Dual photo- and pH-responsive supramolecular nanocarriers based on water-soluble pillar[6]arene and different azobenzene derivatives for intracellular anticancer drug delivery

Article abstract of DOI:10.1002/chem.201405095

Two novel types of supramolecular nanocarriers fabricated by the amphiphilic host-guest inclusion complex formed from water-soluble pillar[6]arene (WP6) and azobenzene derivatives G1 or G2 have been developed, in which G1 is structurally similar to G2 but has an extra phenoxy group in its hydrophobic region. Supramolecular micelles can be initially formed by WP6 with G1, which gradually transform into layered structures with liquid-crystalline properties, whereas stable supramolecular vesicles are obtained from WP6 and G2, which exhibit dual photo- and pH-responsiveness. Notably, the resulting WP6a??G2 vesicles can efficiently encapsulate anticancer drug mitoxantrone (MTZ) to achieve MTZ-loaded vesicles, which maintain good stability in a simulated normal physiological environment, whereas in an acid environment similar to that of tumor cells or with external UV irradiation, the encapsulated drug is promptly released. More importantly, cytotoxicity assay indicates that such vesicles have good biocompatibility and the MTZ-loaded vesicles exhibit comparable anticancer activity to free MTZ, especially with additional UV stimulus, whereas its cytotoxicity for normal cells was remarkably reduced. Flow cytometric analysis further confirms that the cancer cell death caused by MTZ-loaded vesicles is associated with apoptosis. Therefore, the dual pH- and UV-responsive supramolecular vesicles are a potential platform for controlled release and targeted anticancer drug delivery.

Full text of DOI:10.1002/chem.201405095

DOI: 10.1002/chem.201405095
Full Paper
&
Drug Delivery
Dual Photo- and pH-Responsive Supramolecular Nanocarriers
Based on Water-Soluble Pillar[6]arene and Different Azobenzene
Derivatives for Intracellular Anticancer Drug Delivery
[
a]
[b]
[a]
[c]
[a]
[b]
[a]
Xiao-Yu Hu, Keke Jia, Yu Cao, Yan Li, Shan Qin, Fan Zhou, Chen Lin,
[b]
[a]
Dongmei Zhang, and Leyong Wang*
Abstract: Two novel types of supramolecular nanocarriers
fabricated by the amphiphilic host–guest inclusion complex
formed from water-soluble pillar[6]arene (WP6) and azoben-
zene derivatives G1 or G2 have been developed, in which
G1 is structurally similar to G2 but has an extra phenoxy
group in its hydrophobic region. Supramolecular micelles
can be initially formed by WP6 with G1, which gradually
transform into layered structures with liquid-crystalline prop-
erties, whereas stable supramolecular vesicles are obtained
from WP6 and G2, which exhibit dual photo- and pH-re-
sponsiveness. Notably, the resulting WP6ꢀG2 vesicles can
efficiently encapsulate anticancer drug mitoxantrone (MTZ)
to achieve MTZ-loaded vesicles, which maintain good stabili-
ty in a simulated normal physiological environment, whereas
in an acid environment similar to that of tumor cells or with
external UV irradiation, the encapsulated drug is promptly
released. More importantly, cytotoxicity assay indicates that
such vesicles have good biocompatibility and the MTZ-
loaded vesicles exhibit comparable anticancer activity to free
MTZ, especially with additional UV stimulus, whereas its cy-
totoxicity for normal cells was remarkably reduced. Flow cy-
tometric analysis further confirms that the cancer cell death
caused by MTZ-loaded vesicles is associated with apoptosis.
Therefore, the dual pH- and UV-responsive supramolecular
vesicles are a potential platform for controlled release and
targeted anticancer drug delivery.
Introduction
(
DDSs) with stimuli-responsiveness, which can either passively
or actively target cancer cells. The most recent development in
designing responsive nanocarriers has been focused on the mi-
celle- and vesicle-based nanomedicines due to their unique
structures, which can efficiently encapsulate the drug and
shield it from degradation, increase drug bioavailability, and fa-
Cancer remains one of the world’s most devastating diseases,
[
1]
with more than ten million new cases every year. Currently,
chemotherapy is still the most commonly used cancer treat-
ment, which also kills healthy cells and causes toxicity to the
patient. However, drugs encapsulated in nanocarriers, which
are referred to as nanomedicines, have shown therapeutic ad-
vantages including better efficacy against resistant tumors and
[2c,3]
cilitate the targeted delivery.
Up to now, various molecular
building blocks have been exploited for the construction of
[4]
[5]
nanocarriers, and among them supramolecular amphiphiles
[
2]
fewer side effects over conventional free drugs. Therefore, it
is highly desirable to develop “smart” drug-delivery systems
based on noncovalent interactions with stimuli-responsive
properties are the most promising ones, since they can not
only spontaneously aggregate to form different assemblies,
but also undergo structure transitions in response to numer-
[
6]
[6b,7]
[8]
[9]
ous stimuli, such as pH, temperature,
redox, voltage,
[
a] Dr. X.-Y. Hu, Y. Cao, S. Qin, Dr. C. Lin, Prof. Dr. L. Wang
Key Laboratory of Mesoscopic Chemistry of MOE
Center for Multimolecular Organic Chemistry
School of Chemistry and Chemical Engineering
Nanjing University, Nanjing 210093 (China)
E-mail: lywang@nju.edu.cn
[10]
[11]
enzyme, and light. So far, various strategies have been ex-
ploited to fabricate stimuli-responsive supramolecular amphi-
[
12]
philes for the construction of nanocarriers; however, to the
best of our knowledge, most of the reported responsive nano-
carriers only exhibited single stimulus-responsiveness, and
there are only a few reports on multistimuli-responsive supra-
molecular nanocarriers, particularly applied for drug deliv-
[b] K. Jia, F. Zhou, Dr. D. Zhang
State Key Laboratory of Pharmaceutical Biotechnology
School of Life Sciences
Nanjing University, Nanjing 210093 (China)
[6b,13]
ery.
Therefore, the construction of novel multistimulus-re-
[
c] Dr. Y. Li
State Key Laboratory of Bioelectronics
School of Biological Science and Medical Engineering
Southeast University, Nanjing 210009 (China)
sponsive supramolecular drug nanocarriers would be of great
interest in the application of biotechnology and biomedicine.
Controllable switching assembly on/off of the nanocarrier is
a prerequisite to achieve controlled drug release and its poten-
tial applications as a “smart” DDS. Compared with the tradi-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405095.
Chem. Eur. J. 2014, 20, 1 – 14
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[
14]
[6c]
tional polymeric nanocarriers, the supramolecular approach
paves an attractive alternative way to construct micelles, vesi-
cles, and other amphiphilic assemblies, since such assemblies
are held together by multiple weak and reversible noncovalent
interactions, which endow the formed supramolecular nano-
carriers with dynamic properties that can be controllably ma-
nipulated through the assembly/disassembly processes under
ness and are significantly biocompatible in aqueous media,
which is of great importance for their applications in drug de-
livery. In addition, azobenzene derivatives have been con-
firmed to be especially advantageous for controlling the micro-
scopic structures and relevant functions of various supramolec-
[
11a,20]
ular systems due to their photoinduced E/Z isomerization,
and the host–guest recognition motif based on the WP6 and
azobenzene motif has been demonstrated to have a strong
binding affinity in water driven by hydrophobic and electro-
[
15]
external stimuli. Pillararenes, a kind of significant macrocyclic
host with unique symmetric pillar architectures and p-rich cavi-
ties, have received great attention in constructing various in-
[11b,21]
static interactions.
Consequently, we envision that dual
[
16]
teresting supramolecular systems, including nanomaterials,
photo- and pH-responsive supramolecular nanocarriers could
be achieved by designing an appropriate azobenzene-based
amphiphilic guest, which could form a supramolecular amphi-
phile with WP6 based on the host–guest interaction. The ob-
tained supramolecular amphiphile would possibly be able to
form supramolecular micelles or vesicles in water by self-as-
sembly, which could be used as a multiresponsive supramolec-
ular nanocarrier for applications in drug delivery.
[
17]
[18]
[19]
chemosensors, transmembrane channels, and polymers.
So far, although several kinds of noncovalent interactions have
been used to fabricate supramolecular amphiphiles, supra-
molecular nanocarriers constructed by pillararene-based supra-
molecular amphiphiles have been much less frequently ex-
[
6a,b]
plored in the field of drug delivery.
Water-soluble pillar[6]ar-
enes (WP6) have been demonstrated to show pH-responsive-
Scheme 1. Schematic illustration of the construction of supramolecular micelles (WP6ꢀG1) or vesicles (WP6ꢀG2) and the application of supramolecular vesi-
cles in drug delivery.
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Following our recent work on pillararene-based pH- and/or
Results and Discussion
2
+
Ca -responsive supramolecular vesicles for intracellular drug
[
6a,b]
delivery,
herein we report the successful construction of
Host–guest complexation studies in water
two novel types of pillararene-based supramolecular nanocarri-
ers formed from WP6 and different azobenzene derivatives, G1
or G2, in which G1 is structurally similar to G2 but has an extra
phenoxy group in its hydrophobic region (Scheme 1). Supra-
molecular micelles were initially formed by WP6 with G1,
which would gradually be transformed into layered structures
with liquid-crystalline properties, probably due to the presence
of cooperative intermolecular hydrogen-bonding and p–p in-
teractions within the azobenzene and benzamido motifs of G1,
whereas stable supramolecular binary vesicles were obtained
from WP6 and G2, which exhibited dual photo- and pH-re-
sponsiveness. Notably, the resulting WP6ꢀG2 supramolecular
vesicles were able to efficiently encapsulate the anticancer
drug mitoxantrone (MTZ) to achieve MTZ-loaded vesicles,
which showed rapid and efficient MTZ release to an acidic pH
environment and/or upon UV stimulus by in vitro drug release
experiments. More importantly, cytotoxicity assay demonstrat-
ed that the loading of MTZ by WP6ꢀG2 vesicles did not affect
the curative effect of MTZ, and the resulting MTZ-loaded
supramolecular vesicles exhibited a comparable therapeutic
effect for cancer cells to free MTZ, especially with external UV
stimulus, whereas the cytotoxicity of MTZ for normal cells was
remarkably reduced. Flow cytometric analysis further con-
firmed that the molecular mechanism for the cancer cell death
caused by MTZ-loaded vesicles was associated with apoptosis.
Initially, azobenzene-based amphiphilic molecules G1 and G2
were designed, which contained an azobenzene-based quater-
nary ammonium group as the hydrophilic head and a flexible
alkyl phenyl ether segment (G1) or alkyl chain (G2) as the hy-
drophobic part as shown in Scheme 1. The G1 and G2 exhibit
quite poor water solubility, so model guests G3 and G4 were
used to investigate the host–guest complexation of WP6ꢀG1
1
1
1
and WP6ꢀG2 by H NMR, H– H COSY, and 2D ROESY spectros-
1
copy, respectively, in D O. From the H NMR spectra of WP6
2
and G3 (Figure 1), remarkable upfield chemical shifts of the
azobenzene protons (H , H , H , and H ), methylene protons
c
d
e
f
(H ), and methyl protons (H ) of the quaternary ammonium
b
a
group were observed upon addition of WP6 due to the shield-
ing effect of the electron-rich cavities of WP6 toward G3. The
peaks of protons H , H , and H on G3 shifted downfield slight-
g
h
i
ly. The above results revealed that WP6 was fully threaded by
guest G3 with the protons H , H , H , H , H , and H in the hy-
a
b
c
d
e
f
drophobic WP6 cavity and other protons H , H , and H out of
g
h
i
the cavity. The assignment of these proton signals could be
1
1
substantiated by the analysis of the H– H COSY data (Fig-
ure S2 in the Supporting Information). Moreover, the spatial
conformation of such an inclusion complex was further con-
firmed by the 2D ROESY spectrum (Figure S4 in the Supporting
Information), from which intermolecular correlations were ob-
1
Figure 1. Partial H NMR (400 MHz, D
2
O, 298 K) spectra: a) model compound G3 (4.0 mm); b) G3 (3.0 mm) and WP6 (6.0 mm); c) WP6 (6.0 mm).
Chem. Eur. J. 2014, 20, 1 – 14
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served between proton H on the azobenzene moiety and pro-
d
tons H and H of WP6, respectively. The results suggested that
1
2
the linear model compound G3 was threaded through the hy-
drophobic WP6 cavity to form a [2]pseudorotaxane with its
positive azobenzene-based quaternary ammonium head close
to the negative carboxylate groups of WP6, thereby generat-
ing an inclusion complex in the aqueous solution. Similarly, the
formation of WP6ꢀG4 inclusion complexes with the positive
azobenzene-based quaternary ammonium head of G4 close to
the negative carboxylate groups of WP6 was also confirmed
by NMR spectroscopy (for details see the Supporting Informa-
tion, Figures S1, S3, and S5).
Figure 2. TEM images of spherical micelles formed from WP6 and G1 gradu-
ally converted to layered structures: the samples prepared after a) 0, b) 3,
c) 7, and d) 14 days. e) Representative POM image under crossed polarizers
The stoichiometry of complexation for both WP6ꢀG3 and
WP6ꢀG4 in water was further investigated by the Job’s plot
method using UV/Vis spectroscopy, which confirmed the 1:1
binding stoichiometry for both WP6ꢀG3 and WP6ꢀG4 (Figur-
es S6 and S7 in the Supporting Information). The association
constants for WP6ꢀG3 and WP6ꢀG4 in water were deter-
ꢂ6
ꢂ5
of d). [WP6]=4.35ꢁ10 m and [G1]=5.0ꢁ10 m. Scale bars: a) 50 nm, b)
100 nm, c) 200 nm, d) 0.5 mm.
4
ꢂ1
mined to be (1.03ꢁ0.24)ꢁ10 m (Figure S8 in the Supporting
It was found that adding different amounts of WP6 to the
4
ꢂ1
Information) and (2.06ꢁ0.88)ꢁ10 m (Figure S9 in the Sup-
above G1 solution led to obvious changes of opalescence in-
1
[6b]
porting Information), respectively, by using H NMR titration
tensity in aqueous solution. Therefore, it was necessary to
experiments. The binding affinity for such host–guest systems
should be attributed to the cooperative electrostatic and hy-
drophobic interactions. All of the above host–guest investiga-
tions indicated that WP6 formed a “tadpole-like” 1:1 inclusion
complex with azobenzene-containing guest G1 or G2 as
shown in Scheme 1.
determine the best molar ratio between WP6 and G1 for con-
structing supramolecular aggregates, which was determined
by UV/Vis titration experiments. As shown in Figure 3, upon
gradually increasing the amount of WP6 that was added, the
absorbance evolution at 550 nm underwent a sharp increase
and then an inverse decrease upon further addition of WP6.
The rapid increase of the absorbance intensity indicated that
WP6 and G1 formed a higher-order complex with a tendency
toward amphiphilic aggregation, whereas it underwent disas-
sembly upon further addition of WP6, generating a simple 1:1
supramolecular inclusion complex. Thus, the best molar ratio
of 13.5:1 ([G1]/[WP6]) for the formation of supramolecular mi-
celles was observed at the inflection point. Based on this best
Construction of supramolecular nanocarriers in water based
on the host–guest complexation of WP6 with G1 or G2
After establishing the above amphiphilic WP6ꢀG1 and
WP6ꢀG2 supramolecular complexes, we further investigated
the construction of supramolecular nanocarriers from
WP6ꢀG1 and WP6ꢀG2 based on their amphiphilic property.
Initial investigation of the self-assembly behavior of G1 and G2
indicated that both of them could assemble into spherical mi-
cellar structures (Figure S12a and S14a in the Supporting Infor-
mation) with an average diameter of about 37 and 45 nm (Fig-
ure S13a and S14b in the Supporting Information), respectively.
The critical aggregation concentrations (CACs) for G1 and G2
molar ratio, the CAC for WP6ꢀG1 was determined to be 3.28ꢁ
ꢂ6
10
(Figure S18b in the Supporting Information), which
showed a pronounced decrease compared with the CAC value
of free G1 due to the formation of WP6ꢀG1 inclusion
complex.
Subsequently, based on z-potential experiments, we chose
the molar ratio of 11.5:1 ([G1]/[WP6], z-potential=ꢂ30.2 mV)
for further investigation of its drug-loading property (Fig-
ure S15 in the Supporting Information), since under the above
conditions, further agglomeration of the obtained supramolec-
ular micelles might not occur due to the repulsive forces-in-
ꢂ4
ꢂ4
were determined to be 1.49ꢁ10 and 1.33ꢁ10 m, respec-
tively, by using concentration-dependent conductivity meas-
urements (Figures S18a and S19a in the Supporting Informa-
ꢂ6
[22]
tion). Notably, when WP6 (4.35ꢁ10 m) was added to the
transparent G1 (5.0ꢁ10 m) solution (TEM and dynamic light
duced increasing stability. Unfortunately, all of the tested hy-
ꢂ5
drophobic anticancer drugs, such as cisplatin, carboplatin, and
adriamycin, could not be successfully loaded by such supra-
molecular micelles. The above problem had puzzled us for
a long time until we surprisingly found an interesting phenom-
enon that such micellar structures were not stable, and gradu-
ally converted to supramolecular layered structures with in-
creasing incubation time indicated by their TEM images
(Figure 2), which makes them unsuitable as a drug nanocarrier.
From the TEM images shown in Figure 2, it was clearly found
that the spherical micellar structures became irregular and
their boundary became blurred after 3 days (Figure 2b). After
scattering (DLS) confirmed that no micelle was observed),
a light blue opalescence and clear Tyndall effect were ob-
served, thus indicating that microaggregates were formed
based on the host–guest interactions between WP6 and G1.
The size and self-assembly morphology of WP6ꢀG1 aggre-
gates in water were determined by TEM and DLS experiments,
which showed that the WP6ꢀG1 complex formed a solid
spherical micellar structure (Figure 2a) with an average
diameter of about 220 nm (Figure S21a in the Supporting
Information).
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such a supramolecular system might have potential applica-
tions for the construction of liquid-crystal materials. We know
that it is the cooperative intermolecular hydrogen-bonding
and p–p interactions within the azobenzene and benzamido
motifs of G1 that endow it with such unique self-assembly be-
havior; therefore, we envisaged that the cooperative interac-
tions might be weakened by removing the benzene ring from
the hydrophobic region of G1, and the designed azobenzene
guest G2 might be suitable to form stable supramolecular
nanocarriers with WP6 by self-assembly.
Along this line, we further investigated whether the estab-
lished amphiphilic WP6ꢀG2 complex could be used to con-
struct supramolecular nanocarriers. Similar to the above
WP6ꢀG1 solution, a clear Tyndall effect could be observed
ꢂ5
ꢂ4
from a mixture of WP6 (1.11ꢁ10 m) and G2 (1.0ꢁ10 m) in
aqueous solution, which suggested the formation of microag-
gregates. The best molar ratio of WP6 and G2 for such amphi-
philic aggregation was determined to be 11:1 ([G2]/[WP6]) by
using similar UV/Vis titration experiments (for details see the
Supporting Information, Figure S16). The CAC for WP6ꢀG2
ꢂ6
was determined to be 2.52ꢁ10 m (Figure S19b in the Sup-
porting Information), which also showed a pronounced de-
crease compared with the CAC value of free G2. Furthermore,
the size and morphology of this nanostructured aggregate
formed by WP6ꢀG2 supramolecular amphiphile in aqueous
solution are shown in Figure 4 by DLS and TEM measurements,
respectively. The DLS results showed that the WP6ꢀG2 com-
plex formed aggregates with a narrow size distribution, giving
an average diameter of 160 nm (Figure 4a), and TEM images
revealed a hollow spherical morphology with a diameter rang-
ing from 100 to 200 nm, thus indicating convincingly the for-
mation of supramolecular vesicles (Figure 4b), which were dif-
ferent from the micellar structures formed from WP6ꢀG1.
Moreover, the thickness of the hollow vesicles was calculated
to be approximately 5 nm from their TEM image (Figure 4c).
Considering that the extended length of two molecules of
WP6ꢀG2 inclusion complex calculated by Chem3D is about
Figure 3. a) UV/Vis absorption of a mixture of WP6 and G1 in water with
constant G1 concentration (0.02 mm) on increasing the concentration of
WP6 (0.05–0.33 equiv) at 258C. b) Dependence of the relative absorption in-
tensity at 500 nm on the WP6 concentration with a fixed concentration of
G1 (0.02 mm) at 258C.
4.9 nm (Figure S11 in the Supporting Information), the ob-
7
days, there was almost no micellar structure, and only some
layer-like structures were observed (Figure 2c). With increasing
incubation time, large numbers of layered structures were ob-
served after 14 days (Figure 2d). Moreover, DLS results also
demonstrated that the micelles gradually became large aggre-
gates with diameters of approximately 4 to 6 mm (Figure S21
in the Supporting Information). We speculated that such
a gradual morphological change, which was similar to that of
free G1 (Figures S12 and S13 in the Supporting Information),
might be associated with the liquid-crystalline behavior of G1,
since the azobenzene motif, especially linking with an extra
phenylamide group, has been widely used as a building block
[
23]
to construct various liquid-crystal materials. Further investi-
gation using polarized optical microscopy (POM) suggested
that the layered structures formed from WP6ꢀG1 complex ex-
hibited predominantly a mosaic texture (Figure 2e), further
suggesting that the above morphological change was associat-
ed with the liquid-crystalline property of G1. This means that
Figure 4. a) DLS data of the WP6ꢀG2 aggregates. b)–e) TEM images:
b) WP6ꢀG2 aggregates; c) enlarged image of b); d) WP6ꢀG2 aggregates
after the solution pH was adjusted to 6.2; e) WP6ꢀG2 aggregates after the
ꢂ5
solution pH was adjusted back to 7.4. [WP6]=1.11ꢁ10 m and
ꢂ4
[G2]=1.0ꢁ10 m.
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tained vesicles could possess a bilayer structure with two hy-
drophilic carboxylate shell layers and one hydrophobic alkyl
chain core layer as shown in Scheme 1. In addition, z-potential
experiments were also performed to examine the stability of
the obtained vesicles with different G2/WP6 molar ratios (Fig-
ure S17 in the Supporting Information). The results suggested
that the z-potential of the obtained vesicles changed gradually
from positive (28.5 mV, [G2]/[WP6]=12:1) to negative
(
ꢂ59.1 mV, [G2]/[WP6]=9:1) upon the addition of WP6. In par-
ticular, the z-potential of the vesicles formed at the best molar
fraction ([G2]/[WP6]=11:1) was close to 0 mV, which means
that the vesicles obtained at the best molar ratio were not
stable due to the absence of repulsive forces among vesicles.
Therefore, considering the repulsive forces-induced increasing
Figure 5. a) UV/Vis spectra of the vesicular solution of WP6ꢀG2 [initial
(
black line) and after irradiation with UV light at 365 nm for 15 min (red
line), and then after irradiation with visible light at 435 nm for 15 min (blue
ꢂ5
ꢂ4
line)]. [WP6]=1.11ꢁ10 m and [G2]=1.0ꢁ10 m). b) WP6ꢀG2 aggregates
after irradiation with UV light at 365 nm for 15 min; c) WP6ꢀG2 aggregates
after irradiation with UV light at 365 nm, and then further irradiation with
visible light at 435 nm for 15 min.
[
22]
stability of vesicles, a molar ratio of 9:1 ([G2]/[WP6], z-po-
tential=ꢂ59.1) was chosen for further investigation of their
stimuli-responsive behavior as well as their application in drug
delivery.
solution with visible light at 435 nm, large numbers of vesicles
were observed again (Figure 5c), which was also supported by
DLS experiments (Figure S22c in the Supporting Information).
All the above results clearly demonstrated that such WP6ꢀG2
supramolecular vesicles exhibited good pH- and UV-respon-
siveness, which endows them with the ability to encapsulate
substrates under neutral conditions and release them in re-
sponse to acidic conditions or upon UV stimulation.
Photo- and pH-responsiveness of the WP6ꢀG2 supramolec-
ular vesicles
The multistimulus-responsive properties of supramolecular
vesicles are of extreme importance for their applications in
[
24]
DDSs. Compound WP6 has been demonstrated to show pH-
[
6a]
responsiveness in aqueous solution,
and the azobenzene
motif is a perfect photoresponsive building block for the con-
struction of various photoresponsive supramolecular sys-
Encapsulation of hydrophilic drug MTZ by WP6ꢀG2 vesicles
[
11a,25]
tems.
As expected, the Tyndall effect for WP6ꢀG2 solu-
and its photo- and pH-responsive in vitro release
tion disappeared after adjusting the solution pH to 6.2; mean-
while, no vesicle was observed in the TEM image (Figure 4d)
due to the precipitation of WP6 as its acid form out of the
acidic aqueous solution. Moreover, when the pH value of the
above solution was adjusted back to 7.4, vesicle re-formation
was observed (Figure 4e), which was also verified by DLS ex-
periment (Figure S22b in the Supporting Information), clearly
confirming the pH-responsive behavior of the WP6ꢀG2 vesi-
cles due to the reversible assembly/disassembly process of the
amphiphilic host–guest complex.
The hollow vesicles prefer to load hydrophilic drugs, so MTZ,
a chemotherapy drug that is widely used to treat a broad
[26]
range of solid malignant tumors, was chosen as a model
drug to investigate the encapsulation efficiency of WP6ꢀG2
vesicles as well as their release behavior upon external stimula-
tion. Compared with the pale yellow WP6ꢀG2 vesicular solu-
tion, the MTZ-loaded vesicular solution turned light blue after
removing the excess unloaded MTZ molecules by dialysis,
which indicated that MTZ was successfully encapsulated into
the WP6ꢀG2 vesicle cavities (Figure 6a). Meanwhile, the UV
absorption of MTZ-loaded vesicular solution became much
stronger from 580 to 660 nm than for the unloaded vesicular
solution (Figure 6b), which represents the characteristic ab-
sorption of MTZ in aqueous solution. Moreover, DLS results
(Figure 6c) and the TEM image (Figure 6d) showed that MTZ-
loaded vesicles were much larger in size (ꢃ364 nm) than the
unloaded vesicles (ꢃ160 nm), which further confirmed that
MTZ molecules were successfully loaded into the vesicle cavi-
ties. Based on the UV/Vis absorption spectra, the MTZ encapsu-
lation efficiency was calculated to be 76%, thus indicating that
such supramolecular vesicles have a very efficient drug-loading
Besides the pH-responsiveness, more importantly, these ob-
tained WP6ꢀG2 supramolecular vesicles exhibited good pho-
toresponsive properties due to the UV-induced E/Z isomeriza-
tion of G2. As shown in Figure 5a, upon irradiation of the
WP6ꢀG2 vesicular solution with UV light at 365 nm, the ab-
sorption band at around 360 nm decreased remarkably, thus
indicating the photoisomerization of the trans-G2 to the cis-G2
state induced by UV irradiation. Conversely, upon further irradi-
ation of the above solution with visible light at 435 nm, the ab-
sorption peak at 360 nm attributable to trans-G2 increased to
almost its original intensity, which suggests a reversible
change from the cis to the trans form; such reversible transfor-
1
[9,10]
mation was also supported by a H NMR spectroscopic study
capability.
(
Figure S10 in the Supporting Information). Furthermore, the
Subsequently, the release behavior of MTZ-loaded supra-
molecular vesicles was investigated under an acidic pH envi-
ronment and/or upon UV stimulation. As shown in Figure 7a,
under simulated physiological conditions (pH 7.4), the cumula-
tive leakage of MTZ from MTZ-loaded vesicles was less than
TEM images showed that when the WP6ꢀG2 vesicular solution
was irradiated with UV light, most of the vesicles collapsed
and only a few irregular aggregates could be observed (Fig-
ure 5b). On the contrary, upon further irradiation of the above
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the first 2 h from the profiles. The microenvironment of tumor
cells is acidic due to the presence of excessive lactic acid and
CO as metabolites, so the rapid release of MTZ from MTZ-
2
loaded vesicles can be selectively triggered in tumor tissues,
which is extremely significant for specific targeted therapy,
shortening the administration time and greatly increasing the
[27]
curative effect. On the other hand, when the MTZ-loaded ve-
sicular solution was irradiated with UV light, the absorption
peak at around 360 nm attributable to trans-G2 decreased dra-
matically, whereas the absorption band at 660 nm correspond-
ing to the characteristic absorption of MTZ gradually increased
with the irradiation time (Figure 7b). This suggested the gradu-
al disassembly of vesicles due to the configuration change of
trans-G2 to cis-G2, which resulted in the gradual release of
MTZ from MTZ-loaded vesicles, and the cumulative amount of
released drug was about 60% after 4 h (Figure 7c).
Thereafter, the dual pH-/UV-responsive properties of MTZ-
loaded vesicles were also investigated. When low pH and UV
irradiation were simultaneously exerted on the MTZ-loaded
vesicles, a burst release of MTZ was observed during the initial
first hour because of the quick disaggregation of vesicles, and
the cumulative amount of released drug was up to 100%
(pH 4.4) and 74% (pH 6.2) after 4 h (Figure 7d). This is consis-
tent with the results of acid-stimulated precipitation of WP6 as
its acid form and photoinduced disassembly of the supra-
molecular vesicles. Overall, these results showed that such
Figure 6. a) Color change of MTZ-loaded vesicles (A) compared with unload-
ed vesicles (B). b) UV/Vis absorption spectra of the solution of MTZ, unload-
ed vesicles, and MTZ-loaded vesicles at 258C in water. c) DLS data of MTZ-
loaded vesicles. d) TEM image of MTZ-loaded vesicles.
8
% within 12 h. However, when the solution pH was adjusted
to acidity, MTZ was released in significant amounts with a cu-
mulative release amount of 44% at pH 6.2 and 93% at pH 4.4
within 12 h. In particular, rapid MTZ release was observed in
Figure 7. a) pH-responsive MTZ release profiles of MTZ-loaded vesicles in release media with different pH values. b) UV/Vis absorption spectra of the solution
of MTZ-loaded vesicles in water (pH 7.4) after irradiation with UV light at 365 nm for different times (0–240 min). c) Release of MTZ from MTZ-loaded vesicles
in water (pH 7.4) after irradiation with UV light at 365 nm for different times (0–240 min). d) Dual pH-/UV-responsive MTZ release profiles of MTZ-loaded vesi-
cles.
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MTZ-loaded vesicles were able to realize a quick and efficient
release of the encapsulated drugs when they were synchro-
nously triggered by low pH and UV light.
Cytotoxicity and anticancer efficiency of MTZ-loaded vesi-
cles
Subsequent stability investigations of the WP6ꢀG2 vesicles in
phosphate-buffered saline (PBS) or under physiological condi-
tions, such as 150 mm NaCl solution and 10% serum, showed
that the cumulative leakage of MTZ from MTZ-loaded vesicles
was less than 5% within 48 h in PBS buffer or 10% serum.
Even in the hypertonic NaCl solution (150 mm), the cumulative
leakage of MTZ from MTZ-loaded vesicles was only about 7%
within 48 h (Figure 8), which indicated that MTZ-loaded vesi-
Figure 8. Stability of the MTZ-loaded WP6ꢀG2 vesicles in PBS buffer
(pH 7.4), 150 mm NaCl solution, and 10% serum at 378C (0–48 h, vesicles
were prepared in 0.01m PBS buffer).
cles have good stability under physiological conditions and
they are ideal candidates for DDSs. Moreover, as DDS materials,
the biocompatibility of the carrier is also a key index for its bio-
medical application. Compound WP6 has been demonstrated
to be significantly biocompatible in aqueous media, and there-
fore the cytotoxicity of guest G2 and unloaded vesicles was
evaluated by the MTT method against NIH3T3 normal cells.
Figure 9a shows the cell viability after 24 h of incubation with
G2 or unloaded vesicles at a concentration varying from 5 to
Figure 9. a) In vitro cytotoxicity of guest G2 and unloaded vesicles against
NIH3T3 cells after 24 h of incubation. b) Effect of MTZ-loaded vesicles and
free MTZ on the viability of NIH3T3 cells at different times. c) Anticancer ac-
tivity of unloaded vesicles, MTZ-loaded vesicles, and free MTZ with or with-
out UV irradiation against MCF-7 cells at different times. Statistically signifi-
cant differences were observed (p<0.05) (*).
100 mm. Clearly, the cell viability remains above 80% even
when the concentration of G2 or unloaded vesicles increases
to 100 mm. Consequently, WP6ꢀG2 supramolecular vesicles
that have good biocompatibility are a suitable material for
DDSs. In addition, it was found that the relative viability of
NIH3T3 cells incubated with MTZ-loaded vesicles was always
much higher than that in the free MTZ from 48 to 96 h (p<
assay against MCF-7 cancer cells, for which the unloaded vesi-
cles and free MTZ were used as controls. As shown in Fig-
ure 9c, it was found that the relative cell viability of the MTZ-
loaded vesicle group against cancer cells with UV irradiation
was only 20% after 96 h, which showed similar therapeutic ef-
fects to the free MTZ group (17%), whereas that without irradi-
ation was about 27% (Figure 9c), which indicated that addi-
tional UV stimulation facilitated drug release compared with
the single pH stimulus. Additionally, for the unloaded vesicle
group, the cell viability without UV irradiation retained about
0.05, Figure 9b). Also, the morphology of living cells in the
MTZ-loaded vesicle groups was better than that in the free
MTZ after 96 h (Figure 10), which indicated that the toxicity of
MTZ was remarkably reduced upon its encapsulation by the
WP6ꢀG2 vesicles.
Moreover, the anticancer activity of MTZ-loaded vesicles
with or without UV irradiation was also investigated by MTT
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totic cells of the whole tested MCF-7 cells was about 47.2%,
and the early apoptotic cells took a percentage of 37.5%
(
Figure 11). When the incubation time was extended to 96 h,
the ratio of apoptotic cells significantly increased to 70.2%,
and the majority of apoptotic cells were still the early apoptot-
ic cells (Figure 11). Therefore, the above flow cytometry data
demonstrated that MTZ-loaded vesicles could induce apoptosis
in MCF-7 cells and the apoptosis effect was time-dependent.
Figure 10. Images of living NIH3T3 cells in a) blank, b) G2, c) unloaded vesi-
cle, d) MTZ-loaded vesicle, and e) MTZ groups after 96 h. Images of living
MCF-7 cells in f) blank, g) MTZ, h) MTZ-loaded vesicle, i) MTZ-loaded vesicle
with UV irradiation, and j) unloaded vesicle with UV irradiation groups after
Conclusion
9
6 h.
Two novel types of pillararene-based supramolecular nanocarri-
ers were successfully constructed from an amphiphilic host–
guest inclusion complex in water: 1) supramolecular micelles
were formed from WP6 and azobenzene guest G1, which
gradually transformed into layered structures with liquid-crys-
talline properties, probably due to the presence of cooperative
intermolecular hydrogen-bonding and p–p interactions within
the azobenzene and benzamido motifs of G1; and 2) stable
supramolecular vesicles were obtained from WP6 and G2,
which displayed a significant dual photo- and pH-responsive
behavior. Significantly, drug loading and in vitro release experi-
ments demonstrated that MTZ could be successfully encapsu-
lated into the WP6ꢀG2 supramolecular vesicles, and the re-
sulting MTZ-loaded vesicles exhibited an excellent responsive-
ness and rapid release of the encapsulated MTZ in an acid en-
vironment similar to that of tumor cells or with external UV ir-
radiation; in a simulated normal physiological environment,
such supramolecular vesicles maintain good stability. More im-
portantly, cytotoxicity tests and flow cytometric analysis further
demonstrated that the loading of MTZ by WP6ꢀG2 supra-
molecular vesicles did not affect the therapeutic effect of MTZ
on cancer cells, especially with additional UV stimulus, whereas
its cytotoxicity for normal cells was remarkably reduced, and
the cancer cell death caused by MTZ-loaded vesicles was asso-
ciated with apoptosis. The present supramolecular vesicle
system with dual pH- and UV-responsiveness is expected to
have great potential applications for the development of
“smart” drug-delivery systems. The anticancer activity of such
MTZ-loaded vesicles against a human melanoma cell line and
8
6%, and only a slight decrease (4%) was found after irradia-
tion, thus indicating that the effect of irradiation was negligi-
ble, which could also be observed from the morphology of
living cells (Figure 10). All of the above results implied that
loading of MTZ by WP6ꢀG2 vesicles reduced its cytotoxicity
for normal cells but kept the therapeutic effect of MTZ for
cancer cells, especially with external UV stimulus. A rational ex-
planation is that compared with normal cells, tumor cells have
strong phagocytosis and an acidic microenvironment to cause
the disassembly of MTZ-loaded vesicles to release MTZ, espe-
[
28]
cially with external UV stimulus. Therefore, such supramolec-
ular vesicles based on the host–guest complexation of WP6
and azobenzene derivatives are promising DDSs.
Cell apoptosis
To further understand the molecular mechanism of the thera-
peutic effect of MTZ-loaded vesicles on cancer cells, flow cyto-
metric analysis based on the fluorescein isothiocyanate (FITC)-
Annexin V/propidium iodide (PI) method was used to investi-
gate whether the antitumor effect of MTZ-loaded vesicles was
associated with apoptosis. MCF-7 cells were incubated with
MTZ-loaded vesicles for 48 and 96 h, and then they were sub-
jected to FITC-Annexin V/PI staining. The cells without treat-
ment by MTZ-loaded vesicles were used as control. After incu-
bating with MTZ-loaded vesicles for 48 h, the quantity of apop-
Figure 11. Flow cytometric analysis of MCF-7 cells treated with MTZ-loaded vesicles (0.94 mm) after incubation for different times. Q1, necrotic cells; Q2, late
apoptotic cells; Q3, living cells; Q4, early apoptotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area.
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animal testing are in progress in our laboratory to investigate
their potential application for the therapy of skin carcinoma.
tive-ion mode with direct infusion. UV/Vis spectra were recorded in
a quartz cell (light path 10 mm) on a PerkinElmer Lambda 35 UV/
Vis spectrometer. The critical aggregation concentration (CAC) de-
termination of G1, WP6ꢀG1, G2, and WP6ꢀG2 was carried out on
a DDS-307A instrument according to procedures reported in the
literature. TEM images were recorded on a JEM-2100 instrument.
Samples were prepared by dropping the solution onto a carbon-
coated copper grid. DLS measurements were performed with
a Brookhaven BI-9000AT system (Brookhaven Instruments Corpora-
tion, USA) equipped with a 200 mW laser light and operating at
l=514 nm. z-potential measurement was performed at 258C on
a Zetasizer Nano Z apparatus (Malvern Instruments Ltd., UK) using
the Smoluchowski model for the calculation of the z-potential
from the measured electrophoretic mobility.
Experimental Section
[6c]
Materials
Mitoxantrone (MTZ; >98%) was provided by Melone Pharma
(
(
Dalian, China). Tetrachloromethane (CCl4), dichloromethane
CH Cl ), and acetonitrile (CH CN) were dried according to proce-
2
2
3
dures described in the literature, and other solvents were used as
received without further purification unless otherwise stated.
Column chromatography was performed with silica gel (200–300
mesh) produced by Qingdao Marine Chemical Factory, Qingdao
(
China). All the other chemicals were purchased from commercial
suppliers, were of analytical grade, and used without further
purification.
Synthetic procedures
All reactions were performed under an ambient atmosphere unless
otherwise stated. Compound WP6 was synthesized and purified
according to previously reported procedures (Scheme S1 in the
Measurements
[12f]
Supporting Information).
The detailed synthetic procedures for
NMR spectra were recorded on a Bruker DPX 300 MHz spectrome-
ter (or Bruker DPX 400 MHz spectrometer) with internal standard
tetramethylsilane (TMS) and solvent signals as internal references
at 258C, and the chemical shifts (d) were expressed in ppm and
J values given in Hertz. Low-resolution electrospray ionization mass
spectrometry (LR-ESI-MS) was performed on Finnigan Mat TSQ
azobenzene derivatives (G1 and G2) are shown in Scheme 2, and
the synthetic routes of the model compounds (G3 and G4) are
shown in Scheme S2 in the Supporting Information.
Synthesis of compound 1: Ethyl 4-hydroxybenzoate (3.32 g,
20 mmol) was dissolved in CH CN (200 mL), K2CO3 (16.6 g,
3
7
000 instruments. High-resolution electrospray ionization mass
120 mmol) and 1-bromodecane (6.64 g, 30 mmol) were added to
the above solution, and then the reaction mixture was stirred at
reflux temperature overnight. After filtering, the solvent was re-
spectrometry (HR-ESI-MS) was performed on an Agilent 6540 Q-
TOF LC/MS system equipped with an ESI probe operating in posi-
Scheme 2. Synthesis of the azobenzene derivatives G1 and G2. NBS=N-bromosuccinimide, BPO=benzoyl peroxide.
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moved under vacuum to give the crude product. Column chroma-
pressure. The residue was diluted with chloroform (20.0 mL), the
orange precipitate was collected by filtration and washed with
CH Cl to remove 5, and the target product G1 was obtained as an
tography was used in the final separation with hexane/CH Cl2
2
(
(
(
20:1–1:1, v/v) as the eluent, to give compound 1 as a colorless oil
2
2
1
1
6.1 g, 19.9 mmol, 99%). H NMR (300 MHz, CDCl ): d=8.01–7.96
orange solid (158 mg, about 86.4%). H NMR (300 MHz, CD OD):
3
3
m, 2H), 6.92–6.87 (m, 2H), 4.34 (q, J=7.1 Hz, 2H), 4.00 (t, J=
d=8.10–7.88 (m, 8H), 7.75 (d, J=8.1 Hz, 2H), 7.05 (d, J=8.8 Hz,
2H), 4.63 (s, 2H), 4.08 (t, J=6.4 Hz, 2H), 3.17 (s, 9H), 1.87–1.76 (m,
2H), 1.51 (brs, 2H), 1.32 (brs, 12H), 0.90 ppm (t, J=6.0 Hz, 3H);
6
6
.6 Hz, 2H), 1.85–1.73 (m, 2H), 1.56–1.15 (m, 18H), 0.88 ppm (t, J=
.7 Hz, 3H).
13
C NMR (75 MHz, CD OD): d=167.1, 162.5, 153.9, 148.6, 142.6,
Synthesis of compound 2: Compound 1 (3.06 g, 10 mmol) was
dissolved in EtOH (50 mL) and KOH (3.37 g, 60 mmol) was dis-
solved in water (5 mL), then the KOH solution was added to the so-
lution of compound 1 and the reaction mixture was stirred at
reflux temperature for 2 h. After the reaction was completed, the
reaction mixture was poured into water (100 mL) and CH Cl
3
1
6
33.7, 129.6, 129.4, 126.3, 123.7, 122.9, 122.0, 120.6, 114.0, 68.7,
8.0, 51.9, 31.7, 29.3, 29.3, 29.1, 29.1, 28.9, 25.8, 22.4, 13.1 ppm; ESI-
+
+
MS: m/z (%): 529.40 [MꢂBr] ; HR-ESI-MS: m/z calcd for [MꢂBr]
C H N O : 529.3537; found: 529.3542.
33 45
4
2
2
2
Synthesis of compound 7: Dodecanoic acid (2.0 g, 10 mmol) was
(
3
100 mL) was added, then the pH of the solution was adjusted to
.0 by using concentrated hydrochloric acid (37%). The organic
layer was separated and dried over Na SO . After removing the sol-
suspended in thionyl chloride (SOCl , 8 mL), then the mixture was
2
stirred at 658C overnight. After evaporating the excess thionyl
chloride, the crude product 7 was obtained as a colorless oil (2.0 g,
9.14 mmol, 91.4%).
2
4
vent under vacuum, the product 2 was obtained as a colorless oil
1
(
2.6 g, 9.34 mmol, 93.4%). H NMR (300 MHz, CDCl ): d=11.01 (brs,
3
1H), 8.05 (d, J=8.8 Hz, 2H), 6.92 (d, J=8.8 Hz, 2H), 4.01 (t, J=
6.5 Hz, 2H), 1.86–1.73 (m, 2H), 1.46–1.43 (m, 2H), 1.27 (s, 13H),
0.88 ppm (t, J=6.0 Hz, 3H).
[29]
Synthesis of compound 8: Compound 4 (0.62 g, 2.9 mmol) was
dissolved in dry CH Cl2 (15 mL) and Et N (0.59 g, 5.8 mmol) was
added to the solution. Compound 7 (0.97 g, 4.4 mmol) was dis-
2
3
Synthesis of compound 3: Compound 2 (2.6 g, 9.32 mmol) was
solved in dry CH Cl (5 mL) and then it was added dropwise to the
2
2
suspended in thionyl chloride (SOCl , 6.6 mL). Then the mixture
was stirred at 658C overnight. After evaporating the excess thionyl
chloride, the crude product 3 was obtained as a colorless oil
above solution of compound 4. After stirring overnight at 258C,
the solvent was removed under vacuum to give a yellow solid.
Column chromatography was used in the final separation with pe-
troleum ether/CH Cl (1:1, v/v) as the eluent, to give a yellow solid
2
(
2.37 g, 7.98 mmol, 85.6%).
2
2
[29]
1
Synthesis of compound 5: Compound 4 (1.06 g, 5.0 mmol) was
8 (1.61 g, 4.09 mmol, 93%). H NMR (300 MHz, CDCl ): d=7.92 (d,
3
dissolved in dry CH Cl2 (30 mL) and Et N (0.76 g, 7.5 mmol) was
added to the solution. Compound 3 (2.23 g, 7.5 mmol) was dis-
J=8.8 Hz, 2H), 7.82 (d, J=8.3 Hz, 2H), 7.70 (d, J=8.7 Hz, 2H), 7.43
(s, 1H), 7.30 (d, J=8.2 Hz, 2H), 2.43 (s, 3H), 2.42–2.34 (m, 2H),
1.81–1.67 (m, 2H), 1.44–1.17 (m, 16H), 0.88 ppm (t, J=6.7 Hz, 3H);
2
3
solved in dry CH Cl (5 mL) and then it was added dropwise to the
2
2
13
above solution of 4 in 30 min. After stirring overnight at 258C, the
solvent was removed under vacuum to give a yellow solid. Column
chromatography was used in the final separation with petroleum
ether/CH Cl (20:1, v/v) as the eluent, to give yellow solid com-
C NMR (75 MHz, CDCl ): d=171.6, 150.5, 148.8, 141.4, 140.5,
3
129.8, 124.1, 122.8, 119.7, 38.0, 31.9, 29.6, 29.5, 29.4, 29.4, 29.3,
+
25.6, 22.7, 21.5, 14.1 ppm; ESI-MS: m/z (%): 394.25 [M+H] ; HR-ESI-
+
MS: m/z calcd for [M+H]
C H N O: 394.2858; found: 394.2861;
25 36 3
2
2
1
+
pound 5 (2.21 g, 4.69 mmol, 93.7%). H NMR (300 MHz, [D ]DMSO):
[M+Na] C H N ONa: 416.2678; found: 416.2683.
25 35 3
6
d=10.40 (s, 1H), 8.00 (dd, J=12.6, 8.9 Hz, 4H), 7.90 (d, J=8.9 Hz,
Synthesis of compound 9: Compound 8 (0.5 g, 1.27 mmol), NBS
0.34 g, 1.9 mmol), and BPO (15 mg, 0.064 mmol) were dissolved in
CCl (15 mL) under an argon atmosphere, then the reaction mix-
2
8
1
H), 7.79 (d, J=8.3 Hz, 2H), 7.40 (d, J=8.3 Hz, 2H), 7.07 (d, J=
.8 Hz, 2H), 4.06 (t, J=6.5 Hz, 2H), 2.41 (s, 3H), 1.80–1.67 (m, 2H),
.43–1.26 (m, 14H), 0.86 ppm (t, J=6.6 Hz, 3H); H NMR (300 MHz,
(
1
4
1
ture was heated at reflux for 48 h. When H NMR spectroscopy in-
dicated the reaction was almost completed, the mixture was
cooled to 08C and filtered under vacuum to give a yellow powder,
which was washed with cold diethyl ether (3ꢁ20 mL). Finally, the
desired compound 9 was obtained as a mixture with a small
amount of the starting material 8 (0.22 g). The two compounds
CDCl ): d=7.94 (d, J=8.6 Hz, 3H), 7.87–7.77 (m, 5H), 7.30 (d, J=
3
8
3
0
1
6
.1 Hz, 2H), 6.97 (d, J=8.7 Hz, 2H), 4.02 (t, J=6.5 Hz, 2H), 2.43 (s,
H), 1.87–1.74 (m, 2H), 1.52–1.40 (m, 2H), 1.28–1.25 (m, 12H),
13
.88 ppm (t, J=6.4 Hz, 3H); C NMR (75 MHz, CDCl ): d=165.2,
3
62.4, 141.3, 140.5, 129.7, 128.9, 126.5, 123.9, 122.8, 120.0, 114.6,
8.3, 31.9, 29.7, 29.6, 29.4, 29.3, 29.1, 26.0, 22.7, 21.5, 14.1 ppm; ESI-
+
+
have the same R value based on TLC, which was confirmed by the
f
MS: m/z (%): 472.30 [M+ H] ; HR-ESI-MS: m/z calcd for [M+H]
C H N O : 472.2964; found: 472.2966; [M+Na] C H N O Na:
1
+
H NMR spectrum (Figure S32 in the Supporting Information).
3
0
38
3
2
30 37
3
2
4
94.2784; found: 494.2788.
Synthesis of compound 6: Compound 5 (236 mg, 0.5 mmol), NBS
143 mg, 0.55 mmol), and BPO (4.6 mg, 0.09 mmol) were dissolved
Synthesis of azobenzene derivative G2: A solution of compound
9
(220 mg, also having a small amount of compound 8) in ethanol
(
(
5.0 mL) and Me N (30% in water, 2.0 mL) was stirred at reflux tem-
3
in CCl (5 mL) under an argon atmosphere, then the reaction mix-
ture was heated at reflux for 24 h. When H NMR spectroscopy in-
dicated the reaction was completed, the mixture was cooled to
0
was washed with cold CH Cl (3ꢁ20 mL). Finally, the desired com-
pound 6 was obtained as a mixture with a small amount of the
starting material compound 5 (0.17 g). The two compounds have
4
perature for 24 h. The solution was concentrated under reduced
1
pressure. The residue was diluted with diethyl ether/CH Cl (v/v=
2
2
5
:1, 20.0 mL), the orange precipitate was collected by filtration and
8C and filtered under vacuum to give a yellow powder, which
washed with diethyl ether/CH Cl (v/v=5:1) to remove compound
2
2
2
2
8, and then the desired product G2 was obtained as an orange
1
solid (150 mg, about 95%). H NMR (300 MHz, CD OD): d=8.03–
3
7
.93 (m, 4H), 7.86–7.65 (m, 4H), 4.64 (s, 2H), 3.13 (s, 9H), 2.41 (d,
the same R value based on TLC, which was confirmed by the
f
J=6.6 Hz, 2H), 1.72 (s, 2H), 1.33 (brs, 16H), 0.89 ppm (brs, 3H);
1
H NMR spectrum (Figure S27 in the Supporting Information).
13
C NMR (75 MHz, CD OD): d=173.6, 153.9, 148.5, 142.3, 133.7,
3
Synthesis of azobenzene derivative G1: A solution of compound
129.8, 123.7, 122.8, 119.6, 68.5, 51.9, 36.7, 31.7, 29.3, 29.2, 29.1,
+
6
(165 mg, also having a small amount of compound 5) in ethanol
28.9, 25.4, 22.3, 13.0 ppm; ESI-MS: m/z (%): 451.35 [MꢂBr] ; HR-
+
(
3.0 mL) and Me N (30% in water, 1.0 mL) was stirred at reflux tem-
ESI-MS: m/z calcd for [MꢂBr] C H N O: 451.3431; found:
3
28 43
4
perature for 24 h. The solution was concentrated under reduced
451.3435.
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Fabrication of the MTZ-loaded vesicles
MTT assay: The relative cytotoxicities of G2, unloaded vesicles,
MTZ, and MTZ-loaded vesicles against NIH3T3 normal cells and
MCF-7 cancer cells were evaluated in vitro by 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the
MTZ-loaded vesicles were prepared as follows. A certain amount of
MTZ was added to an aqueous solution of G2 (1% EtOH was
added to improve the solubility of G2), then a certain concentra-
tion of WP6 solution was added quickly, and finally some water
was added until the volume of the solution reached 25 mL. The ul-
timate concentrations of MTZ, G2, and WP6 were 0.018, 0.09, and
5
cells were seeded in 96-well plates at a density of 1.0ꢁ10 cells per
well in complete DMEM (200 mL) containing 10% FBS supplement-
ꢂ1
ꢂ1
ed with penicillin (50 UmL ) and streptomycin (50 UmL ), and
cultured in 5% CO at 378C for 24 h. Then NIH3T3 cells were incu-
2
0
.01 mm, respectively. After standing overnight, the prepared MTZ-
loaded vesicles were purified by dialysis (molecular weight cutoff
0000) in distilled water several times until the water outside the
bated with G2, unloaded vesicles, MTZ, and MTZ-loaded vesicles.
MCF-7 cells were subsequently incubated with unloaded vesicles,
MTZ, and MTZ-loaded vesicles. The ultimate concentrations of
MTZ, WP6, and G2 were 0.94, 0.7, and 6.3 mm, respectively. For UV-
stimulus experiments, the cells were irradiated under UV light at
1
dialysis tube exhibited negligible MTZ fluorescence. For the cell ex-
periments, MTZ-loaded vesicles were prepared in PBS buffer by
using the same procedure as mentioned above.
3
65 nm (25 W) for 30 min after incubation with MTZ-loaded vesi-
cles for 3 h and cultured for another 24 h, and then the UV irradia-
tion procedure was repeated. At the end of each incubation (24,
48, 72, or 96 h), the cells were washed, replenished with fresh cul-
ture medium, and further incubated for 2 h. Subsequently, MTT so-
lution (20 mL) was added to each well and incubated for another
The MTZ encapsulation efficiency was calculated by the following
[7e]
equation [Eq. (1)],
in which mMTZ-loaded and mMTZ are the mass of
MTZ encapsulated in vesicles and mass of MTZ added, respectively.
The mass of MTZ was measured by a UV spectrophotometer at
6
60 nm and calculated relative to a standard calibration curve at
4
h. After that, the medium containing MTT was removed and di-
ꢂ1
concentrations from 0.184 to 6.68 mgmL in water.
methyl sulfoxide (DMSO, 100 mL) was added to each well to dis-
solve the MTT formazan crystals. Finally, the plates were shaken for
1
4
0 min, and the absorbance of formazan product was measured at
90 nm by a microplate reader (BioTek ELx808). Untreated cells in
Encapsulation efficiency ð%Þ ¼ ðmMTZ-loaded=mMTZÞ ꢄ 100
Stimuli-responsive behaviors of the MTZ-loaded vesicles
ð1Þ
media were used as the blank control. All experiments were carried
out with three replicates. The cytotoxicity was expressed as the
percentage of the cell viability relative to the blank control.
pH-Responsive drug release in vitro: Tris-HCl buffer solutions
0.01m, pH 7.4), citrate buffer solutions (0.05m, pH 6.2), and
Apoptosis assay: MCF-7 cells were seeded in 12-well plates at
a density of 1.0ꢁ10 cells per well in DMEM culture medium
(
5
sodium acetate buffer solutions (0.05m, pH 4.4) were used as drug
release media to simulate normal physiological conditions and the
intracellular conditions of tumors. In a typical release experiment,
MTZ-loaded vesicles (15 mL) were added to an appropriate release
medium (10 mL) at 378C. At selected time intervals, a portion
(
1 mL), cultured for 24 h, and then the culture medium was re-
moved. MTZ-loaded vesicles dissolved in DMEM culture medium at
a
final concentration of 0.94 mm were added to the wells
ꢂ1
(1 mLwell ) and the cells were incubated at 378C for 48 and 96 h.
MCF-7 cells without incubation of MTZ-loaded vesicles were used
as control. After incubation for 48 and 96 h, the cell solutions were
centrifuged for 5 min at 2000 rpm. Culture medium was removed
and the cells were washed with cold PBS twice. After removal of
the supernatants, the cells were resuspended in binding buffer
(
2 mL) of the release medium was taken out for measuring of the
released MTZ concentration by the UV/Vis absorption technique. A
nearly 100% release of MTZ from MTZ-loaded vesicles was ob-
tained by using a very low pH medium (solution of HCl, pH 3) in
which most of the vesicles collapsed.
(500 mL). The apoptotic cells were determined by staining using an
UV-Responsive drug release in vitro: To investigate the UV-re-
sponsive behaviors of the WP6ꢀG2 supramolecular vesicles, the
MTZ-loaded vesicular solution was placed vertically under a high-
pressure mercury lamp (365 nm, 200 W) at a distance of 10 cm. In
the cell experiments, an ultraviolet lamp (365 nm, 25 W) was used.
Annexin V-FITC apoptosis detection kit according to the manufac-
turer’s protocol: Annexin V-FITC (5 mL) and PI (5 mL, provided in the
kit) were added to the cell suspensions; after incubation in the
dark for 15 min, data for 1.0ꢁ10 gated events were collected and
analyzed by a BD FACSCalibur flow cytometer and CELLQuest
software.
4
Dual pH- and UV-responsive drug release in vitro: For the dual
pH- and UV-responsive release experiments of MTZ-loaded vesicles,
the detailed procedures were almost the same as those for the pH-
responsive experiments mentioned above, but additionally, the ve-
sicular solution was placed vertically under a high-pressure mercu-
ry lamp (365 nm, 200 W) at a distance of 10 cm, and detected at
selected time intervals. The concentration of MTZ was determined
by measuring the absorbance at 660 nm by using a standard ab-
sorbance versus concentration curve constructed for MTZ in the
corresponding release buffer.
Statistical analysis
Differences between treatment groups were analyzed statistically
using Student’s t-test. A statistically significant difference was re-
ported if p<0.05. The data were expressed as the mean ꢁ stan-
dard deviation from at least three separate experiments.
Acknowledgements
Cytotoxicity measurement
This work was supported by the National Basic Research Pro-
gram of China (2014CB846000, 2013CB934400), and National
Natural Science Foundation of China (Nos. 91227106,
Cell culture: MCF-7 cancer cells (a human breast cancer cell line)
and NIH3T3 normal cells (a mouse embryonic fibroblast cell line)
were cultivated, respectively, in Dulbecco’s modified Eagle’s
medium (DMEM) supplied with 10% fetal bovine serum (FBS) and
21202083, 21302092). We wish to thank Professor Dongzhong
ꢂ1
ꢂ1
antibiotics (50 UmL penicillin and 50 UmL streptomycin) at
78C in a humidified atmosphere containing 5% CO2.
Chen from Nanjing University for fruitful discussions and
suggestions.
3
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www.chemeurj.org
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Received: September 2, 2014
Published online on && &&, 0000
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
FULL PAPER
On the double: Two types of nanocarri-
ers are fabricated: supramolecular mi-
celles formed from water-soluble pil-
lar[6]arene (WP6) and azobenzene de-
rivative G1, which gradually transform
into layered structures, and supramolec-
ular vesicles formed by WP6 and guest
G2, which exhibit dual photo- and pH-
responsive behavior (see figure). Mitox-
antrone (MTZ)-loaded vesicles show
a similar therapeutic effect for cancer
cells to free MTZ and reduced damage
to normal cells.
&
Drug Delivery
X.-Y. Hu, K. Jia, Y. Cao, Y. Li, S. Qin,
F. Zhou, C. Lin, D. Zhang, L. Wang*
&
& – &&
Dual Photo- and pH-Responsive
Supramolecular Nanocarriers Based on
Water-Soluble Pillar[6]arene and
Different Azobenzene Derivatives for
Intracellular Anticancer Drug Delivery
&
&
Chem. Eur. J. 2014, 20, 1 – 14
www.chemeurj.org
14
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!