5
37 ± 3
28 ± 4
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3
.4.2 Morphological change and cellular uptake
The fluorescence microscopic images were used to visualize the specific targeting ability,
as well as the anticancer activity of Au(drug)@DFMSNs and Au(drug)@DFMSNs against the
MCF-7 cancer cells with overexpressed folate receptors via acridine orange staining. At first, the
cells were grown on a 6-well plate and incubated with two samples (100 and 200 μg/mL) of
Au(drug)@DFMSNs and Au(drug)@DFMSNs for 4 h. The microscopic views of MCF-7 cancer
cells after incubation show the decreasing in the number of viable cells compared to the control
samples (Fig. 10). As can be seen, the high concentrations of drug-loaded mesoporous cause
membrane damage and the following apoptosis. The morphological variations of the MCF-7
cancer cells treated with Au(drug)@DFMSNs were also seen by acridine orange staining. In Fig.
1
0, the breast cancer cells treated with Au(drug)@DFMSNs show more morphological changes,
including decreased cell volume and cell shrinkage with a concentration-dependent manner,
compared to the cells treated with Au(drug)@DAMSNs. The results are in good agreement with
the MTT assay study. Therefore, the experimental results demonstrate the excellent capability of
Au(drug)@DFMSNs as an efficient drug nanocarrier for the breast cancer cells treatment in a
dosage-dependent manner.
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Fig. 10. The fluorescence microscopic images of the MCF-7 cells stained by acridine orange
with different consecration of Au(drug)@DFMSNs (B and E) and Au(drug)@DAMSNs (C and
F). The green staining represents viable cells and the yellow staining represents late apoptotic
cells.
3
.4.3 Folate receptor targeting study and cellular imaging
The targeting effect of folic acid in this drug delivery system was proved in two human
cancer cell lines, including MCF-7 and A549, through DAPI staining. The cellular imaging and
folate receptor targeting efficiency of the free Au(drug), Au(drug)@DAMSNs, and Au(drug)@
DFMSNs in A549 (FR negative cells) [54,55] and MCF-7 (FR positive cells) [56-57] are shown
in Fig. 11. It is worth mentioning that a noticeable uptake of Au(drug)@DFMSNs and the
following cell apoptosis cannot occur in the A549 cells, due to their deficiency of the folate
receptors, while the MCF-7 cells showed high cellular uptake of Au(drug)@DFMSNs and
significant cell apoptosis compared to the A549 cells. In addition, the results reveal that the entry
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of Au(drug)@DFMSNs into the MCF-7 cells increases, due to the folic acid conjugation onto the
nanocarrier. Subsequently, the rate of cellular uptake enhances and more cell apoptosis can occur
in the MCF-7 cells. The cellular uptake efficiency of the drug-loaded nanocarrier without the
folate conjugation (Au(drug)@DAMSNs) and drug-loaded folate-conjugated nanocarrier
(Au(drug)@DFMSNs) in the MCF-7 and A549 cells were also investigated. The results show
that the Au(drug)@DFMSNs nanocomposite has more cellular uptake and cytotoxic effect in
both cell lines compared with Au(drug)@DAMSNs. There is a good agreement between the
results obtained from the MTT assay and DAPI staining.
Fig. 11. DAPI staining assay for targeted apoptosis detection in A549 and MCF-7 cells.
3
.4.4 Flow cytometry study
The cell apoptosis and necrosis of MCF-7 cells treated with Au(drug)@DAMSNs and
Au(drug)@DFMSNs were evaluated by flow cytometry (Fig. 12). The Au(drug)@DAMSNs
nanoparticles show little influence on the apoptosis and necrosis of MCF-7. However, the free
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Au(drug)@DFMSNs nanoparticles with folic acid conjugation at the same concentration can
simultaneously induce both apoptosis and necrosis of MCF-7. The flow cytometry study proves
that the Au(drug)@DFMSNs nanoparticles are significantly taken up by the FR-positive MCF-
7
cells via folate receptors-mediated endocytosis and induced more apoptosis compared with
Au(drug)@DAMSNs.
Fig. 12. Flow cytometry dot plot of A) Control, B) Au(drug)@DAMSNs, and C)
−
−
Au(drug)@DFMSNs in MCF-7 cancer cells. In flow cytometry plot dot, AV /PI (Q3), intact
+
+
+
−
−
+
live cells; AV /PI (Q1), necrotic cells; AV /PI (Q4), early apoptotic cells; AV /PI (Q2), late
apoptotic cells.
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4
. Conclusion
Over the last decade, the development and application of nanotechnology in cancer
detection and therapeutics have grown rapidly. The precise design of new carriers for the
concurrent delivery of chemo- and radio-therapeutics increase the effectiveness of the drug and
reduces the dosage of each particular drug needed to produce a noticeable therapeutic response.
In this work, a new nanocarrier system containing dendritic folic acid-modified mesoporous
silica nanopaeticles (DFMSNs) nanoparticles loaded with a cyclometallated gold(III) complex
([Au(bzpy)Cl2]) as a cytotoxic agent was prepared. The results demonstrated the favorable
uptake and then release of the Au(III) drug in the spherical DFMSNs nanoparticles, proving the
potential of DFMSNs nanoparticles as a drug delivery nanocarrier. In addition, the results
showed that the releasing of Au(III) complex is significantly dependent on the pH of the medium
and the nature of surface species on the mesoporous nanoparticles. Because of the presence of
hydrogen-bonding interactions between the chloride ligands in [Au(bzpy)Cl2] and the functional
groups on the surface pores of DFMSNs, the release of [Au(bzpy)Cl2] from the nanocarrier is
pH-controlled. The in vitro drug release tests revealed pH-dependent and sustained drug release
profiles, which can improve the therapeutic anti-cancer effect of the Au(III) drug and reduce the
damage or potential damage to normal cells or tissues due to the acidic microenvironment of the
tumor.
Finally, the in vitro anticancer activity of the free complex ([Au(bzpy)Cl2]), the free
nanocarrier (DFMSNs), Au(drug)@DAMSNs, and Au(drug)@DFMSNs against the A549 and
MCF-7 cancer cells using the MTT assay revealed that Au(drug)@DFMSNs is a notably better
cytotoxic agent than other compounds against both cell lines. In the comparative studies, the
fluorescence microscopic images and different staining methods were used to visualize the
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cellular uptake, anticancer activity, and specific targeting ability of Au(drug)@DFMSNs toward
the MCF-7 cancer cells. The results proved that Au(drug)@DFMSNs is an efficient candidate
for breast cancers targeted therapy via folate receptors-mediated endocytosis.
Acknowledgment
This research was financially supported by Isfahan University of Technology (IUT-
IRAN). We thank our colleagues from the Ecole Polytechnique Fédérale de Lausanne (EPFL)
chemistry and chemical engineering faculty for DLS and Zeta potential analyses and especially
Daniel John Gilles Marchand, for his comments that greatly improved the manuscript.
Supporting Information
The supplemental file contains the spectroscopic characterization of the cyclometallated
gold(III) complex and zeta potential measurement in different pH conditions.
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