Synthesis of silver nanoparticles for the dual delivery of doxorubicin and alendronate to cancer cells

Article abstract of DOI:10.1039/c5tb00994d

We present the synthesis of a silver nanoparticle (AgNP) based drug-delivery system that achieves the simultaneous intracellular delivery of doxorubicin (Dox) and alendronate (Ald) and improves the anticancer therapeutic indices of both drugs. Water, under microwave irradiation, was used as the sole reducing agent in the size-controlled, bisphosphonate-mediated synthesis of stabilized AgNPs. AgNPs were coated with the bisphosphonate Ald, which templated nanoparticle formation and served as a site for drug attachment. The unreacted primary ammonium group of Ald remained free and was subsequently functionalized with either Rhodamine B (RhB), through amide formation, or Dox, through imine formation. The RhB-conjugated NPs (RhB-Ald@AgNPs) were studied in HeLa cell culture. Experiments involving the selective inhibition of cell membrane receptors were monitored by confocal fluorescence microscopy and established that macropinocytosis and clathrin-mediated endocytosis were the main mechanisms of cellular uptake. The imine linker of the Dox-modified nanoparticles (Dox-Ald@AgNPs) was exploited for acid-mediated intracellular release of Dox. We found that Dox-Ald@AgNPs had significantly greater anti-cancer activity in vitro than either Ald or Dox alone. Ald@AgNPs can accommodate the attachment of other drugs as well as targeting agents and therefore constitute a general platform for drug delivery.

Full text of DOI:10.1039/c5tb00994d

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Journal of Material Chemistry B
ARTICLE
Synthesis of Silver Nanoparticles for the Dual Delivery of
Doxorubicin and Alendronate to Cancer Cells
a
a
b
a
a
b
c
d
F. Benyettou, R. Rezgui, F. Ravaux, T. Jaber, K. Blumer, M. Jouiad, L. Motte, J.-C. Olsen, C.
e
a
a
Received 00th January 20xx,
Accepted 00th January 20xx
Platas-Iglesias, M. Magzoub, and A. Trabolsi *
We present the synthesis of a silver nanoparticle (AgNP) based drug-delivery system that achieves the simultaneous
intracellular delivery of doxorubicin (Dox) and alendronate (Ald) and improves the anticancer therapeutic indices of both
drugs. Water, under microwave irradiation, was used as the sole reducing agent in the size-controlled, bisphosphonate-
mediated synthesis of stabilized AgNPs. The AgNPs were coated with the bisphosphonate Ald, which templated
nanoparticle formation and served as a site for drug attachment. The unreacted primary ammonium group of Ald
remained free and was subsequently functionalized with either Rhodamine B (RhB), through amide formation, or Dox,
through imine formation. The RhB-conjugated NPs (RhB-Ald@AgNPs) were studied in HeLa cell culture. Experiments
involving the selective inhibition of cell membrane receptors were monitored by confocal fluorescence microscopy and
established that macropinocytosis and clathrin-mediated endocytosis were the main mechanisms of cellular uptake. The
imine linker of the Dox-modified nanoparticles (Dox-Ald@AgNPs) was exploited for acid-mediated intracellular release of
Dox. We found that the Dox-Ald@AgNPs had significantly greater anti-cancer activity in vitro than either Ald or Dox alone.
The Ald@AgNPs can accommodate the attachment of other drugs as well as targeting agents and therefore constitute a
general platform for drug delivery.
DOI: 10.1039/x0xx00000x
www.rsc.org/
vehicle that could facilitate simultaneous intracellular uptake of Dox
and other drugs could lead to a highly effective cancer therapy.
1
. Introduction
Cytotoxic drugs are used in cancer therapy to inhibit tumor growth Silver nanoparticles (AgNPs) have been used as effective drug
1
and metastasis. One of the most prescribed anticancer drugs is delivery agents and have attracted special attention because of
5
doxorubicin (Dox), which acts by intercalating DNA and inhibiting their intrinsic anticancer activity. Various strategies have been
macromolecular biosynthesis. However, Dox has limited cellular developed for the preparation of biocompatible AgNPs, including
2
6
7
8
uptake and a strong tendency to bind off-target macromolecules,
micro-emulsion, organic-water two-phase synthesis, radiolysis,
9
These drawbacks lead to low therapeutic indices and necessitate and, the most common protocol, reduction in aqueous solution.
the use of large doses and repeated administration to achieve Despite its eco-friendly potential, the latter method uses an excess
1
0
11
therapeutic efficacy. Unfortunately, such intensive treatment of reducing agent, such as citrate or sodium borohydride, which
12
regimes cause severe side effects. One way to minimize the can be difficult to remove and/or cytotoxic. Consequently, the
required dosage of Dox and to limit the severity of its side effects is development of AgNP syntheses that are both “green” and non-
to improve its cellular uptake. Another strategy is to administer Dox toxic remains an open challenge.
3
with drugs that act synergistically. For example, Dox is significantly
Here, we present a simple synthetic route to AgNPs that effectively
more effective against breast cancer when combined with
deliver a synergistic combination of Dox and Ald to HeLa cells
3
,4
alendronate (Ald), a potent bisphosphonate. Furthermore, the
(
Scheme 1). Our AgNPs have a coating of Ald molecules that are
use of Dox as part of a synergistic drug combination has the
attached by chelating bisphosphonate moieties. This mode of Ald
binding leaves the ammonium group of the drug free in solution
and is supported by Fourier transform infrared (FTIR) spectroscopy
as well as density functional theory (DFT) calculations. The
ammonium group serves as an attachment site for either
Rhodamine B (RhB), a hydrophilic, drug-like molecule, or Dox. RhB is
attached by amide formation, and Dox, by imine formation. Both
types of bond are pH sensitive and allow for intracellular release of
RhB or Dox within the acidic microenvironment of late endosomes
and lysosomes.
4
potential to delay the onset of Dox resistance. Thus, a delivery
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To assess the efficacy of the system, we studied the in vitro cellular spherical nanoparticles of 11 nm diameter. The particles are highly
uptake of RhB-conjugated AgNPs (RhB-Ald@AgNPs) in HeLa cells monodispersed and uniformly distributed as a result of fast and
and tracked the particles by monitoring RhB fluorescence. We then uniform microwave heating. High-resolution TEM (Fig. 1C and
determined the toxicity of Dox-Ald@AgNPs in HeLa cells and Supporting Information) confirmed the size of the particles and
quantified the synergistic effect of the Dox-Ald drug combination.
indicated that they are crystalline with the typical face-centered
cubic structure of bulk silver.
Fig. 1. Characterization of Ald@AgNP samples synthesized under
MW irradiation and with [Ag] = 0.85 M, [Ald] = 0.15 M, t = 15 min,
pH = 4 and at 70 C°. (A) Absorption spectrum, (B) SEM and (C) high-
resolution TEM of Ald@AgNPs. (Inset of C: Fourier transform of
particle along the (110) zone axis of face-centered cubic silver).
We measured the hydrodynamic size and the surface charge of the
particles by using DLS. Both size and charge are good indicators of
16
the aggregation state of NPs. The hydrodynamic diameter at
physiological pH (7.4) was found to be 20 nm, which is suitable for
1
7
intracellular delivery applications. The ζ-potential was found to be
37 mV as a consequence, in part, of the negatively charged state
–
of Ald at this pH. The negative ζ-potential stabilizes the NPs by
creating inter-particle electrostatic repulsions that prevent
aggregation. This type of stability is a key feature. To avoid
thrombosis upon intravenous injection the nanoparticles must not
aggregate.
Scheme 1. (A) Schematic representation of Ald@AgNP formation
and dye/drug conjugation. (B) Schematic representation of the
uptake of drug-Ald@AgNPs into cells and drug cargo release.
2
. Results and discussion
2.2. Surface characterization of Ald@AgNPs
3
1
2
.1. Synthesis and characterization of Ald@AgNPs
FTIR and P NMR spectroscopies, and DFT calculations were used
to determine the mode of attachment of Ald to the surface of the
NPs. Absorptions that correspond to the phosphonic acid group
Previously reported syntheses of AgNPs typically require
a
10
stoichiometric amount of reducing agent, such as citrate or
–1
1
1
appear in the 850–1300 cm fingerprint region and give an
indicatation of the group’s hydrogen-bonding and metal-binding
sodium borohydride. In this work, we used water as both solvent
and reductant. The optimized synthetic procedure involves addition
of an aqueous solution of silver nitrate (0.85 M, 1 mL) to an
aqueous solution of Ald (0.15 M, 1 mL). The solution is heated by
microwave (MW) irradiation for 15 minutes at pH 4 at a target
temperature of 70 °C. A change in color of the solution from
colorless to yellow indicates the formation of nanoparticles. The
product is dialyzed for two days to remove unreacted starting
materials.
1
8
interactions. In the FTIR spectrum of free Ald (Fig. 2A), absorption
1
9
bands consistent with phosphonic acid are present at 1234, 1172,
–
1
–1
and 1127 cm
(
ν
, P=O), at 1047 and 1011 cm
s 3
(ν , νas of PO ),
–
1
and at 913 cm
(ν, POH), and the P=O and POH absorption bands
are sharp. In contrast, in the spectrum of Ald@AgNPs (Fig. 2B) the
P=O and POH absorptions merge to form one broad band centered
–1
at 1097 cm and indicate that the phosphonate groups of Ald are
strongly surface-associated. Such broadening and shifting is
consistent with the results of previous studies that involved
hydroxyapatite and silver and established that the P=O absorption
band is quite sensitive to hydrogen bonding and metal
UV–Vis spectroscopy, scanning electron microscopy (SEM),
transmission electron microscopy (TEM), and dynamic light
scattering (DLS) confirmed the formation of Ald@AgNPs and were
employed to characterize the formation of metallic NPs in aqueous
20
coordination.
1
3,14
solution and to assess their stability.
reliable technique to authenticate the formation and stability of
UV–Vis spectroscopy is a
The spectra also indicate that the ammonium group of Ald is largely
unaffected by adsorption of the drug. The N–H bending bands
1
5
AgNPs in aqueous solution. AgNPs usually exhibit a strong, broad
absorption peak as a result of surface plasmon resonance. Our
AgNPs showed an absorption maximum at 412 nm (Fig. 1A), which
corresponds to a surface plasmon resonance typical of 11 nm NPs.
The presence of this absorption maximum is also strong evidence
for lack of NP aggregation. A SEM micrograph (Fig. 1B) shows
–1
appear at 1641 and 1540 cm in the spectrum of free Ald and at
–
1
1
636 and 1535 cm in the spectrum of Ald@AgNPs. These data are
consistent with a free ammonium group that projects away from
the silver surface into solution.
2
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3
1
–1
Solution
P
NMR experiments (Supporting Information) frequency calculations (half width at half height 25 cm ) is
–1
demonstrated that all Ald molecules on the surface of the dominated by two absorption maxima at 1001 and 1181 cm for
nanoparticles are chemically equivalent: only one phosphorus P-O stretching vibrations that are similar to the experimental values
–1
–1
resonance, which appears at 19.2 ppm, is present in the of 978 and 1097 cm . The band calculated at 688 cm that arises
Ald@AgNPs spectrum. For free Ald, the resonance appears at 17.7 from C-O-H and P-O-H bending vibrations (experimental value 669
–
1
+
ppm.
cm ), and the bending vibrations of the -NH
3
group calculated at
–1
1
675 and 1533 cm are also close to the experimental values (1636
–1
and 1535 cm ). These in silico results must be considered carefully
because of the complexity of the actual system and the simplicity of
the model used. However, the overall good agreement between the
observed and calculated IR spectrum in terms of shape and position
of the main absorption bands suggests that Ald interacts with the
AgNPs surface through the negatively charged bisphosphonate unit.
Calculations performed on a model system that contained an amine
group bound to Ag provide poor agreement with the experimental
spectrum.
Fig. 2. Comparison of FTIR spectra of (A) free Ald and (B)
Ald@AgNPs.
To gain greater insight about the interactions between AgNPs and
Ald, we performed DFT calculations by using the hybrid meta
generalized gradient approximation (hybrid meta-GGA) with the
2
1
TPSSh exchange-correlation functional. In these calculations (Fig.
) we used the standard 6-311G(d,p) basis set for C, H, N, O, and P
atoms, and Ag was described by using the ECP28MWB relativistic
3
2
2
pseudopotential of Dolg et al., which includes 28 electrons in the
core, together with the associated (8s7p6d2f1g)/[6s5p3d2f1g] basis
2
3
set. Bulk solvent effects (water) were included by using the
integral equation formalism variant of the polarizable continuum
model (IEFPCM). The selection of a reasonable model to gain
information on the binding mode of small molecules to
2
4
Fig. 3. Calculated FTIR spectra of (A) free Alendronate, (B) a model
system of Ald@AgNPs with the ammonium group of Ald interacting
with the AgNP surface and (C) a model system of Ald@AgNPs with
the phosphonate groups of Ald interacting with the AgNP surface.
nanoparticles is not straightforward. Several test calculations
showed that the presence of two or three Ag atoms bound to Ald
was an adequate approximation. The protonation state of Ald upon
binding to the NPs is also not known, although the negative ζ-
potential suggests the Ald moieties are negatively charged.
Different test calculations performed with different protonation
states of Ald indicated that the best agreement between
experimental and calculated IR spectra were obtained when both
the biphosphonate moiety and the amine group were
monoprotonated, which leads to an overall –2 charge for the Ald
We quantified the number of Ald molecules on the AgNP surface
using thermogravimetric analysis (TGA) which indicated
a
composition of 85.93% Ag and 9.07% Ald and which corresponds to
an average of 4115 Ald molecules per NP and 84% surface coverage
(
see the Supporting Information for TGA and calculations). Such
coverage density is in accordance with results previously reported
for the adsorption of bisphosphonate onto the surface of iron-oxide
2
–
unit. Full geometry optimization of the Ald@Ag2 system provided
a molecular structure in which each Ag atom is bound to three
oxygen atoms of the Ald moiety with Ag–O distances of 2.32–2.56
Å, and a Ag–Ag distance of 2.92 Å. The latter value is in excellent
agreement with those obtained experimentally by using EXAFS
2
6
NPs of similar diameter.
2
.3. Mechanism of Ald@AgNP formation
Ald@AgNPs are formed in our reaction mixtures despite the
absence of stoichiometric amounts of conventional reducing
25
measurements (2.88 Å). The simulated IR spectrum obtained with
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agents. In our method, we believe that microwave-irradiated water charged silver clusters, and the peak at 411 nm represents the
2
7
acts as the reducing agent. Vaks et al. have proposed that water larger AgNPs.
dissociates homolytically under the influence of 2.5–10 GHz MW
To further investigate the involvement of the Ald phosphonate
radiation according to Equation 1. They suggest that, in contrast to
conventional heating, MW irradiation produces shear and other
mechanochemical forces in hydrogen-bonded networks of water
molecules, and that these forces are necessary to produce the
detected bond cleavage:
functionality in the NP templation process, we conducted three
31
control experiments. In the first, we used a diesterified Ald, in
which the phosphonate groups are protected with phenyl groups
and the ammonium group is free. In the second experiment, we
performed the synthesis at pH 2, an acidity at which Ald has only
one negatively charged phosphonate oxygen which is tied-up by its
H
2
O ꢀ H•aq + OH•aq
(1)
3
2
strong electrostatic interaction with the ammonium group. In a
third experiment, we adjusted the pH to 4 with triethylamine rather
than NaOH. In this case, the negatively charged oxygen atoms of Ald
are sterically hindered by ammonium cations and are not free to
Among the several recombination reactions that they propose, the
one described by Equation 2 is of particular interest:
H•aq + OH−aq ꢀ (H
O)liq + е−aq
(2)
2
In this step, a highly reducing (–2.7 V) solvated electron is complex silver metal ions. In all three control experiments, no AgNP
produced. Such an electron would be capable of reducing formation was observed. Together, these results argue against the
0
+
0
28
monovalent silver (E Ag /Ag = –1.8 V). Linnert et al. have shown direct involvement of an Ald’s ammonium group in AgNP
that, under similar conditions, it is possible to reduce silver ions templation and bolster the case for the involvement of its
29
with solvated electrons generated by radiolysis. These authors bisphosphonate.
suggest that silver atoms thus formed (Equation 3) react rapidly
The experiments just described also rule-out the involvement of the
Ald ammonium group in silver(I) reduction. In order to show that
the bisphosphonate group of Ald is not involved in the reduction of
+
+
with Ag ions to form Ag
2
species (Equation 4), which subsequently
dimerize (Equation 5):
Ag + е−aq ꢀ Ag
+
0
(3)
(4)
(5)
We propose that steps similar to those described by Linnert et al.
the silver ions, we analyzed by P NMR spectroscopy crude colloid
31
0
+
+
solutions immediately after Ald@AgNP synthesis. If the
bisphosphonate were to act as a reducing agent, it would degrade
to form phosphates. However, no phosphates were detected in the
reaction mixtures. Only one phosphorus resonance, at 19.2 ppm,
Ag + Ag ꢀ Ag
2
+
2+
2
2 4
Ag ꢀ Ag
2
8
30
and Ershov et al. occur during the formation of Ald@AgNPs, in was observed, and this signal corresponds to the two equivalent
+
particular, that metallic Ag ions are reduced by solvated electrons phosphorus atoms of the chelated bisphosphonate moiety. (See the
generated from water under MW irradiation.
Supporting Information for more details.)
In our control experiments, with conventional rather than The evidence suggests that Ald acts as a “microreactor” in which
microwave heating, the color change characteristic of NP formation complexed silver ions are reduced. Silver(I) bisphosphonate
was not observed after 15 minutes at 70 °C. Furthermore, no NPs complexes act as nucleation sites in which silver clusters and NPs
were formed when Ald was absent from the reaction mixture. Both grow. The bisphosphonate group of Ald controls the growth and
MW irradiation and Ald appear to be necessary for Ald@AgNP size of the NPs and stabilizes the particles by chelation. This process
formation.
is also consistent with the FTIR data (Fig. 2) which indicates the Ald
molecules are adsorbed onto the surface of AgNPs through
chelation by the bisphosphonate group.
2
8
Linnert et al. have reported that AgNP nucleation is greatly
accelerated by polyphosphate (Pp), which increases the lifetime of
+
Ag
2
by acting as a nucleating site for the formation of the metal 2.4. Cell penetration studies
(
Equation 6). In the presence of Pp, stabilized small clusters form
To study the mechanisms of cellular uptake and cargo release, we
attached the fluorescent dye RhB to the Ald@AgNPs.
and give rise to an absorption band at 470 nm in the UV-visible
spectrum. These small clusters grow into larger ones that have
quasi-metallic properties and ultimately become AgNPs. Similarly,
2
.4.1. RhB conjugation
30
33
Ershov et al. established that upon radiation-induced reduction of Under previously described conditions, the carboxylic acid of the
+
Ag ions, the emergence of NPs is preceded by the appearance of RhB was activated using carbodiimide chemistry and reacted with
stable, positively-charged clusters. In the absence of a stabilizing the ammonium group of surface-bound Ald (Fig. 4). The reaction
additive such as Pp, clusters exist in aqueous solution for only a mixture was purified by dialysis to remove any unreacted dye from
short time (milliseconds to seconds) and do not lead to formation of the solution.
NPs.
TGA was used to estimate the mass of RhB attached to the NPs and
2
+
+
2+
PpAg
4
+nH
2
+ 2nAg ꢁPpAg2n+4
(6)
from this measurement it was determined that an average of 1054
RhB molecules were conjugated onto the surface of each particle
The UV-visible spectrum of Ald@AgNPs generated under our
standard conditions in which Ald acts as a stabilizing additive is
similar to those previously reported. Two absorption peaks (see the
Supporting Information) confirm the stepwise mechanism of AgNP
(
see the Supporting Information for calculations). With an average
of 4115 Ald molecules per particle the conjugation efficiency of RhB
was ~25%.
formation. The peak at 470 nm represents the small, positively Unlabeled Ald@AgNPs are not intrinsically fluorescent at the RhB-
specific excitation wavelength of λex = 510 nm (see the Supporting
4
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Information for details). At pH 7.4 and at λex = 510 nm, the labeled 2.4.3. Internalization pathway studies in HeLa cells
sample of Ald@AgNPs (RhB-Ald@AgNPs) fluoresced less than an
In vitro experiments with RhB-Ald@AgNPs were performed to
aqueous sample of free RhB of the same dye concentration (Fig. 4).
determine the mechanism and kinetics of NP uptake into HeLa cells,
This indicates that fluorescence quenching occurs within the RhB-
which are a commonly used model for primary and metastatic
Ald@AgNPs construct. Such quenching can be attributed to
human tumors.
electronic interactions between the excited dye molecules and the
34
2.4.3.1. Kinetics of cellular uptake
NPs, or to self-quenching of the dye on the surface of the particles
3
5
where the effective concentration of the dye is relatively high.
.4.2. pH Triggered RhB release in solution
Acidic pH is a useful trigger for the selective release of anticancer
HeLa cells were incubated with RhB-Ald@AgNPs ([Ag] = 0.1  M,
[
RhB] = 2 μM), and cellular uptake of NPs was monitored by
2
measuring the total amount of fluorescence accumulated within the
38
cells over time using confocal microscopy. Immediately following
3
6
drugs from NPs. The pH of both primary and metastasized tumors
addition of the RhB-Ald@AgNPs to the cell medium (t = “0”),
fluorescence of the dye was undetectable as a result of quenching
at the NP surface. However, once the NPs were internalized,
fluorescence increased over time and reached a plateau after 2 h
3
7
is lower than that of normal tissue. Furthermore, pH can change
in cancer cells during endocytosis, dropping to 5.0–6.0 in late
3
7
endosomes, or to 4.0–5.0 in lysosomes.
We lowered the pH of a solution of RhB-Ald@AgNPs to 5.4. As (see the Supporting Information). Uptake half-time was estimated
shown in Fig. 4, the solution’s fluorescence was dramatically to be 77 ± 3 min for RhB-Ald@AgNPs. No fluorescence was
enhanced after 24 hours relative to that of a sample that was kept observed within cells incubated with free RhB at the same
at physiological pH (7.4). Such enhancement demonstrates that concentration. These experiments demonstrate that Ald@AgNPs
release of the dye from the silver nanocarriers is pH-sensitive. can function as a vehicle for intracellular delivery of small-molecule
cargo.
Release of the RhB chromophore could be the result of i) cleavage
of the amide linkage that connects RhB and Ald or ii) dissociation of 2.4.3.2. Intracellular delivery and drug release mechanisms
3
1
the bisphosphonate of Ald from the silver surface. P NMR
spectroscopic studies indicated (Figure S5) that acidification can
cause Ald to dissociate from Ald@AgNPs. Either mechanism, amide
cleavage or bisphosphonanate dissociation, would lead to diffusion
of the RhB chromophore away from the surface of RhB-Ald@AgNPs
The mechanisms by which macromolecules enter cells can be
divided into two broad categories: i) passive mechanisms of
internalization, such as direct translocation, which are energy
39
independent, and ii) active mechanisms, such as endocytosis,
4
0
which are energy dependent. We determined the relative
and therefore to enhanced fluorescence. Both mechanisms are amounts of RhB-Ald@AgNPs internalized by passive versus active
probably occurring.
mechanisms by measuring the fluorescence intensity emitted from
cells that were incubated with NPs for 2 h at either 4 or 37 °C. At 4
°
C, all active cellular processes, including endocytosis, are inhibited.
At 4°C the fluorescence intensity emitted from incubated cells was
8% of that emitted from cells incubated at 37 °C (Fig. 5 and the
1
Supporting Information). This difference indicates that, although a
significant fraction of RhB-Ald@AgNPs is internalized by passive
mechanisms, the predominant mechanisms are active ones.
Fig. 4. Top: Schematic representation of the attachment of RhB to
Ald@AgNPs
using
coupling
agents
(EDC)
1-ethyl-3-(3-
and N-
dimethylaminopropyl)carbodiimide
hydroxysuccinimide (NHS). Bottom: Fluorescence emission spectra
of RhB-Ald@AgNPs at pH 7.4 (solid curve) and after 24 hours of
acidic hydrolysis at pH 5.4 (dashed curve). Inset: Fluorescence
Fig. 5. Mechanism(s) of cellular internalization of RhB-Ald@AgNPs
in HeLa cells. A) Effect of endocytotic inhibitors on cellular uptake of
RhB-Ald@AgNPs. B) Confocal images after 2 h incubation ([Ag] = 0.1
emission spectrum of RhB in water at room temperature with λex
10 nm.
=
ꢀ
M, [RhB] = 2
μM). The experiment at 4 °C indicates an active
5
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internalization pathway. Methyl-β-cyclodextrin (MBCD) and filipin
(
FLP) did not affect NP uptake. The inhibition of fluorescence by
cytochalsin D (CLN), chlorpromazine (CPZ) and ammonium chloride
AMCl) indicates that macropinocytosis and chlathrin-mediated
(
endocytosis are the major pathways of RhB-Ald@AgNP
internalization. Red fluorescence in the confocal images indicates
that the RhB cargo was efficiently released into lysosomes.
To determine the nature of the active entry mechanism(s) and their
relative contribution to overall cellular uptake, chemical inhibitors
for the various endocytic pathways were used (Fig. 5). The cells
were treated with various inhibitors for 30 minutes prior to addition
of NPs. The amount of fluorescence accumulated within the cells
was monitored over 2 h following incubation with the fluorescent
NPs and subsequently imaged with confocal microscopy. Fig. 5A
shows the effect of the endocytic inhibitors on the amount of
intracellular fluorescence relative to the control cells. Methyl-β-
Fig. 6. In vitro Dox release profiles for Dox-Ald@AgNPs conducted
cyclodextrin and filipin, which are inhibitors of lipid raft synthesis
and caveolin-dependent endocytosis, respectively, did not affect
uptake of NPs because the total intracellular fluorescence value was
close to that obtained for the control cells. However, addition of
over seven days at 37 °C and either pH 7.4 (red curve) or pH 5.4
(
black curve). Schematic representations show the imine linkage
that breaks under acidic conditions to release Dox.
chlorpromazine, which is an inhibitor of clathrin-mediated Dox release from Dox-Ald@AgNPs was monitored over time by
endocytosis, resulted in a decrease in the overall fluorescence measuring Dox fluorescence in solution. Two aqueous samples were
(
30%) for RhB-Ald@AgNPs, which indicates that NP internalization analyzed: one at physiological pH (7.4) and the other at a pH
occurs partly through clathrin-dependent endocytosis. Addition of typically found within late endosomes and lysosomes (5.4). Fig. 6
cytochalsin D, an inhibitor of macropinocytosis, resulted in the shows the drug release profiles of the two samples. A small amount
greatest reduction in intracellular fluorescence (60%) relative to (~9%) of Dox was released over seven days at pH 7.4, whereas up to
control cells. This observation indicates that macropinocytosis is the 95% of the drug was released at pH 5.4 over the same period.
4
1
major pathway for internalization of NPs. Greulich et al. also
showed that macropinocytosis and clathrin-dependent endocytosis
are the primary uptake mechanisms of AgNPs.
The release of Dox from the NPs resulted from acid-promoted
hydrolysis of the imine-linkage between Dox and Ald. We anticipate
that the pH-sensitive Dox-Ald@AgNPs will exhibit limited premature
Finally, cells were treated with ammonium chloride, which inhibits drug-release in the bloodstream (pH 7.4), but will effectively kill
lysosomal acidification. In the absence of acidification, intracellular cancer cells following cell internalization and Dox release within
fluorescence is 38% lower relative to the control. This decrease endocytic compartments at pH 6.5–4.5. Such a mechanism has the
indicates that release of RhB molecules from the nanocarriers potential to increase drug efficacy and limit side effects.
occurs as a result of breakage of the amide bond induced by the
Confocal laser scanning microscopy was used to determine the
late endosomes/lysosomes’ acidic (pH 5.0) microenvironment (Fig.
intracellular localization of Dox-Ald@AgNPs following uptake by
5
). Thus, following cellular internalization of the RhB-Ald@AgNPs,
endocytosis. HeLa cells were incubated with Dox-Ald@AgNPs for 3
h at 37 °C. After incubation, Dox was found to be distributed within
the cytoplasm (Fig. 7), which indicates that Dox is released from
endolysosomal compartments after endocytosis. The facile release
mainly through macropinocytosis and clathrin-mediated
endocytosis, the dye is released from the NPs in endo-lysosomal
compartments as a result of their acidic microenvironment.
2
.5. Dox conjugation, pH-triggered release and anti-proliferative of Dox into the cytoplasm is highly advantageous and makes
activity against HeLa cells
Ald@AgNPs well-suited for intracellular drug delivery.
Encouraged by the results of the RhB internalization studies, the
ability of Ald@AgNPs to deliver Dox to HeLa cells was investigated
and cell growth inhibition by the delivered drug was monitored.
Dox was attached to the surface of Ald@AgNPs by imine bond
formation. Imine bond formation between free Ald and free Dox is
supported by FTIR spectroscopy (Figure S1). TGA was used to
estimate the mass of Dox attached to the NPs and an average of
6
82 Dox molecules were calculated to be attached to each particle
see Supporting Information for calculations). With an average of
115 Ald molecules per particle, the conjugation efficiency of Dox
was ~17%.
(
4
Fig. 7. Confocal images of HeLa cells incubated for 3 h at 37 °C with
(
A) phosphate buffered saline as a control or (B) Dox-Ald@AgNPs,
6
| J. Name., 2012, 00, 1-3
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ARTICLE
which are seen distributed evenly in the cytosol and appear red as a 8B, green curve). With respect to the molar concentration of Dox,
result of Dox fluorescence ([Ag] = 0.1 ꢀM, [RhB] = 1.2 μM). Cell the anti-proliferative activity of Dox-Ald@AgNPs (Fig. 8B, blue
membranes are labeled with the green dye Alexa Fluor® 488.
curve) was 30 times greater (IC50 = 0.1 ꢀM) than that of free Dox.
Taken together, these results demonstrate the effectiveness of drug
co-delivery, which in this case significantly increased the
therapeutic indices of both Ald and Dox.
The anti-tumor effect of Dox-Ald@AgNPs on HeLa cells was also
evaluated. Inhibition of HeLa cell growth by free Ald, starch-coated
4
2
AgNPs, Ald@AgNPs or Dox-Ald@AgNPs was measured after 48 h
incubation as a function of either Ald or Dox concentration.
Two drugs are said to act synergistically when their combined effect
is greater than the sum of the effects from each drug individually.
Synergistic combinations allow for reduced drug dosing, minimized
4
4
toxicity and fewer side effects. Chou and Talalay introduced the
concept of the combination index (CI) for evaluating the
effectiveness of drug combinations. On this scale, CI > 1, CI = 1 and
CI
< 1 indicate antagonism, additive effects, and synergism,
respectively. For the Dox-Ald@AgNP system, we calculated (see the
Supporting Information for details) a CI value of 0.05, which reflects
significant synergism.
Conclusions
We have developed a technically simple method for the synthesis of
Ald@AgNPs that act as a pH-responsive dual drug-delivery system.
The synthesis relies on water, activated by microwave radiation, as
the sole reducing agent and avoids the use of cytotoxic or difficult-
to-remove reagents. Ald@AgNPs were characterized by TGA; FTIR
and UV-Vis absorption spectroscopies; fluorescence emission
spectroscopy; and ζ-potential measurements; and in SEM, PXRD,
and EDX experiments. The optimized synthetic method produces
size-controlled Ald@NPs with enhanced colloidal stability.
Ammonium groups decorate the surface of the Ald@AgNPs and
allow for attachment of functional molecules such as dyes and
drugs. Cell-permeability studies involving RhB@AgNPs showed that
the main cellular uptake routes of the particles are
macropinocytosis and clatharin-mediated endocytosis. We attached
Dox to Ald@AgNPs and studied the drug’s pH-triggered release in
HeLa cells. Dox was released in late endosomes/lysosomes and
became evenly distributed throughout the cytosol. AgNPs, Ald and
Dox are, individually, antiproliferative. In the Ald@AgNP and Dox-
Ald@AgNP systems, synergistic effects against HeLa cells were
apparent. The therapeutic effect of Ald was enhanced by a factor of
Fig. 8. (A) Inhibition of HeLa cell proliferation after 48 hours as a
function of molar concentration of Ald, where Ald was added as
free Ald (black curve), Ald@AgNPs (red curve), or Dox-Ald@AgNPs
(
blue curve). (B) Inhibition of HeLa cell proliferation after 48 h as a
5
0 by attachment to AgNPs, and that of Dox was enhanced by a
function of the molar concentration of Dox, where Dox was added
as free Dox (green curve) or Dox-Ald@AgNPs (blue curve).
factor of 30 when attached to Ald@AgNPs. Thus, the Dox-
Ald@AgNP construct could avoid the problem of low therapeutic
index, which hampers the use of many current cancer drugs.
Furthermore, as a combination therapy, Dox-Ald@AgNP could
potentially avoid, or at least delay, the onset of drug resistance. We
are continuing to develop the Dox-Ald@AgNP system as a candidate
anticancer therapeutic. We are also devising methods for attaching
other drugs as well as cell-targeting agents to the Ald@AgNP
delivery platform.
As expected, free Ald exhibited partial inhibition of cancer cell
growth, with a maximum inhibition of 47% at 500 ꢀM (Fig. 8A, black
curve). With respect to the molar concentration of Ald, Ald@AgNPs
(
IC50 = 10.1 ꢀM) were significantly more toxic (Fig. 8A, red curve).
This increase might be due, in part, to the greater lipophilicity and
consequent cellular uptake of Ald@AgNPs relative to free Ald. The
greater toxicity of Ald@AgNPs is also consistent with previous
4
3
results that show that AgNPs have intrinsic anticancer properties
and can induce apoptosis in HeLa cells. In our own experiments, we
found that the IC50 of starch-coated AgNPs was 27 ꢀM (Supporting
Acknowledgements
42
Information). With respect to the molar concentration of Ald, the
anti-proliferative activity of Dox-Ald@AgNPs (Fig. 8A, blue curve)
was 20 times greater (IC50 = 0.5 ꢀM) than that of Ald@AgNPs (Fig. The research described here was sponsored by New York
University Abu Dhabi (NYUAD), UAE. F.B., R.R., T.J., M.M., and
A.T. thank NYUAD for their generous support for the research
8
A, red curve). This enhancement can be attributed to the toxicity
of Dox itself, which had an IC50 of 3 ꢀM against HeLa cells (Figure
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Journal Name
program at NYUAD. This research was carried out by using the 24 X. Lu and E. Masson, Langmuir, 2011, 27, 3051-3058.
Core Technology Platform resources at NYUAD. The authors 25 V. V. Srabionyan, A. L. Bugaev, V. V. Pryadchenko, A. V.
Makhiboroda, E. B. Rusakova, L. A. Avakyan, R. Schneider, M.
Dubiel and L. A. Bugaev, J. Non-Cryst. Solids, 2013, 382, 24-
thank Dr Erwan Guenin for providing the diesterified
Alendronate.
3
1.
2
6 a) F. Benyettou, Y. Lalatonne, I. Chebbi, M. Di Benedetto, J.
M. Serfaty, M. Lecouvey and L. Motte, Phys. Chem. Chem.
Phys.,, 2011, 13, 10020-10027 ; b) Y. Lalatonne, C. Paris, J. M.
Serfaty, P. Weinmann, M. Lecouvey and L. Motte, Chem.
Commun, 2008, 2553-2555; R. Aufaure, Y. Lalatonne, N.
Lievre, O. Heintz, L. Motte and E. Guenin, RSC Advances,
References
1
a) B. A. Teicher, E. A. Sotomayor and Z. D. Huang, Cancer
Res., 1992, 52, 6702-6704; b) A. Yagoda and D. Petrylak,
Cancer, 1993, 71, 1098-1109; c) M. Pegram, S. Hsu, G. Lewis,
R. Pietras, M. Beryt, M. Sliwkowski, D. Coombs, D. Baly, F.
Kabbinavar and D. Slamon, Oncogene, 1999, 18, 2241-2251;
d) T. Browder, C. E. Butterfield, B. M. Kraling, B. Shi, B.
Marshall, M. S. O'Reilly and J. Folkman, Cancer Res., 2000,
2
014, 4, 59315-59322.
2
2
7 V. L. Vaks, G. A. Domrachev, Y. L. Rodygin, D. A. Selivanovskii
and E. I. Spivak, Radiophys Quantum El., 1994, 37, 85-88.
8 T. Linnert, P. Mulvaney, A. Henglein and H. Weller, J. Am.
Chem. Soc., 1990, 112, 4657-4664.
9 J. Jortner and R. M. Noyes, J. Phys. Chem., 1966, 70, 770-774.
0 B. G. Ershov and E. V. Abkhalimov, Colloid J, 2007, 69, 579-
6
0
, 1878-1886.
S. R. MacEwan, D. J. Callahan and A. Chilkoti, Nanomedicine,
010, , 793-806.
J. M. Owens, K. Fuller and T. J. Chambers, J. Cell. Physiol,
997, 172, 79-86.
2
3
2
3
4
2
5
5
84.
3
3
3
3
1 E. Guénin, M. Monteil, N. Bouchemal, T. Prangé and M.
Lecouvey, Eur. J. Org. Chem., 2007, 2007, 3380-3391.
2 M. Meloun, Z. Ferenčíková, L. Netolická and T. Pekárek, J.
Chem. Eng. Data, 2011, 56, 3848-3854.
3 F. Benyettou, E. Guenin, Y. Lalatonne and L. Motte,
Nanotechnology, 2011, 22, 055102.
4 a) B. I. Ipe, K. G. Thomas, S. Barazzouk, S. Hotchandani and P.
V. Kamat, J. Phys. Chem. B, 2002, 106, 18-21; b) S. Ghosh, U.
Anand and S. Mukherjee, Analyst, 2013, 138, 4270-4274.
5 E. Dulkeith, A. C. Morteani, T. Niedereichholz, T. A. Klar, J.
Feldmann, S. A. Levi, F. C. van Veggel, D. N. Reinhoudt, M.
Moller and D. I. Gittins, Phys. Rev. Lett., 2002, 89, 203002.
6 .B. Kang, M. M. Afifi, L. A. Austin and M. A. El-Sayed, ACS
nano, 2013, 7, 7420-7427.
7 T. Sun, Y. S. Zhang, B. Pang, D. C. Hyun, M. Yang and Y. Xia,
Angew. Chem. Int. Ed., 2014, 53, 12320-12364.
8 M. Rodrigues, B. G. de la Torre, D. Andreu and N. C. Santos,
Biochim Biophys Acta, 2013, 1830, 4554-4563.
9 R. D. Phair and T. Misteli, Nature, 2000, 404, 604-609.
0 Y. Zhang, H. Peng, W. Huang, Y. Zhou and D. Yan, J. Colloid.
Interf. Sci., 2008, 325, 371-376.
1 C. Greulich, J. Diendorf, T. Simon, G. Eggeler, M. Epple and
M. Koller, Acta Biomater., 2011, 7, 347-354.
2 Silver nanoparticles coated with starch, which is non-toxic,
were prepared as described in Kahrilas et al. ACS Sustainable
Chem. Eng., 2014, 2, 590–598. The particles were added to
1
M. Salerno, E. Cenni, C. Fotia, S. Avnet, D. Granchi, F. Castelli,
D. Micieli, R. Pignatello, M. Capulli, N. Rucci, A. Angelucci, A.
Del Fattore, A. Teti, N. Zini, A. Giunti and N. Baldini, Curr.
Cancer Drug Tar., 2010, 10, 649-659.
5
a) L. S. Nair and C. T. Laurencin, J. Biomei. Nanotechnol.,
2
Sayed, Plasmonics, 2007,
007,
3
, 301-316; b) P. Jain, X. Huang, I. El-Sayed and M. El-
2, 107-118.
6
7
P. Setua, A. Chakraborty, D. Seth, M. U. Bhatta, P. V. Satyam
and N. Sarkar, J. Phys. Chem. C, 2007, 111, 3901-3907.
3
Q. Yu, C. Liu, Z. Zhang and Y. Liu, J. Phys.Chem. C, 2008, 112
266-2270.
A. Henglein and D. Meisel, Langmuir, 1998, 14, 7392-7396.
H. Wang, X. Qiao, J. Chen and S. Ding, Colloid Surface A,
005, 256, 111-115.
0 A. Henglein and M. Giersig, J. Phys. Chem. B, 1999, 103
533-9539.
1 D. L. Van Hyning and C. F. Zukoski, Langmuir, 1998, 14, 7034-
046.
2 R. Hu, K. T. Yong, I. Roy, H. Ding, S. He and P. N. Prasad, J.
Phys. Chem. C, Nanomaterials and interfaces, 2009, 113
676-2684.
3 R. R. Arvizo, S. Bhattacharyya, R. A. Kudgus, K. Giri, R.
Bhattacharya and P. Mukherjee, Chem. Soc. Rev., 2012, 41
943-2970.
,
2
3
3
3
8
9
2
1
1
1
,
9
3
4
7
,
4
4
2
1
1
,
2
4 A. Panacek, L. Kvitek, R. Prucek, M. Kolar, R. Vecerova, N.
Pizurova, V. K. Sharma, T. Nevecna and R. Zboril, J. Phys.
Chem. B, 2006, 110, 16248-16253.
HeLa cell culture, and after 48 hours of incubation, cell
viability was considerably reduced. The IC50 of the particles
was found to be 27 μM.
3 T. Kim and T. Hyeon, Nanotechnology, 2014, 25, 012001.
4 a) T.-C. Chou, Cancer Res., 2010, 70, 440-446; b) T.-C. Chou
and P. Talalay, Adv. Enzyme Regul., 1984, 22, 27-55.
1
1
5 K. G. Stamplecoskie and J. C. Scaiano, J. Am. Chem. Soc.,
2
4
4
010, 132, 1825-1827.
6 A. E. Nel, L. Madler, D. Velegol, T. Xia, E. M. Hoek, P.
Somasundaran, F. Klaessig, V. Castranova and M. Thompson,
Nat. Mater., 2009,
7 A. Ediriwickrema and W. M. Saltzman, ACS Biomaterials
Science & Engineering, 2015, , 64-78.
8, 543-557.
1
1
1
1
8 P. Fiurasek and L. Reven, Langmuir, 2007, 23, 2857-2866.
9 F. Benyettou, E. Guenin, Y. Lalatonne and L. Motte,
Nanotechnology, 2011, 22, 055102.
2
2
0 X. Liu, Y. Mou, S. Wu and H. C. Man, Appl. Surf. Sci., 2013,
273, 748-757.
1 a) J. Tao, J. P. Perdew, V. N. Staroverov and G. E. Scuseria,
Phys. Rev. Lett., 2003, 91, 146401; b) Frisch, M. J.; Trucks, G.
W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman,
J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.
et al. Gaussian, Inc., Wallingford CT, 2009
.
2
2
2 D. Andrae, U. Häußermann, M. Dolg, H. Stoll and H. Preuß,
Theoret. Chim. Acta, 1990, 77, 123-141.
3 J. M. L. Martin and A. Sundermann, J. Chem Phys., 2001, 114
3
,
408-3420.
8
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