Cationic vesicles based on amphiphilic pillar[5]arene capped with ferrocenium: A redox-responsive system for drug/siRNA Co-delivery

Article abstract of DOI:10.1002/anie.201407272

A novel ferrocenium capped amphiphilic pillar[5]-arene (FCAP) was synthesized and self-assembled to cationic vesicles in aqueous solution. The cationic vesicles, displaying low cytotoxicity and significant redox-responsive behavior due to the redox equili

Full text of DOI:10.1002/anie.201407272

Angewandte
Chemie
DOI: 10.1002/anie.201407272
Drug/siRNA Co-Delivery
Cationic Vesicles Based on Amphiphilic Pillar[5]arene Capped with
Ferrocenium: A Redox-Responsive System for Drug/siRNA Co-
Delivery**
Yincheng Chang, Kui Yang, Peng Wei, Sisi Huang, Yuxin Pei,* Wei Zhao, and Zhichao Pei*
Abstract: A novel ferrocenium capped amphiphilic pillar[5]-
arene (FCAP) was synthesized and self-assembled to cationic
vesicles in aqueous solution. The cationic vesicles, displaying
low cytotoxicity and significant redox-responsive behavior due
to the redox equilibrium between ferrocenium cations and
ferrocenyl groups, allow building an ideal glutathione (GSH)-
responsive drug/siRNA co-delivery system for rapid drug
release and gene transfection in cancer cells in which higher
GSH concentration exists. This is the first report of redox-
responsive vesicles assembled from pillararenes for drug/
siRNA co-delivery; besides enhancing the bioavailability of
drugs for cancer cells and reducing the adverse side effects for
normal cells, these systems can also overcome the drug
resistance of cancer cells. This work presents a good example
of rational design for an effective stimuli-responsive drug/
siRNA co-delivery system.
and 2) units that respond to intracellular stimuli. So far, most
of the reported siRNA cationic systems are limited to the
[7]
protonation of amino groups under acidic conditions and
the stimuli-responsive structural units reported include amino
[
4,8]
groups, hydrazone linkages, disulfide bonds (-SÀS-),
and
[
9]
diselenide bonds (-SeÀSe-). Considering that the concen-
tration of GSH in tumor cells is higher than that in normal
[
10]
cells,
it is ideal and ubiquitous as a trigger for redox-
sensitive system to accomplish rapid drug release in cancer
[
8]
cells. Nevertheless, few GSH-responsive nanocarriers for
[
11]
the drug/siRNA co-delivery have been reported up to date.
[12]
Pillar[n]arenes
are a new class of macrocyclic com-
pounds which possess a hydrophobic core sandwiched
between two functional rims. In the past few years, they
have attracted considerable attention in the fabrication of
nanocarriers for biological applications, because of their
unique rigid pillar architecture and facile chemical modifica-
[
13]
D
rug resistance and similar cytotoxicity in both cancerous
tion. Among various drug nanocarriers,
from amphiphilic pillararenes (APs)
vesicles formed
have shown great
[1]
[14]
and healthy cells are major problems in chemotherapy. One
promising way to overcome these obstacles is the develop-
ment of stimuli-responsive co-delivery systems of drug/
siRNA (small interfering RNA), which possess the ability to
recognize and respond to the specific microenvironmental
potential in drug delivery due to an efficient drug encapsu-
lation in their cavities. To the best of our knowledge, cationic
vesicles assembled with APs for drug/siRNA co-delivery have
not yet been reported.
[
2]
[3]
[15]
changes (such as pH, temperature, and glutathione (GSH)
The ferrocenium cation, sensitive to GSH, undergoes
[
4]
[5]
concentration) associated with neoplastic diseases. Due to
the specific stimulus, the nanocarriers disassemble after
entering cancer cells, leading to a rapid release of encapsu-
lated siRNA and drug. siRNA can knock down drug
resistance genes and the drug sensitivity of cells will be
restored to enable effective chemotherapy. Meanwhile, due to
the absence of the specific stimulus, drug release does not
occur or occurs slowly in healthy cells, thus efficiently
avoiding severe side effects.
a polarity shift (a driving force of assembly and disassembly
[
16]
for constructing vesicles)
when reduced to ferrocene (a
[17]
known biocompatible building block). More importantly, in
comparison to other typically exploited redox-responsive
bonds, the positive charge of ferrocenium makes possible for
the loading of negatively charged siRNA onto nanocarri-
[
4,18]
ers.
Therefore, it is a potential structural unit for the
construction of GSH-responsive drug/siRNA co-delivery
systems. We envision that the conjugation of ferrocenium
cation head groups to pillararenes will lead to an AP with
positive charges as well as redox-active structural units. This
AP will, possibly, be able to self-assemble into cationic
vesicles in aqueous solution and thus be applied in drug/
siRNA co-delivery.
Thus, we synthesized a novel ferrocenium-capped amphi-
philic pillar[5]arene (FCAP), which was further applied to
construct nanoscale cationic vesicles for drug/siRNA co-
delivery as depicted in Scheme 1. This is the first report of
redox-responsive cationic vesicles for drug/siRNA co-deliv-
ery and it is the first use of ferrocenium as GSH-responsive
structural unit in a drug delivery system. The cytotoxicity,
redox-responsive behavior, drug encapsulation and release
rate, and bioavailability of the cationic vesicles were studied.
Moreover, as a proof-of-concept, doxorubicin hydrochloride
(DOX) and drug resistance gene silencing siRNA (MRP1
The indispensable structural features of a material used
for such systems are: 1) positive charges, which allow effective
[6]
loading of polyanionic siRNA by electrostatic interaction,
[+]
[+]
[
*] Y. Chang, K. Yang, P. Wei, S. Huang, Prof. Dr. Y. Pei, W. Zhao,
Prof. Dr. Z. Pei
College of Science, Northwest A&F University
Yangling, Shaanxi 712100 (P.R. China)
E-mail: peiyx@nwafu.edu.cn
peizc@nwafu.edu.cn
+
[
] These authors contributed equally.
[
**] This research work was supported by the National Natural Science
Foundation of China (NSFC 21174113 and NSFC 31270861). We
thank Yihan Pei (Clare College, Cambridge) for language help.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201407272.
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Figure 1. a) SEM image of the cationic vesicles formed from FCAP.
b) TEM image. c) Tyndall effect of FCAP aggregation (left) and
precipitates formed in the presence of excess GSH (right). d) The view
of samples in Figure 1c under normal light. e) TEM image of FCAP in
the presence of GSH.
were 91.9 nm and 0.298, respectively. The spherical assem-
blies were stable in solution at 300 K for one week, implying
the vesicles had a good stability. The critical aggregation
concentration (CAC) of FCAP was found to be 58 mm by the
[
14a]
water surface tension method (Figure S12).
The redox-responsiveness of the vesicles formed from
FCAP was investigated by exposing the solution to the
reductant GSH or the oxidant FeCl . As shown in Figure 1c,
3
a clear Tyndall effect was observed with FCAP aqueous
solution (60 mm), indicating the existence of abundant nano-
particles. However, accompanied by the disappearance of the
Tyndall effect, yellow precipitates formed gradually from the
transparent light green solution of FCAP upon addition of
GSH (Figure 1d). TEM studies, as expected, found no
vesicles accordingly (Figure 1e). All these results suggested
that the vesicles were disassembled when exposed to GSH,
corresponding to the reduction of the light green ferrocenium
cation to the yellow ferrocenyl groups. Further studies using
UV/Vis spectroscopy confirmed that the reversible trans-
formation between ferrocenium cations and ferrocenyl
groups upon the presence of reductant (GSH) or oxidant
Scheme 1. Illustration of the synthesis of FCAP, formation of cationic
vesicles, and their redox-responsive drug/siRNA release.
siRNA) were selected as the anticancer drug/siRNA pair for
[
1b,c]
a co-delivery study in human ovarian SKOV-3 cancer.
[19]
To synthesize FCAP, 1
was first functionalized with
sodium azide. Azide-modified pillar[5]arene 2 was obtained
in 92% yield, and subjected to copper-catalyzed azide-alkyne
cycloaddition (CuAAC) to form pillar[5]arene 3 in a yield of
8
8% (Scheme 1). Oxidation of the ferrocenyl groups on 3 by
FeCl gave FCAP stoichiometrically, accompanying a polarity
(FeCl ; Figure S13). Therefore, the vesicles based on FCAP
3
3
shift from hydrophobicity of 3 to amphipathy of FCAP which
could lead to regular aggregates in water. For details of the
synthesis and characterization of 1–3 and FCAP, see the
Supporting Information (SI).
By subjecting a solution of FCAP to sonication for 30 min,
spherical vesicles were formed and confirmed by scanning
electron microscopy (SEM, Figure 1a). Negative-stained
transmission electron microscopy (TEM) images suggested
that the thickness of the vesicles was about 3.2 nm from the
distinguishably dark periphery to the light central part
can be used as redox-responsive nanocarriers capable
responding to GSH.
Cytotoxicity of unloaded vesicles to normal cells was
evaluated by the methyl thiazole tetrazolium (MTT) cell-
survival assay. As shown in Figure S14, the relative cell
viability of 293T cells (normal cells) incubated with unloaded
vesicles was over 85% in the concentrations of 10 mm FCAP
after 24 h, indicating that the cationic vesicles based on FCAP
developed in this work had low cytotoxicity to normal cells,
which was essential for the design of drug nanocarriers.
To study the drug release behavior of the redox-respon-
sive cationic vesicles, doxorubicin hydrochloride (DOX),
a fluorescence dye and anticancer drug molecules, was
(
Figure 1b), which is close to the molecular length of 3
[
2.9 nm calculated with the energy-minimized structure (Fig-
ure S10)]. This indicates that the vesicles were assembled by
FCAP in a monolayer packing mode. Further analysis with
dynamic light scattering (DLS, Figure S11) showed that the
average diameter and polydispersity index of the vesicles
[
20]
selected as model drug. SEM disclosed that the surface of
DOX-loaded vesicles (see SI for preparation in details)
became quite rough (Figure S15a). DLS analysis showed that
2
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[
22]
the DOX-loaded vesicles had an average diameter of 79 nm
and a polydispersity index of 0.232 (Figure S15b). UV/Vis
spectra of the DOX-loaded vesicular solution confirmed that
DOX molecules were successfully encapsulated in the
vesicles. In addition, the color of the unloaded vesicular
solution changed from light green to brown upon DOX-
loading into the vesicles (inset of Figure S15c). From UV/Vis
absorption spectra, the DOX encapsulation and loading
efficiency were calculated to be 67.0% and 9.1%, respec-
tively, indicating a good drug-loading capability of the
cationic vesicles.
compared to HeLa cells.
In addition, the enhanced
apoptotic effects of DOX-loaded vesicles on cancer cells
were also confirmed in the cases of SKOV-3 and KM-12 cells
(Figure S19). All these results suggested that the encapsula-
tion of DOX by the vesicles could enhance the drugꢀs
efficiency against cancer cells while effectively reducing its
cytotoxicity to normal cells.
Synthetic siRNAs fail to cross biological membranes by
passive diffusion because of their high molecular weight and
polyanionic nature; therefore, they generally require delivery
systems to be positively charged for loading of siRNA by
electrostatic interactions. FCAP synthesized in this work
has ten positive charges due to the ten capped ferrocenium
cations in its molecular structure. When mixed with FAM-
siRNA (a fluorescent labeled siRNA) at a ferrocenium
cation/P ratio of 5:1 (defined as the molar ratio of ferroce-
nium cation units in the cationic vesicles to phosphate units in
siRNA), the cationic vesicles self-assemble to FAM-siRNA/
cationic vesicle complexes through electrostatic interactions.
The SEM image of the complexes showed nanoparticles with
an average diameter of 60 nm (Figure 3a). The cellular
uptake and intracellular distribution of the FAM-siRNA/
cationic vesicle complexes were studied by flow cytometry
and CLSM. As shown in Figure 3b, HeLa cells transduced
with FAM-siRNA/cationic vesicle complexes showed ca.
54% cellular internalization, a similar result to Lipofect-
amine 2000 (58%) which had been used as siRNA trans-
[
6]
DOX release profiles from DOX-loaded vesicles with or
without exposure to GSH in water were shown in Figure S16.
1
0 mm GSH was used to trigger the responsive release of
DOX, which was relevant to the concentration of GSH in
[
21]
cancer cells (1–11 mm).
The amount of DOX release
reached a maximum of 92% with 10 mm GSH, resulting
from the disassembly of DOX-loaded vesicles when the
ferrocenium cations were reduced to ferrocenyl groups by
GSH. In contrast, the DOX-loaded vesicles without GSH
exposure remained relatively stable and only 39% of DOX
was released in the same period. The rapid release behavior
resulting from the redox-responsive DOX-loaded vesicles
responding to GSH makes the DOX-loaded vesicles ideal
nanocarriers for drug delivery.
The cellular uptake of DOX-loaded vesicles and free
DOX was investigated by confocal laser scanning microscope
[23]
(
CLSM) for comparison. Red fluorescence could be observed
fection agent.
The results were further confirmed by
clearly in the nuclei of HeLa cells after 1 h incubation with
DOX-loaded vesicles (Figure S17a), whereas the red fluores-
cence of HeLa cells incubated with free DOX was weaker
confocal microscopy. Strong green fluorescence was found
(
Figure S17) under the same conditions, indicating that DOX
loaded by vesicles could enter the cells more rapidly than free
DOX.
To evaluate the anticancer efficiency of DOX-loaded
vesicles, 293T cells and HeLa cells were incubated with DOX-
loaded vesicles and free DOX for 24, 48, and 72 h, respec-
tively. For 293T cells (Figure 2a), the relative viabilities after
incubation with DOX-loaded vesicles at all three tested time
periods were visibly higher than those with free DOX
(
especially at 24 h). In contrast, HeLa cells under the same
conditions showed lower relative viabilities after incubation
with DOX-loaded vesicles than with free DOX (Figure 2b).
This is also supported by the images of normal (293T) and
cancer (HeLa) cells treated with DOX-loaded vesicle for 24 h
(
Figure S18), which showed better growth of 293T cells
Figure 3. a) SEM image of FAM-siRNA/cationic vesicle complexes.
b) Flow cytometry analyses of HeLa cells after incubation with FAM-
siRNA/cationic vesicle complexes at ferrocenium cation/P ratio of 5:1.
c) CLSM images of HeLa cells, after being treated with FAM-siRNA/
cationic vesicles complexes; scale bar 50 mm.
Figure 2. Comparison of DOX and DOX-loaded vesicles on viabilities
of 293T cells (a) and HeLa cells (b) at different times.
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in the cytoplasm of the cells incu-
bated with FAM-siRNA/cationic
vesicle complexes, demonstrating
successful delivery (Figure 3c).
As a proof-of-concept, multi-
drug-resistant
protein
siRNA
(
MRP1 siRNA) was selected to
fabricate DOX-loaded MRP1
siRNA/cationic vesicle complexes,
which were used for the co-delivery
study to assess the drug resistance
[
1b,c]
gene silencing of SKOV-3 cells.
A negative siRNA was used as the
control. For siRNA loading, freshly
prepared DOX-loaded vesicles
were first diluted to the desired
ferrocenium cation/P ratio, fol-
lowed by the addition of the
siRNA solution. The mixture was
allowed to stand for 20 min before
use. The internalization of MRP1
siRNA into SKOV-3 cells is shown
in Figure 4a. After incubating
SKOV-3 cells with DOX-loaded
FAM-MRP1 siRNA/vesicles com-
plexes for 6 h, both green and red
fluorescence were observed in the
cells. This suggested that both
DOX and FAM-MRP1 siRNA
were internalized into the cells by
the cationic vesicles (Figure 4a).
The gene silencing efficiency was
investigated at both mRNA and
protein level. The transcriptional
MRP1 mRNA was detected by
quantitative real-time polymerase
chain reaction (RT-PCR) (Fig-
ure 4b) and MRP1 protein was
determined by ELISA (Figure 4c). Figure 4. a) CLSM images of SKOV-3 cells after being treated with DOX-loaded FAM-siRNA/vesicles
The DOX-MRP1 siRNA/vesicles complexes for 6 h; scale bar 50 mm. b) Gene silencing efficiency of SKOV-3 cells at the mRNA level
determined by RT-PCR. c) Gene silencing efficiency of SKOV-3 cells at the MRP1 protein expression
(
DOX-loaded MRP1 siRNA/cat-
level determined by ELISA. d) The cell death and apoptosis of SKOV-3 cells after staining with
annexin V–FITC and propidium iodide (PI) by flow cytometry. Untreated SKOV-3 cells were used as
a control. Cells that were negative for both annexin V–FITC and PI staining were classified as alive,
whereas cells stained positively for annexin V–FITC and negatively for PI were classified as apoptotic.
Cells that stained positively for PI were classified as necrotic.
ionic vesicles complexes) showed
marked down-regulation of MRP1
mRNA and a decrease in the
MRP1 expression compared to
the negative control (DOX-loaded
negative siRNA/cationic vesicle
complexes). Then the annexin V conjugate staining assay
was performed to evaluate the cytotoxicity of the DOX-
loaded MRP1 siRNA/cationic vesicle complexes. As shown in
Figure 4d, the cell death and apoptosis of cells incubated with
DOX-loaded negative siRNA/cationic vesicle complexes was
of the cancer cells was re-sensitized after being transfected
with DOX-loaded MRP1 siRNA/cationic vesicles complexes,
and chemotherapeutic efficacy was enhanced to a certain
extent by the synergistic effects of MRP1 siRNA and DOX.
In summary, novel cationic vesicles resulting from the self-
assembly of FCAP have been developed for GSH-triggered
drug and siRNA co-delivery. Drug release studies in vitro
demonstrated that DOX-loaded cationic vesicles can improve
the inhibition of cancer cell growth resulting from subsequent
GSH-triggered rapid drug release in cancer cells. Further-
more, DOX-loaded vesicles are markedly less toxic to normal
cells than free DOX. More importantly, the co-delivery of
3
7.9%, whereas that with DOX-loaded MRP1 siRNA/
cationic vesicles complexes reached 52.8%. By the MTT
method, the IC50 value in SKOV-3 after 48 h incubation with
DOX-loaded MRP1 siRNA/cationic vesicles complexes was
calculated to be 2.1 mm, whereas it was 4.9 mm with DOX-
loaded negative siRNA/cationic vesicles complexes (Fig-
[24]
ure S20). These results suggested that the DOX resistance
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Communications
Drug/siRNA Co-Delivery
Simultaneous delivery of an anticancer
drug and siRNA was achieved with cat-
ionic vesicles self-assembled from a novel
ferrocenium-capped amphiphilic pil-
lar[5]arene. These systems exhibit low
cytotoxicity to healthy cells and are redox-
responsive in the presence of a reduc-
tant/oxidant.
Y. Chang, K. Yang, P. Wei, S. Huang,
Y. Pei,* W. Zhao, Z. Pei*
&&&& — &&&&
Cationic Vesicles Based on Amphiphilic
Pillar[5]arene Capped with Ferrocenium:
A Redox-Responsive System for Drug/
siRNA Co-Delivery
6
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