An iGlu Receptor Antagonist and Its Simultaneous Use with an Anticancer Drug for Cancer Therapy

Article abstract of DOI:10.1002/chem.201406527

Glutamate receptor antagonists have been known to play a crucial role in the treatment of many neuronal diseases. Recently, these antagonists have also shown therapeutic effects in the treatment of cancer. In this study, an ionotropic glutamate (iGlu) receptor antagonist, 4-hydroxyphenylacetyl spermine (L1), was used concurrently with a common anticancer drug, doxorubicin (Dox), for simultaneous cancer therapy. Mesoporous silica nanoparticles (MSNPs) were employed as the delivery vehicle for both L1 and Dox by conjugating the iGlu receptor antagonist on the surface and encapsulating Dox within the mesopores. Dox was then trapped within the mesopores by functionalizing a redox-cleavable capping group on the MSNP surface, and it could be released upon exposure to the reductive glutathione. In vitro studies on B16F10 and NIH3T3 cell lines revealed that the iGlu receptor antagonist L1 exhibited therapeutic as well as targeting effects. In addition, the simultaneous use of therapeutic L1 and Dox proved to be synergistic in the treatment of cancer. The present work demonstrated the feasibility of employing a delivery system to deliver both neuroprotective drug and anticancer drug for efficient anticancer treatment.

Full text of DOI:10.1002/chem.201406527

DOI: 10.1002/chem.201406527
Full Paper
&
Antitumor Agents |Hot Paper|
An iGlu Receptor Antagonist and Its Simultaneous Use with an
Anticancer Drug for Cancer Therapy**
[
a]
[a]
[a]
[a]
[a]
Si Yu Tan, Chung Yen Ang, Zhong Luo, Peizhou Li, Kim Truc Nguyen, and
[a, b]
Yanli Zhao*
Abstract: Glutamate receptor antagonists have been known
to play a crucial role in the treatment of many neuronal dis-
eases. Recently, these antagonists have also shown thera-
peutic effects in the treatment of cancer. In this study, an
ionotropic glutamate (iGlu) receptor antagonist, 4-hydroxy-
phenylacetyl spermine (L1), was used concurrently with
a common anticancer drug, doxorubicin (Dox), for simulta-
neous cancer therapy. Mesoporous silica nanoparticles
was then trapped within the mesopores by functionalizing
a redox-cleavable capping group on the MSNP surface, and
it could be released upon exposure to the reductive gluta-
thione. In vitro studies on B16F10 and NIH3T3 cell lines
revealed that the iGlu receptor antagonist L1 exhibited
therapeutic as well as targeting effects. In addition, the
simultaneous use of therapeutic L1 and Dox proved to be
synergistic in the treatment of cancer. The present work
demonstrated the feasibility of employing a delivery system
to deliver both neuroprotective drug and anticancer drug
for efficient anticancer treatment.
(MSNPs) were employed as the delivery vehicle for both L1
and Dox by conjugating the iGlu receptor antagonist on the
surface and encapsulating Dox within the mesopores. Dox
[7]
Introduction
(
AMPA), N-methyl-d-aspartate (NMDA), and kainite. These re-
ceptors play a key role in many of the central nervous system
processes. Hence, substrates that target these receptors are
potential therapeutic agents for the treatment of those neuro-
logical diseases. For instance, Harsing Jr. et al. adopted a 2,3-
benzodiazepine-type AMPA receptor antagonist to prevent
neuronal excitotoxicity and to protect the brain cells from
Glutamate plays a very important role in the synaptic transmis-
[
1]
sion of the mammalian central nervous systems. Owing to
this fact, glutamate receptors are commonly known to be
[2]
highly expressed on the membrane of neuronal cells. In the
neuronal cells, glutamate binds to the ionotropic glutamate
[8]
(
iGlu) receptors and regulates the amount of calcium ions in
apoptosis. Another work from Nishizawa et al. revealed that
perampanel, an antiepileptic agent currently in phase III clinical
trials, showed a broad spectrum of anticonvulsant activity in
[
3]
the postsynaptic cleft. Overstimulation of these receptors
2
+
causes an overload of Ca ions in cells, which could result in
[
4]
[9]
the cell damage and subsequent cell death. The iGlu recep-
tors are essential in memory and learning. The dysfunction of
such receptors are related to many neurogenerative
preclinical models. In a work by Quack et al., aminoadaman-
tanes were adopted as an NMDA receptor antagonist and the
results exhibited that this class of antagonists possessed neu-
roprotective properties against the overstimulation of the
[
5]
disorders, such as epilepsy, lateral sclerosis, Parkinson’s and
[
6]
[10]
Alzheimer’s diseases .
NMDA receptors. An interesting class of iGlu receptor antag-
iGlu receptors can be classified into three types: (R,S)-2-
onists that have been gaining a lot of popularity for their uses
[11]
amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic
acid
as neuroprotective drugs is polyamine toxins.
They are
obtained from the venom of spiders and wasps, and are low-
molecular-weight compounds, which block the receptors by
+
+
[
a] S. Y. Tan, C. Y. Ang, Z. Luo, P. Li, K. T. Nguyen, Prof. Dr. Y. Zhao
Division of Chemistry and Biological Chemistry
School of Physical and Mathematical Sciences
Nanyang Technological University
[12]
an uncompetitive mechanism. As polyamine toxins are selec-
2+
[13]
tive inhibitors of Ca permeable glutamate receptors, they
are effective anticonvulsants in animal seizure models and
2
1 Nanyang Link, Singapore 637371 (Singapore)
E-mail: zhaoyanli@ntu.edu.sg
[14]
show important functions in polytherapies
.
Homepage: Homepage: http://www.ntu.edu.sg/home/zhaoyanli/
In recent years, there has been a growing trend in the usage
of glutamate receptor antagonists for the treatment of
[
b] Prof. Dr. Y. Zhao
School of Materials Science and Engineering
Nanyang Technological University
Singapore 639798 (Singapore)
[15]
cancer. Ikonomidou et al. observed that iGlu antagonists, di-
zocilpine and GYKI5246, were able to prevent the proliferation
of colon adenocarcinoma, astrocytoma, breast and lung carci-
+
[
] These authors contributed equally to this work.
[15e]
noma, as well as neuroblastoma cells.
Another work by Yu
[
**] iGlu=Ionotropic glutamate.
and co-workers reported the use of various types of isoquino-
lines, which are known to be non-competitive inhibitors of
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201406527.
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AMPA, for the treatment of five human cancer cell lines in
drugs for the treatment of B16F10 cells were investigated in
vitro.
[
16]
vitro. Although the results of such treatments seem to be ef-
[
17]
fective in cancer therapy, reports for the simultaneous use of
a neuroprotective drug and an anticancer drug on a metastatic
cancer model are still scarce. Thus, in this study, we synthe- Results and Discussion
sized a neuroprotective drug, which is an iGlu receptor antago-
Synthesis and characterization of functionalized MSNPs
nist, and investigated its therapeutic effect on a metastatic
[
18]
melanoma cancer model (B16F10) .
Although there are
The use of MSNPs as drug delivery vehicles has shown a lot of
promise in the treatment of many types of cancers. Due to the
easy synthesis and functionalization, in the present work,
MSNPs were adopted as a platform for the dual delivery of the
iGlu receptor antagonist L1 and Dox. The integration of these
two drugs in a single nanoparticle system enhances the effi-
ciency for the cancer treatment. Briefly, the MCM-41-type silica
nanoparticles were synthesized through a template-directed
a number of iGlu receptor antagonists available, we selected
N-(4-hydroxylphenylacetyl) spermine (L1) for its easy synthesis
and functionalization. To extend the scope of this research, an
anticancer drug, doxorubicin (Dox), was employed along with
the prepared neuroprotective drug to investigate their
simultaneous therapeutic efficiency.
To allow these two drugs to be concurrently delivered to
the B16F10 cells, mesoporous silica nanoparticles (MSNPs)
[25]
co-condensation method before grafting 3-chloropropyltrie-
thoxysilane onto the surface of the obtained MSNPs
[
19]
were employed as the delivery vehicles (Scheme 1). MSNPs
(
Scheme 2). The chloride moiety
was then substituted with an
azide group followed by grafting
3
-mercaptopropyltriethoxysilane
and 2-[methoxy(polyethylene-
oxy)-propyl] trimethoxysilane.
The surfactant was later re-
moved to yield MSNPs-4. To
anchor iGlu receptor antagonist
L1 onto the surface of MSNPs-4,
an alkyne moiety was introduced
onto L1 to yield molecule D. A
“
click” reaction was then per-
formed between molecule
D
and MSNPs-4 to yield MSNPs-5.
The final deprotection of the
tert-butyl groups from the D
moiety on MSNPs-5 afforded
MSNPs-6.
To characterize the surface
area and pore volume of the
MSNPs-0, the surfactant was re-
moved and Brunauer–Emmett–
Teller (BET) analysis was per-
Scheme 1. Schematic illustration of the different components used for the preparation of multifunctional MSNPs
as the drug carriers with subsequent therapeutic application in vitro.
formed. A type IV isotherm was
À1
have been commonly used in the drug delivery due to their
obtained for the MSNPs with a pore volume of 1.123 mLg
[
20]
2
À1
large surface area and easy functionalization. Furthermore,
their large volume allows a high drug-loading capability within
the mesopores, which can be released upon a specific stimu-
and a surface area of 1084.843 m g (see Figure S1 in the
Supporting Information). The pore size was determined to be
2.79 nm by using the Barret–Joyner–Halenda (BJH) model.
After the functionalization with different moieties on the sur-
face to obtain MSNPs-6, the pore volume and surface area de-
[
21]
lus. In this work, multifunctional MSNPs were designed by
conjugating the iGlu receptor antagonist, N-(4-hydroxylphenyl-
acetyl) spermine, onto the surface of MSNPs through a “click”
À1
2
À1
creased to 0.296 mLg and 543.903 m g respectively with
a pore size remaining almost the same at 2.95 nm (see Fig-
ure S1 in the Supporting Information). Powder X-ray diffraction
(XRD) spectrum of MSNPs-6 also showed a characteristic hex-
agonal and well-ordered porous structure with a pore size of
4.02 nm (see Figure S2 in the Supporting Information). As the
calculation of the pore size from the XRD analysis considers
the thickness of the silica wall around the pore, the pore size
value obtained from the XRD measurement was slightly higher
[
22]
reaction (Scheme 2).
On the other hand, Dox was loaded
within the mesopores of MSNPs and capped by disulfide-
[
23]
bridged adamantane/b-cyclodextrin (b-CD) stopper. As can-
cerous cells express a high concentration of glutathione (GSH),
the successful delivery of the multifunctional MSNPs into the
cancerous cells enables GSH to react with the disulfide bond,
[24]
thereby releasing the loaded Dox from the mesopores. Si-
multaneous delivery and therapeutic efficiency of these two
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Scheme 2. Synthetic route for the preparation of MSNPs-6.
Figure 1. a) TEM and b) SEM images of MSNPs-6.
than that obtained from the BJH model. MSNPs-6 was further
characterized by both transmission electron microscope (TEM)
and scanning electron microscope (SEM), showing a spherical
morphology with a diameter around 200 nm (Figure 1a and b).
Based on these results, the presence of mesoporous structure
for the as-synthesized nanoparticles was confirmed.
Figure 2. FTIR spectra of MSNPs-1–6.
conjugation of molecule D onto the surface of MSNPs-4. Com-
paring the XPS data between MSNPs-1 and MSNPs-2, the dis-
appearance of the Cl 2p peak at 200.47 eV from MSNPs-1 and
the appearance of the N 1 s peak at 398.52 eV in MSNPs-2 indi-
cated the successful conversion of chloride to azide. In addi-
tion, the increase in the nitrogen content from 3.1 to 5.9% for
MSNPs-4 and MSNPs-5 showed the successful conjugation of
molecule D to the surface of MSNPs-5. Apart from the increase
in the nitrogen content that was observed for different MSNPs,
the appearance of an S 2p peak at 163.52 eV in MSNPs-3
revealed the successful grafting of 3-mercaptopropyltriethoxy-
silane.
The changes in the surface functionalities of MSNPs were de-
termined by FTIR spectroscopy and X-ray photoelectron spec-
troscopy (XPS). The examination of the FTIR spectra revealed
À1
that MSNPs-2 contains an azide peak at 2029 cm , due to the
azide substitution that was performed on the chloride moiety
of MSNPs-1. After the conjugation of molecule D onto the sur-
À1
face of MSNPs-4, a strong carbonyl peak at 1571 cm for
MSNPs-5 was observed, which was attributed to the tert-butyl
carbamate functional group of molecule D. The deprotection
of the tert-butyl carbamate protecting group led to a weak
carbonyl signal for MSNPs-6, indicating the successful
deprotection from MSNPs-5 (Figure 2).
The changes of different surface functionalities were also
confirmed by zeta potential measurements (Table 1). Based on
the data obtained, MSNPs-4 gave a positive zeta potential,
which is probably attributed to the protonation of the azide
group. After the treatment of molecule D with MSNPs-4, the
The data obtained from XPS (see Figure S3 in the
Supporting Information) also exhibited similar trends, in which
an increase in the nitrogen content was observed after the
conversion of chloride to the azide moiety as well as after the
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Table 1. Zeta potentials of MSNPs-4, MSNPs-5, and MSNPs-6 with
respective standard deviations.
MSNPs
Zeta Potential
Standard Deviation
MSNPs-4
MSNPs-5
MSNPs-6
22.3
À15.9
43.5
7.06
7.60
5.55
azide moiety formed a 1,2,3-triazole ring, leading to the zeta
potential shift to a negative value. The increase in the zeta po-
tential values from MSNPs-5 to MSNPs-6 was likely caused by
the protonation of the amine groups on the surface, further
demonstrating the successful deprotection of the tert-butyl
carbamate groups.
Ninhydrin detection of primary amines was used to trace
the deprotection of the tert-butyl carbamate group (see Figure
S4 in the Supporting Information). The addition of MSNPs-6 to
ninhydrin showed a drastic increase in absorbance at 496 nm.
This absorbance increase was attributed to the byproduct for-
mation when the deprotected form of molecule D reacted
with ninhydrin. The deprotection of the tert-butyl carbamate
group afforded the amine unit, which reacted with ninhydrin
in solution to give a series of byproducts having the combined
absorbance at 496 nm. After calculations, the amount of ligand
L1 on the surface of the MSNPs-6 was determined to be
Figure 3. Dox release profiles for a) MSNPs-4–Dox and b) MSNPs-6–Dox
without and with GSH at 10 and 5 mm.
0.08 mmol per mg of MSNPs-6.
1
4
0 mm GSH was added to both MSNPs-6–Dox and MSNPs-
–Dox, a significant increase in the Dox concentration was
Drug loading and release studies
observed (Figure 3). The use of 10 mm GSH gave a higher Dox
release than that of 5 mm GSH. Further comparison of two re-
lease profiles at 5 mm revealed that the release of Dox from
MSNPs-6–Dox was almost two times higher than that of
MSNPs-4–Dox. This large difference in the Dox release could
be due to the interaction of the carboxylic acid groups on GSH
with the amino groups on the surface of the MSNPs. This inter-
action might allow GSH to easily access the disulfide bond and
cause its cleavage. The above results indicate the successful
loading and release of drugs by MSNPs-4 and MSNPs-6.
After the characterization of the MSNPs, the drug loading and
release capabilities with the anticancer drug Dox were investi-
gated. Dox was loaded within the mesopores of the MSNPs
capped with the disulfide bond-linked adamantane/b-CD com-
À1
plex. Firstly, Dox (4 mgmL ) was loaded into the pores of the
MSNPs-4 (20 mg) in water. Thereafter, water was evaporated,
and molecule 2 dissolved in ethanol was added to the mixture.
Lastly, ethanol was evaporated and an aqueous solution of b-
CD was added to the mixture. The resulting MSNPs-6–Dox
were then collected by centrifugation and washed extensively
with water. The drug loading capacity of MSNPs-6–Dox was de-
termined to be 2.1%. The relatively low loading capacity is due
to the multiple functionalities present on the surface of the
MSNPs, which hinder the drug-loading process. To compare
the effects of L1 for in vitro cancer therapy, MSNPs-4 was also
loaded with Dox to serve as a control. It was found that
MSNPs-4–Dox had a drug loading capacity of 2.4%. As MSNPs-
Endocytosis and anticancer efficiency towards B16F10
To verify the targeting ability and toxicity of polyamine toxin
L1 towards the B16F10 melanoma cell line, cell viability assays
of MSNPs-6 and MSNPs-4 were evaluated. As L1 is a glutamate
receptor antagonist and can be adopted for the treatment of
cancer, it was hypothesized that MSNPs-6 might have the abili-
ty to inhibit the progression of B16F10 cell line. This hypothe-
sis was tested by adding different concentrations of MSNPs-4
4
does not contain L1, the hindrance present on the surface of
MSNPs-4 is lower, resulting in a slightly higher loading
capacity.
À1
and MSNPs-6 (0.2, 0.1, 0.05, 0.025 mgmL ) into cultured
4
Thereafter, the Dox release behavior from MSNPs-4–Dox and
MSNPs-6–Dox was investigated (Figure 3). The fluorescence of
B16F10 cells (1ꢁ10 ) followed by the incubation for 4 h. Com-
paring the effects of the two types of MSNPs on B16F10 cell
line revealed that MSNPs-6 gave 50% cell viability, which is sig-
nificantly lower than the 70% cell viability of MSNPs-4
(Figure 4). There may be two main reasons for this trend. First-
ly, the polyamine groups on L1 being positively charged in
phosphate buffered saline (PBS) might disrupt some cell mem-
Dox was measured at 593 nm (l =480 nm), and the concen-
ex
tration of Dox released with respect to the amount of MSNPs
was calculated with reference to a standard curve. In the
absence of the reducing agent, release of the drug was not
observed in the two sample solutions. However, when 5 and
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cytoplasm of the cells, but not in the nucleus. MSNPs-4 and
MSNPs-6 were also investigated using NIH3T3 cell line, and the
cell images showed minimal green fluorescence in both cases.
The fluorescence microscopy images further confirmed the
selective uptake of L1 containing MSNPs-6 by the B16F10
melanoma cells over the NIH3T3 normal cells. The uptake of
[27]
these nanoparticles occurs by endocytosis after the binding
of the L1 ligand to the cell surface. Since B16F10 cells contain
[18a]
more iGlu receptors,
the uptake of MSNPs-6 is proportion-
ally higher, leading to the significant green fluorescence
observed.
Anticancer efficiency of MSNPs-4–Dox
After screening the therapeutic efficiency of MSNPs-4 and
MSNPs-6, Dox was loaded into MSNPs-4 to obtain MSNPs-4–
Dox for investigating its anticancer property. Cell viability
Figure 4. Cell viability of MSNPs-4 and MSNPs-6 using cancerous cells
B16F10) and non-cancerous cells (NIH3T3).
(
(
Figure 6) showed that there was a decrease of the cell viability
branes, causing the cell death. Secondly, the presence the
for both cell lines when increasing the Dox concentrations of
MSNPs-4–Dox. The results revealed a lower cell viability
(p<0.05) for B16F10 cancer cells as compared with NIH3T3
2
+
polyamine tail on L1 allows it to bind and antagonize Ca
permeable iGlu receptors. The binding of L1 results in a volt-
-
[28]
age-dependent block of inward current, which decreases the
cell division and enhances the cell death. Thus, L1-containing
MSNPs-6 exhibits some cytotoxic effect towards the B16F10
cell line, which is consistent with our hypothesis as well as the
normal cells. This is due to the regulation of GSH within can-
cerous cells, which causes more disulfide bonds to be cleaved
for increased drug release. This observation was further sup-
ported by fluorescence microscopy images (Figure 7), whereby
the cells were incubated with MSNPs-4–Dox at a Dox concen-
[
26]
literature reports.
À1
To ensure that the nanoparticles exhibit less cytotoxic effects
to normal cells, the same experiment was conducted on
mouse embryonic fibroblast NIH3T3 cells, a normal cell line,
using the same concentrations of MSNPs-4 and MSNPs-6
tration of 1.25 mgmL for 4 h. Red fluorescence from Dox was
localized in B16F10 cells but not in NIH3T3 cells, indicating the
selective drug release of MSNPs-4–Dox within the cancerous
cells as compared with the non-cancerous cells.
(Figure 4). Based on the results obtained, both samples gave
a cell viability of 70% with a negligible difference in the cyto-
toxic effects. Comparing the results with the B16F10 cell line,
the data obtained were surprising, as it indicates that L1-con-
taining MSNPs-6 has a selective cytotoxic effect to B16F10 mel-
anoma cell line over the NIH3T3 normal cell line. As B16F10
cell line is overexpressed with iGlu receptors as compared to
Inhibitory effects and intracellular drug release of
MSNPs-6–Dox
To probe the therapeutic effect of the L1 ligand together with
Dox, MSNPs-6 was loaded with Dox and then capped with a di-
sulfide-bridged adamantane/b-CD complex. Encapsulating Dox
within the nanoparticles enables us to study the dual effects of
anticancer and neuroprotective drugs. MSNPs-6–Dox was incu-
bated with both B16F10 and NIH3T3 cell lines for 4 h before
the cell viabilities of both cell lines were tested. The data in
Figure 8a showed that an increase in the Dox concentration of
MSNPs-6–Dox led to a decrease of the cell viability for both
B16F10 and NIH3T3 cell lines. The cell viability of the B16F10
cell line was significantly lower (p<0.01) than that of the
NIH3T3 normal cell line. This significant difference is attributed
to the presence of both glutamate receptor antagonist as well
as Dox. The glutamate receptor antagonist provides a targeting
ability along with some therapeutic properties. On the other
hand, the selective release of Dox from MSNPs-6–Dox was
realized by the cleavage of the disulfide bond by GSH.
[
26, 29]
the NIH3T3 cell line,
by the B16F10 cell line. An insignificant difference in the cy-
totoxicity of MSNPs-4 towards both cell lines was observed
Figure 4), whereas MSNPs-6 showed a marked difference of
there was selective uptake of MSNPs-
6
(
the cytotoxicity to B16F10 over NIH3T3 cells. This comparison
further supports the above observation that L1-containing
MSNPs-6 is therapeutically selective towards the B16F10 cell
line.
Fluorescence microscopy images were then obtained to
qualitatively determine the amount of MSNPs endocyctosed by
the cells (Figure 5). To determine the localization of the MSNPs
within the cells, fluorescein isothiocyanate (FITC) was incorpo-
rated into the nanoparticles during the synthesis. B16F10 and
NIH3T3 cells were incubated with MSNPs-4 and MSNPs-6
À1
(
0.2 mgmL ), respectively, for 4 h before fixing and staining
To compare the targeting and therapeutic efficiency be-
tween MSNPs-6–Dox and MSNPs-4–Dox on both cell lines (Fig-
ure 8b), a t-test was performed to show the difference in the
cell viability. The results revealed a significant difference in the
t-test values between two cell lines using MSNPs-6–Dox, and
the t-test value of MSNPs-6–Dox was 5-fold lower than that of
with 4’,6-diamidino-2-phenylindole (DAPI). When B16F10 cells
were incubated with MSNPs-4, there was a substantially low
amount of MSNPs-4 uptake into the cells, indicated by the
weak and non-localized green fluorescence. When MSNPs-6
was used, significant green fluorescence was observed in the
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Figure 5. Fluorescence microscopy images of B16F10 and NIH3T3 cell lines with MSNPs-4 and MSNPs-6. A) B16F10 cell line incubated with MSNPs-6;
B) B16F10 cell line incubated with MSNPs-4; C) NIH3T3 cell line incubated with MSNPs-6; D) NIH3T3 cell line incubated with MSNPs-4. The nucleus was stained
with DAPI, and green fluorescence was attributed to the FITC-labeled MSNPs-4 and MSNPs-6. Scale bar: 20 mm.
This therapeutic effect was also supported from the
fluorescence microscopy images, in which a positive uptake
was observed from B16F10 cells but not from NIH3T3 cells
when incubated with MSNPs-6–Dox at a Dox concentration of
À1
1
.25 mgmL (Figure 9). The intense-green fluorescence, caused
by FITC co-condensed within the MSNPs, was visible in B16F10
cells but not in NIH3T3 cells. This observation is consistent
with the result obtained from MSNPs-4–Dox, reflecting the
uptake of the MSNPs within the cells. The red fluorescence
within the B16F10 cells was attributed to the successful release
of Dox from MSNPs-6–Dox into the intracellular environment.
In addition, the red fluorescence was also observed in the nu-
cleus of the B16F10 cells, indicating that released Dox entered
the nucleus to intercalate with the DNA of the cells. This inter-
calation process inhibits the proper cellular functions and
causes apoptosis. Based on the cell viability data and fluores-
cence microscopy images, it could be concluded that MSNPs-
Figure 6. Cell viability of MSNPs-4–Dox with B16F10 cancerous cells and
non-cancerous NIH3T3 cells.
6–Dox contains both targeting and therapeutic properties,
MSNPs-4–Dox. This observation further confirms the anticancer
property of L1. With the dual selectivity, a synergistic effect
consisting of better targeting and therapeutic properties was
achieved using MSNPs-6–Dox.
presenting a promising potential for cancer therapy.
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Figure 7. Fluorescence microscopy images of B16F10 and NIH3T3 cell lines with MSNPs-4–Dox. A) B16F10 cells incubated with MSNPs-4–Dox and B) NIH3T3
cells incubated with MSNPs-4–Dox. The nucleus was stained with DAPI, green fluorescence was attributed to the FITC-labeled MSNPs-4–Dox, and red
fluorescence was derived from anticancer drug Dox. Scale bar: 20 mm.
alization of the neuroprotective drug on the surface of the
MSNPs has shown unexpected targeting as well as anticancer
properties. The functionalized MSNPs loaded with Dox and
capped with thiol cleavable groups have demonstrated selec-
tive drug release to cancer cells over normal cells. When both
the neuroprotective drug and the selective release of Dox
have been integrated onto the MSNPs, targeted and enhanced
therapeutic efficacy has been observed in cancer cells. This
work has successfully demonstrated the feasibility that the
drugs used for the treatment of neuronal diseases could also
be employed in combination with other anticancer drugs for
the treatment of cancer.
Experimental Section
Synthesis of MSNPs
Synthesis of MSNPs-0: The MSNPs were synthesized according to
[29]
previous reports.
Fluorescein isothiocynate (FITC, 4 mg) was
added to an oven-dried round bottom flask purged with N . The
2
solid was treated with (3-aminopropyl)triethoxysilane (APTES,
4
4 mL) in ethanol (1 mL) in the dark at room temperature for 24 h.
The next day, cetyltrimethylammonium bromide (CTAB, 0.1 g) was
dissolved in distilled water (50 mL), and NaOH (0.35 mL, 2m) was
added to the solution. The mixture was then heated to 808C fol-
lowed by addition of tetraethyl orthosilicate (TEOS, 0.5 mL) and the
above FITC-APTES solution (50 mL). The resulted solution was
stirred for 1 min before the addition of ethyl acetate (0.5 mL). The
mixture was further stirred for 2 h, and the obtained yellow solid
was filtered and washed three times with distilled water and
MeOH. The precipitate was then dried under vacuum to afford the
as-synthesized MSNPs-0.
Figure 8. a) Cell viability of MSNPs-6–Dox with cancerous B16F10 cell line
and normal NIH3T3 cell line; b) Comparison of the cell viability data
between MSNPs-4–Dox and MSNPs-6–Dox when incubated with B16F10 and
NIH3T3.
Conclusion
Synthesis of MSNPs-1: MSNPs-0 (1.5 g) was added to anhydrous
toluene (120 mL), and 3-chloropropyltrimethoxysilane (375 mL) was
added to the solution. The resulting mixture was heated at reflux
under N2 overnight. Thereafter, the precipitate was collected by
centrifugation (10000 rpm, 10 min) and washed with toluene and
EtOH to yield MSNPs-1.
A new type of MSNPs-based delivery system has been
prepared, with the simultaneous use of a neuroprotective drug
(
L1) and an anticancer drug (Dox), for the treatment of meta-
static melanoma cancer. The MSNPs system consisting of the
neuroprotective drug, water-soluble polyethylene glycol chain,
and a thiol-cleavable capping group on the surface presents
synergistic effects in the cancer therapy. The selective function-
Synthesis of MSNPs-2: MSNPs-1 (1.3 g) was added to N,N-dime-
thylformamide (DMF, 15 mL), and sodium iodide (217 mg) was
added to the solution. The solution was stirred for 5 min and NaN3
Chem. Eur. J. 2015, 21, 1 – 10
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Figure 9. Fluorescence microscopy images of B16F10 and NIH3T3 cell lines with MSNPs-6–Dox. Fluorescence microscopy images of A) B16F10 cell line incu-
bated with MSNPs-6–Dox, and B) NIH3T3 cell line incubated with MSNPs-6–Dox. The blue fluorescence observed was due to the DAPI staining of the nucleus,
green fluorescence was derived from FITC-labeled MSNPs-6–Dox, and red fluorescence was derived from anticancer drug Dox. Scale bar: 20 mm.
(
94.3 mg) was added thereafter. The mixture was stirred at room
absorbance wavelength at 496 nm was used to calculate the
temperature overnight and the precipitate was isolated by centri-
fugation (10000 rpm, 10 min). The precipitate was washed with
DMF and EtOH to yield MSNPs-2.
amount of compound D with respect to the amount of MSNPs.
Fluorescence microscopy imaging
Synthesis of MSNPs-3: MSNP-2 (1.0 g) was suspended in anhy-
drous toluene (80 mL), and 2-[methoxy(polyethyleneoxy)-propyl]
trimethoxysilane (0.65 mL) and 3-mercaptopropyltrimethoxysilane
To observe the uptake of nanoparticles in the cells, B16-F10 cells
and NIH3T3 cells were cultured separately in 35 mm glass-bot-
tomed dishes with an initial seeding density of 2ꢁ10 . The cells
5
(
1.5 mL) were added to the solution. The resulting solution was
were maintained in the growth medium for 24 h and were subse-
heated at reflux under N overnight. The precipitate was obtained
2
quently incubated with MSNPs-4 and MSNPs-6 to achieve a final
by centrifugation (10000 rpm, 10 min) and washed with toluene
and EtOH to yield MSNPs-3.
À1
concentration of 0.1 mgmL
for each MSNPs. After 4 h, the
medium was removed and the cells were washed thrice with 1X
PBS (2 mL). Fresh medium was then added and the cell culture
was incubated for another 24 h. The medium was removed and
the cells were washed three times with 1X PBS and fixed with 10%
formaldehyde in 1X PBS for 20 min at 378C. The formaldehyde so-
lution was removed and the cells were again washed with 1X PBS
three times. The cells were then incubated with 4’,6-diamidino-2-
Synthesis of MSNPs-4: MSNP-3 (1.0 g) was added to a flame-dried
round bottom flask, and methanol (160 mL) and HCl (9 mL) were
added. The mixture was heated at reflux overnight, and the precip-
itate was collected by centrifugation (10000 rpm, 10 min) and
washed with MeOH to yield MSNPs-4.
Synthesis of MSNPs-5: Compound D (0.4 g; the synthesis and
characterization of compound D are shown in the Supporting In-
formation, Scheme S1) was dissolved in DMF (3 mL), and then an
À1
phenylindole (DAPI) solution in 1X PBS (1 mL, 1 mgml ) for 20 min,
and were later washed three times with 1X PBS. The cells were im-
mersed in 1X PBS (2 mL) and were ready for fluorescence micros-
copy imaging using Nikon Eclipse TE2000-E confocal fluorescence
microscope with Nikon TE2-PS100W high-pressure mercury lamp.
The above procedure was repeated to obtain the fluorescence mi-
croscopy images for MSNPs-4–Dox and MSNPs-6–Dox at nanoparti-
aqueous solution (1 mL) containing CuSO (28.0 mg) and ascorbic
4
acid (44.4 mg) was added to the mixture. Thereafter, MSNPs-4
(
0.5 g) was added and the mixture was allowed to stir at room
temperature overnight. The precipitate was obtained by centrifu-
gation (10000 rpm, 10 min) and washed extensively with water
and MeOH to yield MSNPs-5.
À1
À1
cle concentrations of 0.23 mgmL and 0.10 mgmL , respectively.
Synthesis of MSNPs-6: MSNPs-5 (0.3 g) was added to dichlorome-
thane (2 mL), and then trifluoroacetic acid (TFA, 2 mL) was added.
The solution was allowed to stir at room temperature overnight,
and the resulting precipitate was obtained by centrifugation
Cell viability assays
A typical cell viability assay was carried by seeding the respective
cell lines into a 96-well plate at a density of 1X 10 cells per well
4
(
10000 rpm, 10 min). The precipitate was washed with MeOH and
dried under vacuum to yield MSNPs-6.
and incubated with Dulbecco’s Modified Eagle’s medium (DMEM,
1
00 mL) for 24 h. The respective sample solutions containing vari-
ous MSNPs (10 mL) were then added to each well with water serv-
ing as the negative control, and these plates were incubated for
Amine quantity tests
4
h. Thereafter, the medium was removed and the cells were
When XPS was used as the method for the determination of suc-
cessful ligand conjugation, the difference in nitrogen content
before and after the conjugation of compound D was obtained.
When ninhydrin was used for the quantification of amine amount
on the surface of MSNPs, a standard curve was prepared with dif-
ferent concentrations of aminopropanol in the presence of ninhy-
drin (4.5 mm) before any measurements were made. Thereafter,
a ninhydrin solution (4.5 mm) containing MSNPs-6 (2 mg) and an-
other solution without MSNPs-6 were measured. The difference in
washed once before fresh medium (100 mL) was added into the
wells. The cells were incubated for another 24 h, and the medium
was replaced with medium containing 3-(4,5-dimethylthiazol-2-yl)-
À1
2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mgmL
)
before a further incubation of 4 h. Then, the medium was removed
and dimethyl sulfoxide (DMSO, 100 mL) was added to dissolve the
purple crystals. Absorbance reading at 560 nm was performed on
a Tecan Infinite 200 PRO microplate reader with the percentage
cell viability determined by [Asample]/[Acontrol], in which [Asample] and
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M. Sifringer, W. Rzeski, C. Staufner, K. S. Brocke, L. Turski, C. Ikonomidou,
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Published online on && &&, 2015
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FULL PAPER
&
Antitumor Agents
S. Y. Tan, C. Y. Ang, Z. Luo, P. Li,
K. T. Nguyen, Y. Zhao*
&& – &&
An iGlu Receptor Antagonist and Its
Simultaneous Use with an Anticancer
Drug for Cancer Therapy
Double agent: The simultaneous use of
a neuroprotective drug and an anti-
cancer drug delivered by mesoporous
silica nanoparticles was investigated for
the treatment of metastatic melanoma
cancer, resulting in improved targeting
and anticancer properties.
A combination therapy……using a neuroprotective
drug, N-(4-hydroxylphenylacetyl) spermine, and an anti-
cancer drug, doxorubicin, integrated with functional
mesoporous silica nanoparticles as carriers has been
developed for improved treatment of metastatic mela-
noma cancer model, as reported by Y. Zhao et al. in
their Full Paper on page && ff.
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!