B. J. Ravoo et al.
tions of surface-bound trans-5 in the intervesicular contact
areas. From our data, it can be concluded that, at high
excess (>10-fold) of competitive guest 5, guest 2 is com-
pletely displaced from the vesicle surface, whereas at low
excess (<3-fold) of competitive guest 5, guest 2 is not dis-
placed to a significant extent. The intermediate concentra-
tion range (around 250 mm of guest 5) reveals the underlying
interplay of rapid, diffusion-controlled dynamics of the in-
clusion complexes at the surface of the vesicles versus slow
adhesion of vesicles by a limited number of intervesicular
links, that is, although most links are broken initially, at
least some will re-form over time, since guest 2 is not com-
pletely displaced from the vesicle surface. These findings
consistently show that the aggregation and adhesion of vesi-
cles of 1a is mediated by the formation of intervesicular in-
clusion complexes, since excess competitive binders (host b-
CD as well as guest trans-5) in solution disrupt inclusion
complexes at the vesicle surface.
Most interestingly, UV irradiation of the ternary mixture
of vesicles of a-CD 1a (30 mm), guest 2 (40 mm), and compet-
itive guest trans-5 (500 mm) at 350 nm for 20 min raises
OD600 from about 0.05 to about 0.37 and the average parti-
cle size from around 150 nm to more than 1000 nm (Fig-
ure 3C and D). UV irradiation at 350 nm induces the photo-
isomerization of trans-5 to cis-5 (Figure S1 in the Supporting
Information). These observations can be taken as further
evidence that the photoinduced aggregation and dispersion
of vesicles is due to photoresponsive interactions of compet-
itive guest 5, since only trans-azobenzenes (but not cis-azo-
benzenes) form inclusion complexes with a-CD. Upon sub-
sequent visible irradiation of the ternary mixture of aggre-
gated vesicles of 1a (30 mm), guest 2 (40 mm), and guest cis-5
(500 mm) at 455 nm for 30 min (to obtain trans-5 from cis-5),
both OD600 decreases from around 0.37 to around 0.11 and
the average particle size decreases from more than 1000 nm
to about 150 nm (Figure 3C and D). In other words, the ag-
gregation of vesicles of 1a is reversible in the presence of
the competitive photoresponsive guest 5. The reversibility of
the competitive photoinduced aggregation is essentially
complete over 5 cycles provided that the irradiation time is
sufficient (20 min at 350 nm and 30 min at 455 nm), the vesi-
cle concentration is limited to 30 mm (so that the maximum
OD600 is less than 0.5), and the concentration of competi-
tive guest 5 is no more than 500 mm. It likely that photoiso-
merization is hindered or even inhibited if the OD600 of the
vesicle solution is higher than 0.5 and/or the concentration
of 5 is more than 500 mm.
Figure 4. Cryo-TEM images of A) vesicles of a-CD 1a, and B) aggrega-
tion of vesicles of a-CD 1a mediated by divalent guest 2. The dark line
at the bottom left corner in A) is the edge of the holey carbon film.
aggregates of (multilamellar) vesicles are observed (Fig-
ure 4B). The vesicles form extensive areas of close contact.
We take these microscopic observations as direct evidence
for adhesion of the vesicles induced by guest 2.
The OD600, DLS, and cryo-TEM data indicate that guest
2 induces rapid aggregation and adhesion of vesicles of 1a.
It should be emphasized that the rate of vesicle aggregation
is determined by the limited mobility of the vesicles, not by
the formation of inclusion complexes. The rate and extent of
vesicle aggregation is concentration dependent: if less guest
2 (20 mm instead of 40 mm) is added to the CD vesicles, it
takes longer before a maximum OD600 is reached and if
more guest 2 (60 mm instead of 40 mm) is added to the CD
vesicles, it reaches a maximum OD600 within a shorter time
(Figure 3A). In analogy to our recent findings,[62] it can be
assumed that inclusion complexation between vesicles of
host a-CD 1a and guest 2 is responsible for the formation
of multiple intervesicular noncovalent links, which leads to
the aggregation of vesicles of 1a. It should be noted that, in
view of the rather low binding constant of host a-CD 1a
and guest 2 (Ka ꢀ100mÀ1), a substantial fraction of guest 2 is
likely to be in solution rather than bound at the surface of
the vesicles. Nevertheless, our data clearly show that the sur-
face coverage of vesicles of 1a with guest 2 is sufficient to
establish a significant number of intervesicular noncovalent
links, even in dilute aqueous solution.
This interpretation is confirmed by the fact that the addi-
tion (after 60 min) of an excess of host b-CD (10 mm) leads
to immediate dispersion of the aggregated vesicles of 1a in
the presence of guest 2 (Figure S4 in the Supporting Infor-
mation). Moreover, the addition (after 60 min) of an excess
of competitive monovalent guest trans-5 (10 mm) also leads
to immediate dispersion of the aggregated vesicles of 1a in
the presence of guest 2 (Figure 3B). The effect of excess
guest at different concentrations (from 60 mm to 10 mm) on
the aggregation of the binary mixture of vesicles of 1a
(30 mm) and guest 2 (40 mm) was also investigated. The addi-
tion of 500 mm of an excess competitive guest trans-5 de-
creases OD600 from about 0.4 to 0.05, in other words, it dis-
perses the aggregated vesicles of 1a despite the presence of
divalent guest 2 (Figure 3B) and the average vesicle diame-
ter of about 150 nm is obtained again (Figure 3C). The addi-
tion of 250 mm of trans-5 results in rapid dispersion of the
vesicles, followed by slow reaggregation. The addition of
125 or 60 mm of trans-5 results in a slight increase of vesicle
aggregation, possibly as a consequence of secondary interac-
The addition of divalent guest 3 (instead of 2) to vesicles
of a-CD 1a leads to very similar results. When 3 is added to
vesicles of 1a at pH 7.4, OD600 increases from around 0.05
to around 0.3 within 30 min (Figure 5A). According to DLS,
the average particle size increases from about 150 nm to
more than 1000 nm (Figure 5B). These observations indicate
that—similar to guest 2—guest 3 induces rapid aggregation
and adhesion of vesicles of 1a. Also in this case, the rate
and extent of vesicle aggregation is concentration depen-
dent. Moreover, the addition of excess host b-CD (10 mm)
10300
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10297 – 10303
Nalluri, Siva Krishna Mohan
