Environmentally Responsive Dual-Targeting Nanoparticles: Improving Drug Accumulation in Cancer Cells as a Way of Preventing Anticancer Drug Efflux

Article abstract of DOI:10.1016/j.xphs.2017.10.029

Drug targeting and stimuli-responsive drug release are 2 active areas of cancer research and hold tremendous potential in the management of cancer drug resistance. In this study, I addressed this issue and focused on the synthesis and characterization of

Full text of DOI:10.1016/j.xphs.2017.10.029

Journal of Pharmaceutical Sciences xxx (2017) 1-8
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Journal of Pharmaceutical Sciences
journal homepage: www.jpharmsci.org
Pharmaceutical Nanotechnology
Environmentally Responsive Dual-Targeting Nanoparticles: Improving
Drug Accumulation in Cancer Cells as a Way of Preventing Anticancer
Drug Efflux
Cenk Daglioglu*
Department of Molecular Biology and Genetics, Faculty of Science, Izmir Institute of Technology, Urla, Izmir 35430, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Drug targeting and stimuli-responsive drug release are 2 active areas of cancer research and hold
tremendous potential in the management of cancer drug resistance. In this study, I addressed this issue
and focused on the synthesis and characterization of pH-responsive Fe3O4@SiO2(FITC)-BTN/folic acid/
DOX multifunctional nanoparticles aiming to increase drug accumulation in malignancies with both dual
active targeting and endosomal drug release properties. Dye-doped silica magnetic-fluorescent com-
posite was constructed by a simple coprecipitation of Feþ2/Feþ3 salts followed by sol-gel formation and
dual-targeting function was obtained by conjugating folate and biotin moieties on the silica surface of
nanoparticles via an esterification reaction. Doxorubicin was then successfully attached on the amine-
functionalized nanoparticles using a pH-sensitive Schiff-base formation. The physicochemical charac-
terization of the structure was performed by dynamic light scattering, zeta potential measurement, X-ray
diffraction, Fourier transform infrared spectroscopy, electron microscopy techniques, and an in vitro pH-
dependent release study. Cellular uptake and cytotoxicity experiments demonstrated an enhanced
intracellular delivery and reduction of cancer cell viability in the cervical carcinoma HeLa cell line.
Furthermore, proapoptotic studies showed that the nanoparticles increased the apoptotic rates within
the same cancer cells. The preliminary cell tests confirm the potential of these multifunctional nano-
particles against the development of drug resistance in cancer cells.
Received 5 June 2017
Revised 15 October 2017
Accepted 17 October 2017
Keywords:
nanoparticles
conjugation
drug resistance
targeted drug delivery
responsive delivery systems
© 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Introduction
DDS to evade drug pump efflux activity as a way of preventing drug
resistance.
Drug resistance is one of the main problems in clinical cancer
treatment. The overexpression of drug efflux transporters, such as
P-glycoprotein (P-gp), plays a significant role in the progression of
resistance.1 To achieve targeted therapeutic concentration, higher
doses or frequency of dosing are required but thus resulting in
greater toxicity.2 In order to address this problem, considerable
attempts have been made to exploit the drug delivery systems
(DDSs) for overcoming drug resistance in cancer therapy.3 Although
most DDSs conferred improved pharmacokinetics and bio-
distribution profiles, the poor cellular uptake and insufficient drug
release inside cells remain rate-limiting steps, which lead to low
intracellular drug concentration below the therapeutic window.4
Therefore, both active targeting capacity and environmentally
responsive drug release character are essential features for an ideal
Among the numerous DDSs, magnetic (Fe3O4) nanoparticles are
promising as drug delivery vehicles for both diagnostic and ther-
apeutic applications.5 The ease of the synthesis of magnetic nano-
particles and subsequent incorporation of various cell-specific
targeting, imaging, and therapeutic functions have enabled these
systems to be employed as smart nanomedicines.6 In this regard,
DDSs endowed with the abilities to deal with efficient accumula-
tion and drug release in cancer cells could lead to easier reaching of
anticancer drug to the effective therapeutic concentration before
the development of drug resistance.
Vitamin receptors are upregulated on a variety of human can-
cers due to their enhanced mitosis rates. Therefore, the
overexpression of these receptors can be exploited to target
vitamin-linked DDSs specifically to tumor cells.7 Considering the
results reported in our previous works, the nanoparticles vector-
ized with biotin (vitamin B7)8 or folate (FA) (vitamin B9)9,10 were
successfully penetrated into the cancer cells via receptor-mediated
endocytosis. In this study, to further enhance the accumulation
level of nanoparticles and to promote fast intracellular drug release
Conflicts of interest: The author reports no declarations of interest.
* Correspondence to: Cenk Daglioglu (Telephone: þ90 232 750 7319; Fax: þ90 232
750 7303).
E-mail address: cenkdaglioglu@iyte.edu.tr (C. Daglioglu).
https://doi.org/10.1016/j.xphs.2017.10.029
0022-3549/© 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

2
C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
in cancer cells in order to prevent cellular efflux, the effect of the
coconjugation of 2 active targeting ligands on the surface of the
nanoparticles in combination with pH-dependent drug release
property was investigated. For this reason, pH-responsive 3 suc-
cessive nanoparticles: (1) Fe3O4@SiO2(FITC)-BTN/DOX nano-
particles containing only biotin; (2) Fe3O4@SiO2(FITC)-FA/DOX
nanoparticles containing only folate; (3) Fe3O4@SiO2(FITC)-BTN/FA/
DOX nanoparticles containing both biotin and folate were fabri-
cated to evaluate accumulation potential of the nanoparticles be-
tween single and dual targeting in inherently drug-resistant HeLa
cells, which conditionally overexpress 21 of the 36 investigated
transporters.11
catalyst at room temperature for 2 h. After this, a mixture of
Fe3O4@SiO2(FITC) nanoparticles (100 mg), BTN-APTES and FA-
APTES conjugate, and free APTES (17
mL) in toluene (160 mL)
was stirred at room temperature for 24 h to introduce BTN-APTES
and FA-APTES conjugate and free APTES on the surface of silica-
coated nanoparticles by hydrolysis and condensation of APTES
through silanization. Final products were collected by a magnet,
washed with toluene and ethanol several times to remove any
unreacted reactants, and dried in vacuum oven at room temper-
ature, overnight. In this step, besides vectorization of nano-
particles, simultaneously the surfaces were modified with
free APTES to form an amine-terminated overlayer for further
functionalization.
Fe3O4@SiO2(FITC)-BTN/DOX, Fe3O4@SiO2(FITC)-FA/DOX, and
Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles were prepared by
conjugating DOX on the surface of Fe3O4@SiO2(FITC)-BTN/NH2,
Fe3O4@SiO2(FITC)-FA/NH2, and Fe3O4@SiO2(FITC)-BTN/FA/NH2
nanoparticles via glutaraldehyde activation, respectively. For pH-
responsive drug release, DOX complex was covalently linked to
amine-functionalized silica surface of nanoparticles via pH-labile
Schiff-base formation from the amino sugar moiety of DOX.12 This
acid-sensitive linkage is stable at natural pH (~7.4), but broken at
mildly acidic pH (~5.0), which allows for the release of DOX in the
more acidic endosome environment (pH 5.0) versus systemic
circulation pH (7.4). Briefly, the surface of Fe3O4@SiO2(FITC)-BTN/
NH2, Fe3O4@SiO2(FITC)-FA/NH2, and Fe3O4@SiO2(FITC)-BTN/FA/
NH2 nanoparticles (10 mg) was activated in 20 mL 1.0% glutar-
aldehyde solution under vigorous mechanical stirring at room
temperature for 1 h. Then, nanoparticles were collected via
centrifugation, and the unreacted glutaraldehyde was removed
by extensive washing with ultrapure water. Glutaraldehyde-
activated nanoparticles were subsequently incubated with DOX
Material and Methods
Materials
Iron (II) chloride tetrahydrate (FeCl2 4H2O) (99%), iron (III) chloride
hexahydrate (FeCl3 6H2O) (98%), tetraethyl orthosilicate 99.9%, fluo-
rescein
isothiocyanate
(FITC),
biotin
(BTN),
FA,
3-
aminopropyltriethoxysilane (APTES), N,N-dicyclohexyl-carbodiimide,
N-hydroxysuccinimide, glutaraldehyde 25% aqueous solution, FT-IR
grade potassium bromide ꢀ99% (KBr), dimethyl sulfoxide (DMSO),
Triton X-100, 3-(4,5-dimethyl-2-thialzolyl)-2,5-diphenyltetrazolium
bromide (MTT), and trypsin were purchased from Sigma-Aldrich
Chemicals. Doxorubicin was obtained from SABA Pharma. Oleic acid
(99%), ammonium hydroxide 25% aqueous solution,1-hexanol (>98%),
€
cyclohexane, and toluene were purchased from Fluka/Riedel-de Haen
Chemicals. 7-aminoactinomycin (7-ADD) and PE-annexin-V were
purchased from BD Biosciences. Dulbecco's Modified Eagle Medium
growth medium, 10% fetal bovine serum, streptomycin, penicillin, and
L-glutamic acid were purchased from Gibco Life Technologies. All other
complex (10
mM) in 20 mL phosphate-buffered saline (PBS) so-
chemicals and reagents were of the highest purity. All the experiments
were performed in deionized Milli-Q water.
lution (pH 7.4) under vigorous mechanical stirring at room tem-
perature for 6 h. The amount of bound DOX was calculated from
the difference between the amount of DOX introduced into the
coupling reaction mixture and the amount of DOX present in the
washing water after immobilization by measuring DOX absor-
bance at 480 nm. The resulting nanoparticles: (1) Fe3O4@SiO2(-
Cell Cultures
HeLa (human epithelial cervical carcinoma) (93021013) cell line
was kindly provided by the Biotechnology and Bioengineering
Research and Application Centre, Izmir Institute of Technology,
Turkey. The cancer cells were cultured in Dulbecco's Modified Eagle
FITC)-BTN/DOX;
(2)
Fe3O4@SiO2(FITC)-FA/DOX;
and
(3)
Fe3O4@SiO2(FITC)-BTN/FA/DOX were magnetically separated and
washed with 1% DMSO in PBS several times to remove any
unreacted reactants and dried under vacuum at room tempera-
ture, overnight.
Medium supplemented with 10% (v/v) fetal bovine serum, 100
mg/
mL streptomycin, 100 U/mL penicillin, and 2 mM -glutamic acid.
L
The cell line was incubated in 5% CO2 and 90%-100% relative hu-
midity at 37ꢁC. Medium renewal was carried out 2 to 3 times per
week, and cells were subcultured when they achieved 80%-90%
confluence. The cell line was discarded after 20 generations, and
new line was obtained from frozen stocks.
Structural and Physicochemical Characterization
Dynamic light scattering (DLS) measurements were performed
at 25ꢁC, using a Malvern Zetasizer Nano ZS compact scattering
spectrometer. Average hydrodynamic diameters, size distributions,
and surface charge analysis of the samples were determined using
Malvern Dispersion Technology Software 7.11. Nanoparticles were
suspended in ultrapure water to give optimum signal intensity. All
measurements were repeated 5 times to verify the reproducibility
of the results.
Synthesis of Multifunctional Nanoparticles
The parental Fe3O4@SiO2(FITC) nanoparticles were synthesized
as described in our previous work.9 They consist of super-
paramagnetic iron oxide nanoparticles (as magnetic contrast
agent), coated with layers of silica shells, encasing FITC within (as
optical contrast agent), for imaging, biocompatibility, and molec-
ular functionalization. To ensure preferential tumor cell uptake of
the nanoparticles, the outermost layer of Fe3O4@SiO2(FITC) was
then functionalized with BTN and/or FA molecules by silanization
with BTN-APTES and/or FA-APTES conjugate. In brief, an APTES
ester of BTN and/or FA (BTN-APTES/FA-APTES) was prepared by
Powder X-ray diffraction (XRD) measurements were per-
formed with “Philips X’Pert Pro,” at room temperature by using
CuK
a
radiation (
l
¼ 1.5405 Å) and BraggeBrentano
q
/2
q
config-
uration. The measurements were performed over the 2
q range of
20-70ꢁ.
The FTIR spectroscopy spectra of the nanoparticles were
collected with a “PerkinElmer Spectrum-100” spectrophotometer
in the range 450-4000 cmꢂ1. The spectra of the dried samples were
obtained by employing a KBr pellet.
mixing biotin (8.0 mg) and/or folate (4.0 mg) with APTES (2.0 mL)
in 40 mL dry DMSO in the presence of N-hydroxysuccinimide
(1.1 mg) and N,N-dicyclohexyl-carbodiimide (4.7 mg) as the

C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
3
were seeded in 12-well plates overnight to allow the adhesion of
the cells before experiments. Next, the nanoparticle formulations
were added to the incubation medium at a concentration of
10
mg/mL for 4 and 24 h of incubation to investigate time-
dependent uptake and accumulation in 5% CO2 at 37ꢁC. After
incubation, all cells were washed thrice with PBS and later micro-
scopic images in the green channel, for detection of the FITC label
encapsulated in nanoparticles, and in the bright-field were ob-
tained by fluorescence microscopy. Internalization of the nano-
particles was visualized using an Olympus IX2-ILL100 fluorescence
microscope equipped with an appropriate filter set. Images were
acquired using a charge-coupled device camera and analyzed using
ImageJ advanced version software. For cellular uptake quantitative
analysis, the cells (2 ꢃ 105 cells/well) were seeded in 6-well plates
overnight before experiments. The medium from each well was
discarded, and cells were treated as described previously. The
samples were trypsinized and harvested to obtain a cell suspen-
sion, which was then analyzed for the distribution of FITC fluo-
rescence by flow cytometry.
Figure 1. Schematic representation of Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles.
*The core-shell structure of the particle (white core-black shell) was obtained from
STEM.
Scanning electron microscopy (SEM) studies were performed
with a high-resolution environmental scanning electron microscope
(FEI Quanta 250 FEG) equipped with a Schottky field emission gun
(FEG) for optimal spatial resolution. Before examination, the lyoph-
ilized nanoparticles were placed on a double stick tape over
aluminum stubs to get a uniform layer of particles.
Scanning transmission electron microscopy (STEM) images of
the nanoparticles were obtained with a “FEI Quanta 250 FEG” mi-
croscope operating with STEM detector. The nanoparticles were
dispersed in water under sonication, and a drop was placed on a
carbon-coated 400 mesh copper grid followed by air drying.
Cytotoxicity of Multifunctional Nanoparticles
The cytotoxicity of the nanoparticle formulations was evaluated
by MTTassay. HeLa cells were seeded into 96-well plates at a density
of 1 ꢃ 104 per well and grown overnight. The cells were then
incubated with increasing concentrations (0.1/0.5/1.0/10/50/100/
200 mg/mL) of nanoparticles and free DOX (1.0/10/100/500/1000
nM) for 48 h at 37ꢁC under 5% CO2. Following this incubation, cells
were incubated in medium containing 0.5 mg/mL of MTT for 4 h. The
medium was discarded, and the precipitated formazan violet crys-
In Vitro Release Studies
tals were dissolved in 150 mL DMSO to solubilize the formazan. After
shaking the plate for 10 min, the absorbance of the sample was
measured at 570 nm by multidetection microplate reader. The
absorbance of dissolved formazan in the visible region correlates
with the number of viable cells. The percentage of viable cells was
calculated according to the following equation:
The release of drugs from the nanoparticle formulation was
examined by the dialysis bag diffusion method. The aqueous sus-
pensions of Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles (1 mg/
mL) were placed into dialysis bag (molecular weight cutoff 3500
Da) and suspended in 25 mL of 1% DMSO in PBS with pH 5.0, 6.0, 7.0,
or 7.4 at 37ꢁC under gentle magnetic stirring, respectively. Then, 1
mL of the each release medium was withdrawn at predetermined
time points and replaced with 1 mL of fresh buffer solution of
relevant pH. The amount of DOX in the withdrawn samples was
determined using UV-Vis spectrometry at 480 nm. All experimental
procedures were repeated 3 times to obtain the average value. The
cumulative release of DOX was calculated according to the
following equation:
ꢀ
.
Cell viability ð%Þ ¼ Mnanoparticles=free drugs ꢂ Mblank
ꢁ
Mcontrol ꢂ Mblank ꢃ 100
where Mnanoparticles/free drugs is the absorbance of the cells, growth
medium, and nanoparticles or free drugs, Mcontrol is the absorbance
of the cell and growth medium, and Mblank is the absorbance of the
The cumulative release of DOX ð%Þ ¼ ðmass of released DOX= total mass of conjugated DOXÞ ꢃ 100
Cellular Uptake Analysis
growth medium alone. Cytotoxicity was evaluated with reference
to the IC50 value that was defined as the concentration of com-
pound causing death in 50% of cells. IC50 values were calculated by
GraphPad Prism 7.0 software using sigmoidal dose-response anal-
ysis obtained in multireplicate experiments.
The cellular uptake behavior of the nanoparticle formulations
was investigated using fluorescence microscopy and flow cytom-
etry. For microscopic observation, HeLa cells (1 ꢃ 105 cells/well)
Table 1
Immobilization Yields, Size, and Zeta Potential of the Nanoparticle Formulations
Nanoparticle Formulations
DOX Immobilization Yield (%)
DOX Immobilization Concentration
M per mg Nanoparticles)
Size (nm)
Zeta Potential (mV)
(
m
Fe3O4@SiO2(FITC)-BTN/NH2
Fe3O4@SiO2(FITC)- FA/NH2
Fe3O4@SiO2(FITC)- BTN/FA/NH2
Fe3O4@SiO2(FITC)-BTN/FA/DOX
91.8
88.3
79.5
e
0.92
0.88
0.79
e
37.5 6.1
43.7 7.7
47.3 8.2
52.8 9.3
þ7.1 2.8
þ5.3 2.3
ꢂ1.7 1.5
ꢂ17.5 2.5

4
C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
Figure 2. Size distributions and zeta potential measurements of (a) Fe3O4@SiO2(FITC)-BTN/NH2; (b) Fe3O4@SiO2(FITC)-FA/NH2; (c) Fe3O4@SiO2(FITC)-BTN/FA/NH2; and (d)
Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles obtained at pH ¼ 7.4 using DLS.
Detection of Apoptotic Cells by Flow Cytometry
The stained cells were first incubated for 15 min at room tem-
perature in the dark, and then analyzed by flow cytometry. The
untreated cells incubated with medium alone were used as the
control. Unstained cells, cells stained with PE-annexin-V alone,
and cells stained with 7-AAD alone were used to set up
compensation and quadrants. Flow cytometric analysis was per-
formed on a FACS (Facscanto; Becton Dickinson, San Jose, CA) by
counting 10,000 events.
The percentage of cells undergoing apoptosis induced by the
nanoparticle formulations was measured using flow cytometry
with 7-ADD and PE-annexin-V double staining. HeLa cells (1 ꢃ 105
cells/well) were seeded in 6-well plates with 2 mL of growth
medium overnight before experiments. Fe3O4@SiO2(FITC)-BTN/
DOX, Fe3O4@SiO2(FITC)-FA/DOX, or Fe3O4@SiO2(FITC)-BTN/FA/DOX
nanoparticles were added into the incubation medium separately
at IC50 concentrations and then the cells were incubated for 4 h in
5% CO2 at 37 ꢁC to allow uptake of the nanoparticles. Before
analysis, the cells were carefully washed with cold PBS, digested
with trypsin, and collected by centrifugation. The cells were
Statistical Analysis
All data were represented as means
SD. Statistical
analysis was performed with the Student t-test, using Excel
Software (Microsoft). A p value of ꢄ0.05 was considered statisti-
cally significant.
washed twice with cold PBS, resuspended in 200
mL of annexin
binding buffer, and stained with 10 L of 7-ADD and PE-annexin-V.
m
Figure 3. (a) Powder XRD patterns of Fe3O4 and Fe3O4@SiO2(FITC) nanoparticles. The position of the broad peak of amorphous silica is marked with an asterisk. (b) FTIR char-
acterizations of Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles. (c) SEM (left panel) and STEM (right panel) images of Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles. (d) Cumulative
percentages of released DOX under pH 7.4, 7.0, 6.0, and 5.0 over time from Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles.

C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
5
Figure 4. (a) Fluorescence microscopy images and (b) cellular uptake quantitative analyses of HeLa cells after incubation with (a) Fe3O4@SiO2(FITC)-BTN/DOX; (b)
Fe3O4@SiO2(FITC)-FA/DOX; and (c) Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles for 4 and 24 h. For columns: 1, bright-field images; 2, fluorescence images; and 3, the merger of
both. The histogram is a representative of n ¼ 3.
Results and Discussion
for
Fe3O4@SiO2(FITC)-BTN/NH2,
Fe3O4@SiO2(FITC)-FA/NH2,
Fe3O4@SiO2(FITC)-BTN/FA/NH2, and Fe3O4@SiO2(FITC)-BTN/FA/
DOX nanoparticles, respectively, with narrow size distribution
(Fig. 2). Particles with a diameter ranging from 10 to 100 nm are
optimal to prevent the uptake of nanoparticles by the reticuloen-
dothelial system, which is responsible for the clearance of nano-
particles and have the most prolonged blood circulation times.13
These long-circulating nanoparticles can efficiently accumulate in
tumors cells due to their dual-targeting function and thus, deliver
their therapeutic payload to cancer cells.
The stepwise conjugation of functional groups on the silica sur-
face was monitored by measuring the surface charges at different
stages of synthesis (Fig. 2 and Table 1). Fe3O4@SiO2(FITC)-BTN/NH2
and Fe3O4@SiO2(FITC)-FA/NH2 nanoparticles exhibited a positive
Synthesis and Characterization of Multifunctional Nanoparticles
In this study, The Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles
consisted of silica-coated luminomagnetic nanomaterials with both
fluorescent and magneticproperties, tripleconjugatedto BTN, FA, and
DOX. To increase the cellular uptake and accumulation, nanoparticles
were covectorized with BTN and FA by silanization BTN-APTES and
FA-APTES conjugates on the silica surface, and simultaneously, the
surfaces were modified with free APTESto form an amine-terminated
overlayer for the subsequent conjugation of DOX via pH-labile Schiff-
base formation for environmentally responsive drug release (Fig. 1).
The quantitative study of DOX conjugation on the silica
surface showed that 0.79
Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles, while coupling yields
of 0.92 М of DOX per mg Fe3O4@SiO2(FITC)-BTN/DOX and 0.88 M of
DOX per mg Fe3O4@SiO2(FITC)-FA/DOX were obtained (Table 1).
The average hydrodynamic diameter of the nanoparticle for-
mulations were 37.5 6.1, 43.7 7.7, 47.3 8.2, and 52.8 9.3 nm
m
M of DOX were conjugated per mg
zeta potential (
z
) value of þ7.1 and þ5.3 (pH 7.4), respectively, due to
presence of excess positively charged amino groups, which nullify
the BTN and FA charge and make the overall surface charge positive.
Modification of the surface by coconjugating BTN and FA groups
reverted the surface charge back to slightly negative value, for
Fe3O4@SiO2(FITC)-BTN/FA/NH2 nanoparticles with average values
m
m

6
C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
Figure 5. Anticancer activities of free DOX, and Fe3O4@SiO2(FITC)-BTN/DOX, Fe3O4@SiO2(FITC)-FA/DOX, and Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles against HeLa cells after
48 h of growth. The results are expressed as percentage of cell viability or cell number obtained in the untreated controls. The nanoparticles concentrations are expressed as
nanoparticles per mL. Each column represents the mean SD of 3 independent experiments performed in triplicate normalized to nontreated cells (taken as 100%).
mg
of ꢂ1.7 mV obtained at pH ¼ 7.4, could be attributed to a function of
the ionizability of both groups on silica surface. Further surface
functionalization of the nanoparticles by conjugating DOX led to an
obvious charge reversion to ꢂ17.5 mV values (pH ¼ 7.4) for
the physiological pH in the blood circulation and the acidic pH in
endosome of tumor cells, and at pH 7.0 and 6.0 to further charac-
terize the pH-responsive nature of the nanoparticles. As shown in
Figure 3d, most of the drug was released from the nanoparticle
formulations within a few hours at pH 5.0, while the nanoparticles
showed no more than 16% of the conjugated drug release at pH 7.4
for 24 h. During the first 4 h under pH 5.0, the release of DOX from
the nanoparticles showed release percentages of 67%, after 24 h, the
accumulated release rates of DOX increased to 77%. Remarkable
differences were observed between the release at pH 7.0 and that at
pH 6.0. As expected, drug release was accelerated at pH 6.0 when
compared with that at pH 7.0 (Fig. 3d). Since DOX was conjugated
with pH-labile Schiff-base formation, this acid-sensitive linkage
was stable at pH 7.4 but broken at pH 5.0, led to the release of DOX
efficiently. This meant that the nanoparticles are able to control
drug release after internalization and improving the accumulation
of anticancer drug at the target site to achieve the effective thera-
peutic concentration.
Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles, attributable to
function of the high ionizability of DOX on silica surface.
a
The bulk crystal structure of the nanoparticles was analyzed by
XRD using the Debye-Scherrer method. For Fe3O4 magnetic particles,
XRD spectra showed the 6 characteristic peaks occurring at 2q of 30.1,
35.5, 43.1, 53.4, 57.0, and 62.6, which are marked by their corre-
sponding indices (220), (311), (400), (422), (511), and (440), respec-
tively (Fig. 3a). These results were in excellent agreement with the
Joint Committee on Powder Diffraction Standards file for Fe3O4 (card
no. #19-629). This analysis revealed that the prepared magnetic
particles are pure Fe3O4 with a cubic spinel structure. For Fe3O4@-
SiO2(FITC) nanoparticles, the same 6 characteristic peaks corre-
sponding to uncoated Fe3O4 were masked and a broad peak ranging
from 20ꢁ to 30ꢁ confirmed the presence of an amorphous silica shell
surrounding the Fe3O4 magnetic core.9 This indicated that the silica
coating did not cause a phase change in the magnetic particles.
Therefore, the magnetic particles could preserve their magnetic
properties for further applications.
The surface conjugation of Fe3O4@SiO2(FITC)-BTN/FA/DOX
nanoparticles with BTN, FA, and DOX were confirmed by FTIR
spectroscopy. The appearance of the absorption peak at 1220 cmꢂ1
in the FTIR spectrum confirms the existence of the biotin moieties.8
The band at 1601 cmꢂ1 corresponding to the stretching vibrations
of C¼C in the backbone of the aromatic ring present in folic acid. On
the other hand, the band at 1568 cmꢂ1 is attributed to the
stretching vibration of 2 carbonyl groups of the anthracene ring of
DOX,9 indicating that DOX were successfully attached to the surface
of the Fe3O4@SiO2(FITC)-BTN/FA/NH2 nanoparticles (Fig. 3b).
SEM and STEM images show that Fe3O4@SiO2(FITC)-BTN/FA/
DOX nanoparticles have core-shell morphology and nearly spher-
ical shape with a narrow size distribution (Fig. 3c). The thickness of
the shell of the nanoparticles in dry state was about 50 nm which is
almost in accordance to those found by DLS, indicating that the
coated nanoparticles are stable and well dispersed in solution
without aggregation or accumulation.
Cellular Uptake
The endocytotic uptake and accumulation of Fe3O4@SiO2(FITC)-
BTN/DOX, Fe3O4@SiO2(FITC)-FA/DOX, and Fe3O4@SiO2(FITC)-BTN/
FA/DOX nanoparticles in HeLa cells were visualized by fluorescence
microscopy and flow cytometry (Figs. 4a and b). The good cellular
localizations of all nanoparticle vectors, obtained by comparing the
fluorescence images with the corresponding bright-field images,
confirmed the persistent intracellular accumulation of the nano-
particles even after 24 h. The fluorescence intensities in the cyto-
plasm
revealed
that
the
Fe3O4@SiO2(FITC)-BTN/FA/DOX
nanoparticles (93.4% for 4 h; 82.2% for 24 h) with both BTN and FA
Table 2
The IC50 Values of the Nanoparticles and Free DOX in HeLa Cells When Incubated
for 48 h
Formulations
IC50
Fe3O4@SiO2(FITC)-BTN/DOX
36.3 5.8 mg/mL
(containing ~28.7 nM conjugated DOX)
47.8 5.4 g/mL
(containing ~37.7 nM conjugated DOX)
23.8 2.1 g/mL
(containing ~18.8 nM conjugated DOX)
342.6 12.5 nM
Fe3O4@SiO2(FITC)-FA/DOX
Fe3O4@SiO2(FITC)-BTN/FA/DOX
Free DOX
m
In Vitro pH-Stimuli Release Study
m
The ability of Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles to
release the conjugated DOX was tested at pH 7.4 and 5.0, mimicking
The values represent the mean SD of 3 independent experiments.

C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
7
Figure 6. (a) Representative images of apoptosis induction by Fe3O4@SiO2(FITC)-BTN/DOX, Fe3O4@SiO2(FITC)-FA/DOX, and Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles in HeLa
cells, measured by flow cytometry. PE-annexin-V/7-ADD double staining of cancer cells treated with IC50 concentration of the nanoparticles. Viable cells labeled with PE-annexin-
V(ꢂ)/7-ADD(ꢂ), early apoptotic cells labeled with PE-annexin-V(þ)/7-ADD(ꢂ), and apoptotic cells labeled with PE-annexin-V(þ)/7-ADD(þ) in flow cytometric graphics. (b) The
phase composition percentage of HeLa cells treated with the nanoparticles. Nontreated cells were used as control. Each column represents the mean
SD of 3 independent
experiments performed in triplicate (n ¼ 3).
targeting ligands show considerably stability and accumulation as
compared to Fe3O4@SiO2(FITC)-BTN/DOX (81.9% for 4 h; 75.7% for
24 h) and Fe3O4@SiO2(FITC)-FA/DOX (78.6% for 4 h; 69.2% for 24 h)
nanoparticles due to the dual drug uptake mechanisms that effec-
tively evaded drug efflux pumps. Consistent with this result, it has
been suggested that the cellular uptake of nanoparticles by means
of receptor-mediated endocytosis leads to the greater drug accu-
mulation due to the avoidance of the drug efflux pump
mechanism.14 Therefore, dual-targeting feature could be of poten-
tial value for increasing the accumulation level of the nanoparticles,
which could minimize anticancer drug expulsion, and thus improve
the anticancer efficacy.
For further cytotoxicity studies, the cells were exposed to
increasing concentrations of freeDOX to determinethe IC50 value. As
shownin Figure5 andTable 2, the percentage ofcell survivalreduced
with increasing DOX dose, however, resulted in 11.9-, 9.1-, and 18.2-
fold increased IC50 values as compared to Fe3O4@SiO2(FITC)-BTN/
DOX, Fe3O4@SiO2(FITC)-FA/DOX, and Fe3O4@SiO2(FITC)-BTN/FA/
DOX nanoparticles, respectively. The IC50 values showed that
Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles (containing approxi-
mately 18.8 nM conjugated DOX) offer 5.3 times higher cytotoxic
potential (lower IC50) than 100 nM free DOX, representing the mean
plasma concentration of doxorubicin in patients under chemo-
therapy,15 which would be significant in a clinical context. DOX is a
common chemotherapeutic used in the treatment of a wide range of
cancers, but the development of resistance by tumor cells limits its
successful treatment.16 Thus, the fast endosomal accumulation and
drug release functions within a single platform may able to over-
come these limitations. These data corroborated that
Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles with enhanced
cellular uptake and drug releaseprofiles could evade an expulsion by
efflux pumps and lead to easier reaching of anticancer drug to the
effective therapeutic concentration in cancer cells.
Cytotoxicity of the Nanoparticle Formulations
The cell inhibitory effect of the nanoparticle formulations was
examined using MTT assay to investigate whether coconjugation of
2 active targeting ligands on the surface of the nanoparticles would
be effective at inhibiting tumor cell proliferation. Cancer cells were
exposed to nanoparticles with increasing concentrations of nano-
particles (0.1-200
mg/mL) and free DOX (1.0-1000 nM) for 48 h.
Among all nanoparticles tested, Fe3O4@SiO2(FITC)-BTN/FA/DOX
nanoparticles showed stronger cell inhibitory effects with
approximately 1.5- and 2.0-fold lower IC50 value as compared to
Fe3O4@SiO2(FITC)-BTN/DOX and Fe3O4@SiO2(FITC)-FA/DOX nano-
particles, respectively (Fig. 5 and Table 2). The IC50 value of
Fe3O4@SiO2(FITC)-BTN/DOX nanoparticles was relatively lower
than Fe3O4@SiO2(FITC)-FA/DOX nanoparticles, demonstrating that
the cellular uptake capacity of BTN is higher than FA in HeLa cells.
Importantly, the Fe3O4@SiO2(FITC)-BTN/FA/DOX nanoparticles via
its dual-targeting function showed considerably cytotoxic activity
at low-dose nanoparticle concentrations in comparison to single-
targeting formulations which demonstrated more than 100% via-
bilities at similar concentrations. This increment was because of the
upregulation of the adenosine triphosphateebinding cassette
proteins (ABC transporters), which triggered a slight increase in the
proliferation rate of cells treated with low-dose drug due to
increased metabolism of the cell,9 confirming the potential of dual-
targeting approach that simultaneously target multiple receptors
on cancer cells resulting in robust antitumor activity due to
increased drug accumulation.
Detection of Apoptotic Cells by Flow Cytometry
To examine whether coconjugation of 2 active targeting ligands
on the surface of the nanoparticles would be effective at increasing
the apoptotic rates, the percentage of cells undergoing apoptosis
was analyzed by flow cytometry with 7-ADD and PE-annexin-V
double staining. The treatment with Fe3O4@SiO2(FITC)-BTN/FA/
DOX nanoparticles increased the number of apoptotic cells by
nearly 12.5% as compared to the other nanoparticles, suggesting
that dual-targeting function enhanced drug accumulation in cancer
cells which then resulted in rapid induction of apoptosis. On the
other hand, Fe3O4@SiO2(FITC)-BTN/DOX and Fe3O4@SiO2(FITC)-FA/
DOX nanoparticles showed similar apoptotic effect with total
apoptotic percentages of 63.8% and 63.6%, respectively (Fig. 6).
These results demonstrated that Fe3O4@SiO2(FITC)-BTN/FA/DOX
nanoparticles clearly advanced drug accumulation in cancer cells
and led to an increase in the rate of total apoptosis that would be
significant against resistance development.

8
C. Daglioglu / Journal of Pharmaceutical Sciences xxx (2017) 1-8
Conclusion
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