Metal ion, light, and redox responsive interaction of vesicles by a supramolecular switch

Article abstract of DOI:10.1002/chem.201304658

Chemical, photochemical and electrical stimuli are versatile possibilities to exert external control on self-assembled materials. Here, a trifunctional molecule that switches between an adhesive and a non-adhesive state in response to metal ions, or light, or oxidation is presented. To this end, an azobenzene-ferrocene conjugate with a flexible N,N-bis(3-aminopropyl)ethylenediamine spacer was designed as a multistimuli-responsive guest molecule that can form inclusion complexes with β-cyclodextrin. In the absence of any stimulus the guest molecule induces reversible aggregation of host vesicles composed of amphiphilic β-cyclodextrin due to the formation of intervesicular inclusion complexes. In this case, the guest molecule operates as a noncovalent cross-linker for the host vesicles. In response to any of three external stimuli (metal ions, UV irradiation, or oxidation), the conformation of the guest molecule changes and its affinity for the host vesicles is strongly reduced, which results in the dissociation of intervesicular complexes. Upon elimination or reversal of the stimuli (sequestration of metal ion, visible irradiation, or reduction) the affinity of the guest molecules for the host vesicles is restored. The reversible cross-linking and aggregation of the cyclodextrin vesicles in dilute aqueous solution was confirmed by isothermal titration calorimetry (ITC), optical density measurements at 600 nm (OD₆₀₀), dynamic light scattering (DLS), ζ-potential measurements and cyclic voltammetry (CV). To the best of our knowledge, a dynamic supramolecular system based on a molecular switch that responds orthogonally to three different stimuli is unprecedented.

Full text of DOI:10.1002/chem.201304658

DOI: 10.1002/chem.201304658
Full Paper
&
Molecular Recognition
Metal Ion, Light, and Redox Responsive Interaction of Vesicles by
a Supramolecular Switch
Avik Samanta and Bart Jan Ravoo*[a]
Abstract: Chemical, photochemical and electrical stimuli are
versatile possibilities to exert external control on self-assem-
bled materials. Here, a trifunctional molecule that switches
between an “adhesive” and a “non-adhesive” state in re-
sponse to metal ions, or light, or oxidation is presented. To
this end, an azobenzene–ferrocene conjugate with a flexible
N,N’-bis(3-aminopropyl)ethylenediamine spacer was de-
signed as a multistimuli-responsive guest molecule that can
form inclusion complexes with b-cyclodextrin. In the ab-
sence of any stimulus the guest molecule induces reversible
aggregation of host vesicles composed of amphiphilic b-cy-
clodextrin due to the formation of intervesicular inclusion
complexes. In this case, the guest molecule operates as
a noncovalent cross-linker for the host vesicles. In response
to any of three external stimuli (metal ions, UV irradiation, or
oxidation), the conformation of the guest molecule changes
and its affinity for the host vesicles is strongly reduced,
which results in the dissociation of intervesicular complexes.
Upon elimination or reversal of the stimuli (sequestration of
metal ion, visible irradiation, or reduction) the affinity of the
guest molecules for the host vesicles is restored. The revers-
ible cross-linking and aggregation of the cyclodextrin vesi-
cles in dilute aqueous solution was confirmed by isothermal
titration calorimetry (ITC), optical density measurements at
600 nm (OD600), dynamic light scattering (DLS), z-potential
measurements and cyclic voltammetry (CV). To the best of
our knowledge, a dynamic supramolecular system based on
a molecular switch that responds orthogonally to three dif-
ferent stimuli is unprecedented.
Introduction
drug and gene delivery systems, light-harvesting systems, and
microreactors.[8–15] The implantation of stimuli-responsive sites
can lead to the preparation of vesicles which are receptive to
light, redox, pH, temperature, or chemical stimuli.[16–18] Similarly,
aggregation, adhesion and fusion of vesicles can be induced
by change of pH, temperature, ionic strength, metal-ion com-
plexation and specific molecular recognition. Several groups
have investigated the interactions of vesicular systems induced
by hydrogen bonding,[19–22] electrostatic attraction[23,24] and
metal-ion coordination.[25–27] In the course of these investiga-
tions, it has become increasingly evident that the understand-
ing and control of multivalent interactions at the surface of the
vesicles is key to the elucidation and mimicking of recognition,
adhesion and fusion of biological cell membranes. In this re-
spect, vesicles and vesicle clusters which respond to external
stimuli, such as light, redox or metal-ion binding are particular-
ly attractive, since these stimuli can be delivered with high
spatial and/or temporal resolution so that dynamic assemblies
may occur that are not available in the absence of the stimu-
lus.
Confinement is instrumental to all biological and physiological
processes. Each living cell has a cell membrane composed of
phospholipids, proteins and carbohydrates which gives rise to
a closed interior separated from the extracellular environment.
Recognition, adhesion and fusion of membranes are crucial to
living cells since these mediate the transport of molecules be-
tween and within cells. The current boom in the field of bio-
mimicry includes the development of complex supramolecular
assemblies and soft materials which replicate and emulate bio-
logical structures and functions.[1] During the last decades,
many studies have been performed to replicate cell–cell inter-
action by constructing artificial nanocontainers, such as lipo-
somes, synthetic vesicles and polymersomes, which contribute
to an understanding of biological processes and provide novel
advances for future therapeutic applications.[2–7] Vesicles are
ubiquitous in living systems and have drawn significant atten-
tion to the advancement of chemistry, biology, and material
science. Fabrication of vesicles with desired size, structure, and
properties facilitates the development of biomimetic systems,
In recent years we have investigated the formation of uni-
lamellar vesicles of amphiphilic cyclodextrins (CDV) and the
molecular recognition of guest molecules at the surface of
such self-assembled nanoscale containers.[28–33] To this end, cy-
clodextrins (CD) are modified with long alkyl chains and short
oligo(ethylene glycol) head groups (Figure 1). These macrocy-
clic amphiphiles form unilamellar bilayer vesicles in aqueous
medium upon hydration of a thin film cast by evaporation
[a] A. Samanta, Prof. Dr. B. J. Ravoo
Organic Chemistry Institute and Graduate School of Chemistry
Westfꢀlische Wilhelms-Universitꢀt Mꢁnster
Corrensstrasse 40, 48149 Mꢁnster (Germany)
E-mail: b.j.ravoo@uni-muenster.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304658.
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induce the aggregation and adhesion of host vesicles. The con-
formation and/or polarity of each guest G1–G3 and hence its
affinity for the CDV can be controlled by metal ion, irradiation
and oxidation. Thus, the aggregation and disaggregation of
CDV can be controlled by any of three external stimuli. The in-
teraction of CDV and guest molecules G1–G3 was investigated
by isothermal titration calorimetry (ITC), optical density meas-
urements at 600 nm (OD600), dynamic light scattering (DLS), z-
potential measurements and cyclic voltammetry (CV). To the
best of our knowledge, a dynamic supramolecular system
based on a molecular switch that responds orthogonally to
three different stimuli is unprecedented.
Results and Discussion
Amphiphilic b-CD was synthesized as described previously.[30]
CDV with an average diameter of about 100 nm were pre-
pared by extrusion in a buffered aqueous solution. Guest mol-
ecules G1, G2 and G3 were prepared by the condensation of
N,N’-bis(3-aminopropyl)ethylenediamine with ferrocenecarbox-
aldehyde, or 4-(phenyldiazenyl)benzaldehyde, or both, respec-
tively, followed by the reduction of the imines with sodium
borohydride. Details of the synthesis are described in the Sup-
porting Information. The spectroscopic and analytic data for
G1–G3 are consistent with their molecular structure. NMR
spectra are provided as Supporting Information.
Figure 1. Schematic representation of a unilamellar b-cyclodextrin vesicle
(CDV) and molecular structures of amphiphilic b-cyclodextrin and divalent
guest molecules G1–G3.
Guest molecules G1 and G2 are homobifunctional noncova-
lent conjugates that carry two hydrophobic recognition units
for b-CD, ferrocene and azobenzene, respectively, which are
connected through the linear hydrophilic spacer N,N’-bis(3-
aminopropyl)ethylenediamine. In contrast, guest G3 is a hetero-
bifunctional noncovalent linker which carries both azobenzene
and ferrocene moieties as supramolecular binding sites sepa-
rated by the same hydrophilic tetraamine spacer. The stimulus-
responsive conformational changes of G3 are illustrated in
Figure 2. The ferrocene and azobenzene units on conjugates
G1–G3 are known to be good inclusion guests for b-CD.[37,38]
The binding affinity of guest to CDV is somewhat weaker than
the affinity for unmodified CDs in aqueous solution due to
presence of oligo(ethyleneglycol) residues on the vesicle sur-
from organic solution and extrusion through a polycarbonate
membrane.[30] The cavities of the CD on the outer surface of
the CDV are accessible to form inclusion complexes with hy-
drophobic guest molecules. The molecular recognition and in-
teraction of vesicles is a simple and versatile model system to
mimic recognition, adhesion and fusion of biological cell mem-
branes.[4] We have recently demonstrated that the adhesion of
CDV can be mediated by light-responsive and metal-respon-
sive divalent guest molecules that act as noncovalent cross-
linkers.[34,35] We have also described the self-assembly of supra-
molecular systems of CDV and azobenzene–spermine or azo-
benzene–carbohydrate
conju-
gates, which can photoreversibly
capture and release DNA or pro-
teins.[36,37]
Herein, we report a dynamic
supramolecular system in which
aggregation and adhesion of bi-
layer vesicles is controlled by
a multistimuli-responsive nonco-
valent cross-linker. We designed
homofunctional as well as
heterobifunctional azobenzene–
ferrocene conjugates (G1–G3,
Figure 1) with a N,N’-bis(3-ami-
nopropyl)ethylenediamine
spacer, which are used as
a
“supramolecular glue” to Figure 2. Stimuli-responsive conformational changes in the molecular structure of G3.
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face.[30] Formation of host–guest complex of G2 and
G3 with CDs should be photoresponsive because the
apolar, rod-like trans isomer of azobenzene favors
complexation with CD, whereas the polar, bent cis
form does not.[34,36,37] The inclusion complex of G1
and G3 with CDs should be redox responsive due to
the fact that the polar, oxidized form of ferrocene
(i.e., the ferrocenium ion) has very low affinity to
form inclusion complex with CD in comparison to
Table 2. Thermodynamic parameters for the interaction of heterobifunctional guest
molecule G3 with b-CD.
Host Guest
n
K1
[mÀ1
K2
[mÀ1
DH1
[kJmolÀ1
DH2
[kJmolÀ1
DG1
[kJmolÀ1
DG2
[kJmolÀ1
]
]
]
]
]
]
b-CD G3 (pH 7.4)
b-CD G3 (pH 9.2)
2
2
812
800
1259 À7.2
1369 À7.9
À14.8
À10.4
À16.6
À16.6
À17.7
À17.9
the apolar, reduced form (i.e., ferrocene).[38] Finally, also the tet-
raamine linker in G1–G3 is critical to the interaction with CDV:
the N,N’-bis(3-aminopropyl)ethylenediamine spacer forms
a highly stable tetradentate coordination complex with suit-
able metal ions (Ka ~1017 mÀ1 for the Cu2+ complex of N,N’-di-
act as independent binding sites. It can be observed that the
second binding constant (K2) is only slightly higher than the
first binding constant (K1; Table 2). For comparison, we also car-
ried out titrations for the complexation of b-CD with ferrocene-
carboxylic acid and azobenzenecarboxylic acid and it can be
seen that in those cases the stoichiometry is 1:1 and the asso-
ciation constants Ka =1.93ꢁ103 mÀ1 for ferrocenecarboxylic
acid and 2.48ꢁ103 mÀ1 for azobenzenecarboxylic acid are in ac-
cordance with the literature.[40] Each titration was performed at
pH 7.4 as well as pH 9.2 and the affinity of guest moieties to-
wards b-CD is very similar at those pH values (Tables 1 and 2).
Having verified the formation of inclusion complex between
guest molecules G1, G2 and G3 and b-CD, we investigated the
stimulus-responsive aggregation of CDV induced by each of
the divalent guest molecules. We first turned our attention to
the metal-ion responsive aggregation of CDV. All experiments
were carried out in dilute aqueous solution at pH 9.2 to ex-
clude the detrimental effect of protonation of G1–G3, which
would reduce the affinity of G1–G3 for metal coordination.
The addition of as little as 15 mm of G1, G2 or G3 to a dilute
solution of CDV (30 mm of amphiphilic b-CD) at pH 9.2 caused
a time-dependent increase of OD600 from about 0.05 to ap-
proximately 0.5 (Figure 3A). The average particle size increased
from about 100 nm to approximately 1000 nm according to
DLS (data shown for G3 in Figure 4D). The rate and extent of
the vesicle aggregation depends on the concentration of
guest molecule: fast and extensive aggregation occurred at
a higher concentration of linker, whereas at low concentration
the rate of aggregation was much slower (data shown for G3
in Figure 3A, inset). The initial OD600 was quickly recovered by
addition of excess host b-CD and a-CD (1 mm; Figure S9 in the
Supporting Information). This result indicates that guest mole-
cules G1–G3 trigger the aggregation of CDV due to the selec-
tive formation of multivalent intervesicular host–guest com-
plexes, which are readily disrupted in the presence of an
excess of host in solution.
benzylated-bis(3-aminopropyl)ethylenediamine
and
Ka ~
1020 mÀ1 for the Cu2+ complex of triethylenetetramine).[39] Due
to the formation of the coordination complex, the conforma-
tion of the guest switches from linear (free linker in the ab-
sence of metal ions) to bent (complex in the presence of metal
ions). The X-ray structures of such complexes are available in
the literature and confirm the bent conformation of the ligand
and a square-planar coordination of Cu
the average distance of azobenzene and ferrocene groups in
G1–G3 should be severely reduced upon coordination of Cu2+
.
2+ [39] As a consequence,
,
resulting in an intravesicular rather than intervesicular interac-
tion with CDV.[35]
The interaction between host b-CD and guests G1, G2 and
G3 was investigated by using ITC (Table 1 and Figures S1–S8 in
Table 1. Thermodynamic parameters for the interaction of homobifunc-
tional guest molecules (G1 and G2) and monomers (ferrocenecarboxylate
and azobenzenecarboxylate) with b-CD.
Host Guest
n
Ka
[mÀ1
DH
[kJmolÀ1
DG
[kJmolÀ1
]
]
]
b-CD G1 (pH 7.4)
b-CD G1 (pH 9.2)
b-CD G2 (pH 7.4)
b-CD G2 (pH 9.2)
b-CD ferrocenecarboxylate (pH 9.2)
b-CD azobenzenecarboxylate (pH 9.2)
2
2
2
2
1
1
2291 À19.5
2303 À22.3
2876 À11.8
À17.8
À19.2
À19.7
À17.9
À24.9
À20.2
2040
1932
2477
À7.1
À7.6
À5.1
the Supporting Information). We performed “reverse titrations”
in which a 10 mm solution of b-CD host was titrated into
a 0.5 mm solution of bifunctional guest. It can be seen from
Table 1 that the thermodynamic parameters of the interaction
are comparable for homobifunctional linkers G1 and G2 and
similar to literature data for inclusion complexes of azoben-
zenes and ferrocenes with b-CD.[40] The complexation of b-CD
to G1 and G2 is of stoichiometry 1:2 with nearly equal affinity
for both ferrocene and azobenzene groups, respectively. In
case of heterobifunctional linker G3, we calculated two differ-
ent binding constants for stepwise binding phenomena
(Scheme S1 in the Supporting Information). The azobenzene
and ferrocene moieties have almost similar affinity toward b-
CD and the groups are distant from each other so that they
Interestingly, when 50 mm of Cu2+ is added to the aggregat-
ed complex of CDV and linkers (G1, G2 and G3), both the orig-
inal OD600 (ca. 0.05; Figure 3B) and the average vesicle diame-
ter (ca.100 nm) were rapidly and efficiently recovered (data
shown for G3 in Figure 4D, blue trace). This observation can
be explained by the fact that all cross-linkers form stable
metal-coordination complexes with Cu2+ and rearrange from
a linear to a bent conformation (Figure 2). We suggest that the
bent conformational isomer of G1, G2 and G3 can only bind to
the surface of one vesicle and intervesicular complexation is
not feasible. For example, in the bent conformation of G3 the
azobenzene and the ferrocene groups are so close to each
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Cu2+ resulted in reaggregation of the CDV, as can be seen
from the increase in OD600 from about 0.05 to 0.3 (Figure 4A
and B) and the average vesicle diameter from about 100 to
900 nm (Figure 4D; turquoise trace). These measurements
show that EDTA scavenges the Cu2+ ion and allows the relaxa-
tion of G3 from a bent conformation to a linear conformation,
which induces the reaggregation of CDV.
In addition, z-potential measurements were performed to
further investigate the interaction of G3 with CDV in the pres-
ence of metal ions. As shown in Figure 4C, the z-potential of
CDV is about À19.5 mV at pH 9.2. The negative surface poten-
tial arises due to the oligo(ethylene glycol) residues on the
vesicle surface.[30] The surface potential increases from about
À19.5 to À11.2 mV after addition of G3. The increase of surface
potential is due to the presence of guest G3 on the surface of
vesicle aggregates. The subsequent addition of Cu2+ induces
the disaggregation of vesicles and the resultant z-potential fur-
ther increases to about À7.2 mV. This increment of surface po-
tential indicates the metal-ion coordination with guest G3 at
the vesicle surface. The addition of excess EDTA decreases the
z-potential from about À7.2 mV to approximately À11.5,
which confirms the removal of Cu2+ from the vesicle surface.
Taken together, the results shown above provide compelling
evidence for the metal-ion responsive aggregation of CDV by
divalent guests G1–G3. In the absence of metal ion, the diva-
lent guest operates as a noncovalent cross-linker that aggre-
gates the CDV. In the presence of metal ion, the divalent guest
folds into a tetradentate complex that cannot act as intervesic-
ular cross-linker. Hence, the metal ion induces a conformational
change that reversibly switches G1–G3 from an “adhesive” to
a “non-adhesive” state.
Figure 3. Metal-ion responsive aggregation of host vesicles of b-CD (CDV)
by divalent guests G1, G2 and G3. A) Time-dependent measurement of
OD600. Inset: concentration-dependent aggregation of CDV and G3. B) Dis-
persion of aggregates in the presence of Cu2+. Typical conditions:
[CDV]=30 mm, [G1]=[G2]=[G3]=6 to 18 mm, [Cu2+]=50 mm in 20 mm car-
bonate/bicarbonate buffer (pH 9.2). Guest was added after 2 min and Cu2+
was added after 30 min.
Next, we investigated the light-responsive interaction of
CDV with G2 and G3. The homobifunctional guest G2 and
heterobifunctional guest G3 both have azobenzene groups
that can form inclusion complexes with b-CD. The formation of
the host–guest complex of azobenzene is light-responsive:
only the trans-azobenzene is an appropriate guest for b-CD,
the cis-azobenzene is not. Hence, G3 can bind two molecules
of b-CD when the azobenzene moiety is in the trans form, but
only one molecule of b-CD when it is in the cis form (ferrocene
still can bind to b-CD in cis-G3 form). On the other hand,
trans-G2 forms an inclusion complex with two molecules of b-
CD but its cis isomer does not bind to any of the b-CDs. As dis-
cussed above, the addition of divalent guests G2 or G3 to
a dilute solution of CDV induces a rapid aggregation of CDV
(Figure 3A). It is likely that both trans-G3 and trans-G2 act as
supramolecular cross-linkers of CDV by forming 2:1 host–guest
inclusion complexes with b-CD on the surface of two different
vesicles, consistent with our earlier observations on divalent
azobenzenes.[34] The photoisomerization of G3 occurs readily
and results in a photoreversible aggregation and dispersion of
CDV, simply by irradiation at 350 nm (to isomerize trans-G3 to
cis-G3) followed by irradiation at 455 nm (to obtain trans-G3
from cis-G3; Figure 5). Selected data for the photoresponsive
aggregation of CDV mediated by G2 are provided in the Sup-
porting Information (Figure S11). Upon 40 min UV irradiation of
a binary mixture of CDV and trans-G3 decreases both the
other that only one of them can form inclusion complex with
CDV. The coordination-induced conformational change of the
linker causes the dispersion of the vesicles. We note that these
observations are fully consistent with our previous report on
comparable noncovalent linkers with tert-butylbenzyl instead
of azobenzene and ferrocene moieties.[35] In that study, we had
also noticed that there is no significant fusion of vesicles while
metal-ion coordination with guest at the vesicle surface.[35]
However, the linker concentration plays a critical role in the
metal responsive dispersion of vesicles. The addition of as
much as 100 mm of Cu2+ to a higher concentration of G1–G3
(12 mm) in the aggregated solution of CDV did not induce any
substantial changes in either OD600 or average particle size
(Figure S10 in the Supporting Information). This phenomenon
is attributed to the fact that at a high concentration of guest
molecule at the vesicle surface, a 2:1 intermolecular metal co-
ordination takes place, which does not lead to the change in
conformation of G1–G3. Furthermore, the metal-ion induced
dispersion of vesicles can be reversed in the presence of
strong metal chelator, such as EDTA. The addition of 200 mm
EDTA to a solution of CDV in the presence of G3-coordinated
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90 nm to approximately 850 nm
(Figure 5C, green trace). The
OD600 and DLS measurements
clearly indicate that the complex
of CDV and cross-linker trans-G3
reassembles upon visible irradia-
tion. The reversibility of photoin-
duced aggregation of vesicles is
evident over three cycles, pro-
vided that the irradiation time is
sufficient (40 min at both 350
and 455 nm) and the vesicle
concentration is limited to
30 mm (Figure 5B).
The results shown in this sec-
tion provide clear cut evidence
for the light-responsive aggrega-
tion of CDV by divalent guests
G2 and G3. Alternating UV and
visible irradiation induces an iso-
merization of the azobenzene
that reversibly switches G2 and
G3 from an “adhesive” trans state
to a “non-adhesive” cis state.
Figure 4. Metal-ion responsive aggregation of host vesicles of b-CD (CDV) by divalent guest G3. A) Aggregation,
disaggregation and reaggregation of CDV induced by G3, Cu2+ and EDTA, respectively, monitored by OD600. G3
was added after 2 min, Cu2+ after 30 min and EDTA after 60 min. B) Reversible metal-ion responsive aggregation
of CDV. C) The z-potential of CDV and CDV clusters. D) Size distribution according to DLS. Typical conditions:
[CDV]=30 mm, [G3]=6 mm, [Cu2+]=50 mm (15 mm for z-potential measurement) and [Na2EDTA]=200 mm in
20 mm carbonate/bicarbonate buffer (pH 9.2).
Finally, we investigated the
redox-responsive interaction of
CDV with G1 and G3. The homo-
bifunctional guest G1 and
heterobifunctional guest G3
both have ferrocene groups that
OD600 from roughly 0.3 to 0.05 (Figure 5A, black trace) and
average particle size from 900 nm to about 90 nm (Figure 5C,
blue trace). UV irradiation (350 nm) induces photoisomerization
of trans-G3 to cis-G3, and the azobenzene moiety (not the fer-
rocene moiety) detaches from the CDV surface, since only
trans-G3 can bind to b-CD. As a result the divalent linker G3
transforms into a monovalent guest molecule, and dispersion
of CDV takes place. Upon subsequent visible-light irradiation
of the dispersed CDV at 455 nm for 40 min (to obtain trans-G3
from cis-G3), OD600 increases from about 0.05 to 0.3 (Figure 5A,
red trace) and the average particle size increases from about
can bind to b-CD. It is well known that ferrocene and b-CD are
a redox-responsive molecular recognition pair: b-CD can form
stable inclusion complexes with the reduced form (ferrocene)
but not with the oxidized form (ferrocenium ion).[38,41] The
redox behavior of G1 and G3 was investigated by CV in the
absence and presence of b-CD (Figure 6). From these voltam-
mograms, the potential of oxidation and reduction peaks (Epa
and Epc, respectively) and peak current at oxidation and reduc-
tion (ipa and ipc, respectively) were determined and are listed in
Table 3. In the absence of b-CD, both guest molecules G1 and
G3 show a single redox wave in the potential range of À0.4 to
Figure 5. Light-responsive reversible aggregation and dispersion of host vesicles of b-CD (CDV) by divalent guest G3. A) Time-dependence of OD600. B) Rever-
sible switching of OD600 under alternate UV (350 nm) and visible (455 nm) light irradiation. C) Size distribution according to DLS. Typical conditions:
[CDV]=30 mm, [G3]=6 mm in 20 mm phosphate buffer (pH 7.4).
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eties in homobifunctional G1. We measured CV at three differ-
ent scan rates and the above observations are essentially iden-
tical in all the experiments. In addition, since the differences
between Epa and Epc are larger than 0.057, the redox reactions
in all cases are not diffusion controlled under the conditions of
the experiment.
As discussed above, the addition of divalent guests G1 or
G3 to a dilute solution of CDV induces a rapid aggregation of
CDV (Figure 3A). The ferrocene groups can be readily oxidized
chemically with sodium hypochlorite solution.[42] To investigate
the redox reversibility of the CDV aggregation, a 30 mm solu-
tion of NaOCl was added to the aggregated mixture of CDV
and ferrocene appended guests G1 and G3. The OD600 decreas-
es from about 0.45 to approximately 0.05 (Figure 7A) and the
average particle size decreases to about 100 nm from approxi-
mately 1000 nm (Figure 7B). The in situ oxidation of ferrocene
moieties to ferrocenium ions transforms the divalent homobi-
functional guest G1 to an inert and highly water soluble com-
pound and divalent heterobifunctional guest G3 to a monova-
lent guest for CDV. The in situ reduction of ferrocenium ion to
ferrocene with excess potassium iodide resulted in renewed
aggregation of the CDV (Figure S12 in the Supporting Informa-
Figure 6. Cyclic voltammetry of guests G1 and G3 in the absence and pres-
ence of b-CD. A) I versus E voltammogram of G1 in the absence (black, deep
grey and light grey traces) and presence (deep green, light green, cyan
traces) of b-CD. B) I versus E voltammogram of G3 in the absence (black,
deep grey and light grey traces) and presence (deep green, light green,
cyan traces) of b-CD. Concentration: [G1]=[G3]=1 mm, [b-CD]=10 mm.
Scan rates: 0.1, 0.5 and 1 VsÀ1
.
Table 3. Results of CV measurements for 1 mm G1 and G3 in the absence
and presence of 10 mm b-CD at 0.1 VsÀ1 scan rate.
Guest
Epa [V]
Epc [V]
ipa [mA]
ipc [mA]
G1
G1+b-CD
G3
0.026
0.095
0.049
0.095
À0.065
À0.042
À0.099
À0.065
15.8
11.3
7.80
6.59
À11.8
À7.68
À6.07
À5.10
G3+b-CD
+0.4 V versus Ag/AgCl. These observation indicate that both
ferrocene moieties in G1 undergo the redox reaction inde-
pendently, presumably because of the relatively long N,N’-
bis(3-aminopropyl)ethylenediamine spacer. The values of Epa
and Epc (for both G1 and G3) increase in the presence of b-CD,
indicating that oxidation of ferrocene moieties requires
a higher potential in the presence of b-CD due to the stable in-
clusion complex formation. The values of ipa and ipc in the pres-
ence of b-CD are smaller than those in the absence, which in-
dicates the fact that b-CD retards redox reaction on the elec-
trode surface. The peak current values of G1 are almost two-
times higher than those of G3 because of two ferrocene moi-
Figure 7. Redox-responsive reversible aggregation dispersion of vesicles of
b-CD (CDV) by divalent guest G3. A) Aggregation and disaggregation of
CDV monitored by OD600. G1 or G3 was added after 2 min, NaOCl after
30 min. B) Size distribution according to DLS. Typical concentrations:
[CDV]=30 mm, [G3]=15 mm, [NaOCl]=30 mm (for G3) or 60 mm (for G1) in
20 mm phosphate buffer (pH 7.4).
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tion). A complete reduction proved to be difficult due to the
competing redox reaction of oxidizing and reducing agents.
We conclude that the guest molecules G1 and G3 induce vesi-
cle aggregation which can be fully disrupted by the addition
of an oxidizing agent, and that in principle the aggregation
can be restored by the addition of excess reducing agent.
Preparation of vesicles
Unilamellar bilayer vesicles (CDV) were prepared by extrusion. Sev-
eral milligrams of amphiphilic b-CD in 2–3 mL of chloroform were
dried by slow rotary evaporation to yield a thin film in a 10 mL
round-bottom flask. Residual solvent was removed under high
vacuum. Aqueous buffer (10 mL; 20 mm phosphate buffer, pH 7.4
or 20 mm carbonate/bicarbonate buffer, pH 9.2) was added and
stirred, overnight. The resulting suspension was repeatedly passed
through a polycarbonate membrane with 100 nm pore size in a Lip-
osofast manual extruder.
Conclusion
A trifunctional molecule with a flexible N,N’-bis(3-amino-
propyl)ethylenediamine spacer was used as a noncovalent
cross-linker that induces aggregation and adhesion of host
vesicles composed of amphiphilic b-CDs by the formation of
host guest inclusion complexes at the surface of the vesicles.
In response to external stimuli (metal ion, light and oxidation)
the guest molecule changes its conformation and polarity and
hence loses its affinity for the host vesicles, which strongly de-
creases intervesicular binding and causes dispersion of vesicle
clusters. The reversible transition from the aggregated state to
the dispersed state of vesicles was mediated effectively by ad-
dressing a single molecular switch selectively with metal ion,
light or redox chemistry. To the best of our knowledge, a dy-
namic supramolecular system based on a molecular switch
that responds orthogonally to three different stimuli is unpre-
cedented. We anticipate that the proof-of-concept demonstrat-
ed here for the aggregation of nanoscale vesicles into micro-
scale vesicle clusters can be easily adapted to the multistimu-
lus responsive assembly of other materials, such as hydrogels,
polymers, colloids and nanoparticles in solution as well as on
surfaces.
Isothermal titration calorimetry (ITC)
ITC was performed by using a Nano-isothermal titration calorime-
ter low volume equipped with a 200 mL gold cell (model SNL
10099; TA Instrument Waters, Lindon, Utah, USA). ITC measure-
ments were performed in 20 mm phosphate buffer or carbonate/
bicarbonate buffer (in 10% DMSO). A 10 mm solution of b-CD was
titrated into a 0.5 mm solution of guest molecules. For each titra-
tion, 20 injections of 2.5 mL were performed with an interval of
300 s. The stirring rate was 300 rpm.
UV/Vis spectroscopy
Optical density measurements at 600 nm were carried out at in
1.5 mL disposable cuvettes with dimensions 12.5ꢁ12.5ꢁ45 mm
and 10 mm path-length using an Uvikon 923 double-beam spec-
trophotometer (Kontron Instruments). Measurements were per-
formed for 30 to 90 min with data points collected every 12 s.
Freshly prepared vesicles and metal ion solutions were used for
each measurement and the measurement procedure was as fol-
lows: for example, 1 mL solutions of CDV (30 mm in 20 mm phos-
phate buffer, pH 7.4 or 20 mm carbonate/bicarbonate buffer,
pH 9.2) were taken in a semimicro disposable cuvette and OD600
was measured for 2 min. After 2 min, 7.5 mL of guest G3 (2 mm so-
lution in DMSO) was added to make the resultant concentration
15 mm (approximately 100% surface coverage of vesicles) in the
cuvette (this addition was done with slight mixing within a single
interval of 12 s) and the measurement was carried out for at least
30 min. After 30 min a few microliters of concentrated metal ion
solution in Millipore water was added to the above solution for
metal-ion responsive experiments. The same measurements were
performed with the other samples by following the above proce-
dure. Typical concentrations: [CDV]=30 mm, [G1]=[G2]=[G3]=5–
50 mm, [CuCl2]=50–100 mm and [Na2H2EDTA]=100–200 mm in cor-
responding buffer solution.
Experimental Section
Materials
All chemicals used in this study were purchased from Acros Organ-
ics (Schwerte, Germany) or Sigma–Aldrich Chemie (Taufkirchen,
Germany) and used without further purification. b-Cyclodextrin
was kindly donated by Wacker Chemie (Burghausen, Germany). All
solvents used in reactions and purification were dried according to
conventional methods. All aqueous solutions were prepared in
Milli-Q water.
Synthesis
The synthesis of G1, G2 and G3 are described in the Supporting In-
formation. The spectroscopic and analytical data for G1–G3 are
consistent with their molecular structure. The synthesis of amphi-
philic b-CD was performed as reported previously.[30] All reactions
were carried out in oven-dried glassware and magnetically stirred
under an inert gas atmosphere. Analytical TLC was performed on
Merck silica gel 60 F254 plates. All compounds were visualized
either by UV light or by dipping in basic permanganate solution.
Column chromatography was carried out by using silica gel 60
(230–400 mesh). 1H and 13C NMR spectroscopic measurements
were carried out by using Bruker ARX 300 MHz or Varian 500 MHz
INOVA spectrometers. Chemical shifts were referenced to internal
Dynamic light scattering (DLS) and z-potential measure-
ments
DLS measurements were performed by using a Malvern Nano-ZS
instrument (Malvern Instruments) with low-volume disposable cuv-
ettes kept at 258C. The average size of CDV and mixtures of vesi-
cles of CDV and guests G1–G3 were measured after mixing the
corresponding components. Immediately after alternate UV
(350 nm, 30 min) and visible light (455 nm, 30 min) irradiations, the
corresponding average size of the binary complex was measured.
Typical concentrations: [CDV]=30 mm, [G1]=[G2]=[G3]=5–
50 mm, [CuCl2]=50–100 mm and [Na2H2EDTA]=100–200 mm in cor-
responding buffer solution. Similarly, z-potential measurements
were performed using a Malvern Nano-ZS instrument (Malvern In-
struments) in disposable folded capillary cells at 258C.
1
standards CDCl3 (d=7.26 ppm for H and 77.0 ppm for 13C) or TMS
(d=0.00 ppm for 1H and 13C). High-resolution mass spectrometry
(HRMS) was performed by using a Bruker MicroTof instrument.
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Keywords: cyclodextrins · host–guest systems · molecular
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Received: November 27, 2013
Revised: January 30, 2014
Published online on March 18, 2014
Chem. Eur. J. 2014, 20, 4966 – 4973
www.chemeurj.org
4973
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim