Hydrogen-bond-regulated distinct functional-group display at the inner and outer wall of vesicles

Article abstract of DOI:10.1002/anie.201500971

A unique supramolecular strategy enables the unidirectional assembly of two bola-shaped unsymmetric π-amphiphiles, NDI-1 and NDI-2, which feature a naphthalene-diimide chromophore connected to nonionic and anionic head groups on opposite arms. The amphiphiles differ only in the location of a hydrazide group, which is placed either on the nonionic or on the anionic arm of NDI-1 and NDI-2, respectively. The formation of hydrogen bonds between the hydrazides, which compensates for electrostatic and steric factors, promotes unidirectional alignment and the formation of monolayer vesicles. The zeta potentials and cation-assisted quantitative precipitation reveal negatively charged and nonionic outer surfaces for NDI-1 and NDI-2, respectively, indicating that hydrogen bonding also dictates the directionality of the monolayer curvature, ensuring that in both cases, the hydrazides remain at the inner wall to benefit from stronger hydrogen bonding where they are in closer proximity. This is reflected in their different abilities to inhibit α-chymotrypsin, which possesses a positively charged surface: NDI-1 induced an inhibition of 80% whereas hardly any inhibition was observed with NDI-2.

Full text of DOI:10.1002/anie.201500971

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Chemie
International Edition: DOI: 10.1002/anie.201500971
German Edition: DOI: 10.1002/ange.201500971
Bolaamphiphiles
Hydrogen-Bond-Regulated Distinct Functional-Group Display at the
Inner and Outer Wall of Vesicles**
Amrita Sikder, Anindita Das, and Suhrit Ghosh*
Abstract: A unique supramolecular strategy enables the
unidirectional assembly of two bola-shaped unsymmetric p-
amphiphiles, NDI-1 and NDI-2, which feature a naphthalene–
diimide chromophore connected to nonionic and anionic head
groups on opposite arms. The amphiphiles differ only in the
location of a hydrazide group, which is placed either on the
nonionic or on the anionic arm of NDI-1 and NDI-2,
respectively. The formation of hydrogen bonds between the
hydrazides, which compensates for electrostatic and steric
factors, promotes unidirectional alignment and the formation
of monolayer vesicles. The zeta potentials and cation-assisted
quantitative precipitation reveal negatively charged and non-
ionic outer surfaces for NDI-1 and NDI-2, respectively,
indicating that hydrogen bonding also dictates the direction-
ality of the monolayer curvature, ensuring that in both cases,
the hydrazides remain at the inner wall to benefit from stronger
hydrogen bonding where they are in closer proximity. This is
reflected in their different abilities to inhibit a-chymotrypsin,
which possesses a positively charged surface: NDI-1 induced
an inhibition of 80% whereas hardly any inhibition was
observed with NDI-2.
bolaamphiphiles. This is of paramount importance as an inner
surface tailored with a suitable functional group can be used
for guest encapsulation mediated by specific interactions
while the functionalized outer wall can regulate communica-
tion with the solution outside the vesicles for sensing,
targeting, and stimuli responsiveness.[2–3] In fact, controlling
the orientational directionality of unsymmetric bolaamphi-
philes[8] is a classical topic, and it is evident from the
literature[8,9] that electrostatic repulsion and/or head-group
volume are the two primary steering factors that determine
which particular groups will converge at the inner wall or
diverge at the outer surface. For example, it has been shown[8c]
that in an unsymmetric bolaamphiphile, if either of the two
different head groups is ionic in nature, the larger head groups
preferentially congregate at the outer wall. Likewise, in
vesicles prepared from a mixture of two diblock copolymers,
namely polystyrene-b-poly(4-vinylpyridine) [PS-b-P(4-VP)]
and polystyrene-b-poly(acrylic acid) (PS-b-PAA), the P(4-
VP) block remains at the outer surface as it consists of longer
chains than the PAA block.[9] However, both of these factors
(electrostatic or steric repulsion) directly depend on the
chemical structure of the head group itself and thereby offer
limited opportunities for the site-specific display of a partic-
ular head group that is designed for functional utility.
Moreover, bolaamphiphiles with one ionic and one nonionic
head group exhibit anti-parallel orientation[5l] to avoid
electrostatic repulsion, and thus the membrane gains an
overall symmetry even though it is made from unsymmetric
bolaamphiphiles. Herein, we describe the first strategy to
address this long-standing problem[8a–b] of controlling the
directionality in the alignment of unsymmetric bolaamphi-
philes by hydrogen bonding among the hydrazide groups
present in the rationally designed bola-shaped twin p-
amphiphiles NDI-1 and NDI-2 (Figure 1).
NDI-1 (Figure 1a) contains a nonionic and an anionic
head group, and the hydrazide is located on the nonionic arm.
High-resolution transmission electron microscopy (HRTEM)
images (Supporting Information, Figure S1) indicate the
formation of hollow spherical structures (70–90 nm in diam-
eter) with a wall thickness of 40 Æ 5 ꢀ, which matches the
estimated length of NDI-1 (Figure S2), thus indicating
monolayer assembly. This was further corroborated by the
small-angle X-ray pattern, which revealed a Braggꢁs diffrac-
tion (Figure S3) that corresponds to d = 36.4 ꢀ (2q = 2.368).
NDI-1 was able to entrap the hydrophilic dye calcein as
confirmed by the emission band at lmax = 509 nm (Figure 1c).
The absorption-normalized emission intensity (Figure S4) of
calcein that was encapsulated in NDI-1 vesicles was signifi-
cantly reduced compared to that of a free calcein solution
owing to self-quenching[7] processes in the confined water-
L
iposomes[1] or polymersomes[2] occupy an eminent place in
biomedical science[3] owing to their promising applications in
diagnostic and therapeutic medicine. A different class of
amphiphiles consisting of hydrophobic p-conjugated chro-
mophores[4,5] has emerged in the recent past as unique self-
assembling systems that combine the colloidal domain with
supramolecular chemistry.[6] We recently described[7] the
hydrogen-bonding-driven structural evolution of vesicles
from bola-shaped naphthalene diimide (NDI) containing
symmetric amphiphiles[7a–b] and also from amphiphilic block
copolymers.[7c] We envisaged that by utilizing the directional
nature of hydrogen bonding, it might be possible to develop
suitable strategies for the preferential segregation of chemi-
cally distinct groups along the inner and outer wall of vesicles
that are assembled from unsymmetric analogues of such
[*] A. Sikder, Dr. A. Das,[+] Dr. S. Ghosh
Polymer Science Unit
Indian Association for the Cultivation of Science
2A and 2B Raja S. C. Mullick Road, Kolkata, India-700032
E-mail: psusg2@iacs.res.in
[+] Present address: Institute for Technical and Macromolecular
Chemistry, University of Hamburg
Bundesstrasse 45, 20146 Hamburg (Germany)
[**] A.S. thanks the CSIR for a research fellowship, and S.G. thanks the
CSIR, New Delhi, India for funding (02(0177)/14/EMR-II).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500971.
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vesicles showed an appreciable kinetic stability as no signifi-
cant leakage of entrapped calcein was observed even after six
days (Figure S9).[12]
However, dynamic-light-scattering studies (for the raw
data, see Table S1–S4) estimated a larger particle size for
NDI-2 (average Dh = 180 nm) than for NDI-1 (Dh = 140 nm;
Figure 1e). Nevertheless, the most pronounced difference is
noticed for the z value of NDI-2, which exhibits a close to
neutral value of À10.0 Æ 2.0 mV, suggesting that the surface is
almost completely nonionic in contrast to the negatively
charged surface of NDI-1. The adsorption of the hydroxide
ions onto the otherwise non-ionic oligooxyethylene (OE)
surface is likely to contribute to such insignificantly negative
z values at basic pH values.[13] To further confirm and quantify
the difference in surface functionality, we conducted cationic
methyl viologen (MV) assisted quantitative precipitation
experiments similar to what is routinely done to probe lectin–
carbohydrate binding.[14] In fact, when an aqueous solution of
NDI-1 (pH 9) was added to MV, the solution instantaneously
turned turbid, and after some time, a precipitate formed. For
NDI-2, however, the solution remained clear for several days
in the presence of MV under identical conditions. After
removing the insoluble solid by centrifugation, the UV/Vis
spectra of the two supernatant solutions differed significantly
(Figure 2). For the NDI-2 solution, almost no change was
noticed in the absorbance spectrum (Figure 2b) whereas for
NDI-1, the intensity was reduced by approximately 80%
(Figure 2a), confirming the presence of effective interactions
of MV selectively with NDI-1 and not with NDI-2. When the
precipitate that was isolated by centrifugation was re-
dissolved in THF (Figure 2a), sharp absorption peaks con-
firmed the presence of NDI-1 in the precipitate. To further
push the extent of precipitation, another experiment was
carried out with FeCl3 as the Fe3+ ion has the ability to form
strong chelate complexes with negatively charged ligands.[15]
The addition of an aqueous FeCl3 solution to a solution of
NDI-1 or NDI-2 (0.5 mm, pH 9) resulted in very different
outcomes (Figure 2c,d). After removal of the precipitate, the
supernatant solution of NDI-1 indicated quantitative precip-
itation (Figure 2c) as its absorption intensity was reduced by
approximately 95% whereas no significant changes were
observed for the NDI-2 solution (Figure 2d), confirming that
Fe3+ selectively interacts with a vesicular assembly of NDI-1.
In case of a 1:1 mixed stacking of NDI-1 and NDI-2 in
alternate fashion, interestingly, almost full precipitation was
observed (Figure S10) as for NDI-1 alone, suggesting that
mostly/fully one type of assembly with an anionic surface
charge is formed by the mixed assembly of NDI-1 and NDI-2.
The model depicted in Scheme 1 can rationalize the
experimental observation presented in the above discussion.
According to this model, the two most prominent common-
alities between the self-assembly of these twin amphiphiles
are: 1) Parallel alignment allows the hydrazides to remain on
the same side of the monolayer. 2) Owing to a preferred
direction of curvature of the monolayer, the hydrazides (and
consequently the functional groups attached to the same arm)
remain at the inner wall of the vesicle. The reason is obvious
as hydrazides, which are known[7] to exhibit strong intermo-
lecular hydrogen bonding, drive the unidirectional alignment
Figure 1. a,b) Structures of NDI-1 and NDI-2. c) Absorption-normal-
ized emission spectra of calcein in NDI-1 (black line) and NDI-2 (gray
line); dashed lines indicate the corresponding emission spectra of
calcein in water without NDI. d) Solvent-dependent (solid line: THF;
dashed line: water, pH 9.0) UV/Vis absorption spectra of NDI-
1 (black) and NDI-2 (gray); c=0.5 mm. e) DLS data of NDI-1 and
NDI-2 (c=0.5 mm).
filled space inside the vesicles. UV/Vis spectra suggest
pronounced solvent effects (Figure 1d); in THF, sharp
absorption bands (lmax = 376 and 383 nm) indicate mono-
meric species while in H2O (pH 9.0), a bathochromic shift of
approximately 7.0 nm with around 60% reduction in absorb-
ance and reversed intensities of the two absorption bands
(lmax = 376 and 383 nm) suggest p–p interactions.[10] Interest-
ingly, zeta potential (z) measurements revealed a highly
negative value of À45.0 Æ 3.0 mV, indicating a negatively
charged outer surface. To investigate the role of the location
of the hydrazide group in displaying the anionic phenoxides
on the outer surface, we compared the self-assembly of
NDI-1 with that of NDI-2 (Figure 1b), which has the same
structure as NDI-1 except for the position of the hydrazide
group, which is now located on the anionic arm. HRTEM
images (Figure S5) along with XRD (Figure S3) confirmed
the presence of monolayer vesicles (diameter ca. 140 nm; wall
thickness: 42 Æ 5 ꢀ) similar to those of NDI-1. Successful
calcein encapsulation (Figure 1c) ascertained the vesicular
structure. Pleasingly, an estimation of the amount of entrap-
ped calcein dye revealed encapsulation efficiencies of
approximately 13% and 10% for the NDI-1 and NDI-2
vesicles, respectively, which are in line with those calculated
for liposomes of comparable dimensions in earlier reports[11]
(see Figure S4 and a related discussion in the Supporting
Information). UV/Vis studies revealed identical p–p inter-
actions (Figure 1d). Variable-temperature UV/Vis studies
indicated a remarkably high thermal stability for the vesicular
assembly of these two p-amphiphiles as their spectral patterns
remained unaltered even at 908C (Figure S6). Concentration-
dependent UV/Vis studies (Figure S7, S8) measured a slightly
higher value for the critical aggregation concentration (CAC)
for NDI-2 (3.3 ꢂ 10À5 m) than for NDI-1 (2.7 ꢂ 10À5 m). Both
2
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towards lower frequencies in D2O ascer-
taining hydrogen bonding. Although the
crowding of multiple peaks in this region
(for the assignment of other peaks, see
Figure S11) together with spectral
broadening does not allow for a precise
estimation of the shift, it was clearly
observable that the extent of the spectral
shift is smaller for NDI-2, suggesting
relatively weaker hydrogen bonding. As
in this case, the negative charges are
encapsulated inside the vesicles, a longer
radius of curvature may be induced to
minimize electrostatic repulsion, which
results in an increased distance between
the hydrazides. Interestingly, this prop-
osition nicely corroborates with the
larger hydrodynamic diameter of the
NDI-2 vesicles compared to that of
NDI-1 (Figure 1e). Further urea-medi-
ated “denaturation”[17] experiments pro-
vided indirect evidence that the hydra-
zide groups are located at the inner wall.
Urea is known to interfere with hydro-
gen-bonded amide groups and destroy
the native secondary structure
Figure 2. Effect of MV (a,b) and FeCl3 (c,d) addition on the UV/Vis spectra of NDI-1 (a,c) and
NDI-2 (b,d) in H2O (0.5 mm, pH 9.0). ppt=precipitate.
of proteins. Likewise, it can
also jeopardize hydrogen-
bonding-driven aqueous self-
assembled structures from
abiotic systems if it can access
the hydrogen-bonding sites.[7a]
However, neither NDI-1 nor
NDI-2 disassembled even
upon addition of an approxi-
mately 2000-fold molar excess
of urea as confirmed by the
almost unchanged absorption
spectra (Figure 3b). This is in
sharp contrast with our recent
report[7a] where prompt urea-
Scheme 1. Proposed model for directional self-assembly and mixed assembly of NDI amphiphiles leading
to contrasting surface charges.
allowing for extended hydrogen bonding even at the cost of
electrostatic repulsion among the anions. Furthermore, in
both cases, the preferred direction of curvature of the
monolayer ensures that the hydrazides remaining at the
inner wall can interact more strongly owing to the shorter
intermolecular distance than at the outer wall, which has
a longer radius of curvature. Importantly, these results imply
that hydrogen bonding dominates over both electrostatic
repulsion and steric demand and dictates the directionality of
both the alignment as well as the curvature. On the other
hand, in mixed assemblies of NDI-1 and NDI-2, the com-
pounds stack in an alternate fashion with antiparallel
orientation to avoid electrostatic repulsion as in this situation,
extended hydrogen bonding is still possible, and thus the
mixed vesicle can be almost fully precipitated from the
solution in the presence of FeCl3.
mediated disassembly of vesicles from symmetric NDI
bolaamphiphiles with hydrazides both at the outer and
inner wall was observed. The lack of such effects in the
present study strongly evokes the inaccessibility of the
hydrazides, indicating that they are buried at the inner wall
for both NDI-1 and NDI-2 and thereby shielded by the rigidly
stacked NDI chromophores, which hinder urea infiltration.
We further considered donor-intercalation-mediated
gating[18] of the membrane and checked whether that would
provide the urea molecules with a pathway for reaching the
hydrogen-bonding sites. When a carboxylate-functionalized,
water-soluble pyrene derivative was mixed with either of the
twin amphiphiles, a new broad absorption band appeared at
560 nm (Figure 3c), which was associated with the intense
green color suggesting the formation of a charge–transfer
(CT) complex[19,20] by donor intercalation in the electron-
deficient NDI membrane. We have previously shown that
hydrogen bonding was a prerequisite for donor intercalation,
In FT-IR studies[16] (Figure 3a), the sharp amide 1 band at
approximately 1700 cmÀ1 in THF showed a pronounced shift
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ure S14). Interestingly, upon urea addition, the intense green
color of the CT solution spontaneously disappeared with
a concomitant decrease in the CT band intensity at 560 nm
(Figure 3c). This observation indicates that the hydrazides,
which were not accessible otherwise, can now be reached by
urea molecules as a result of the donor intercalation, which is
expected to impart polarity to the membrane by partial
charge transfer even in the ground state.[19,22]
We then examined the impact of the contrasting surface
charges of these twin vesicles on the electrostatic interac-
tion[23] driven reorganization of the positively charged surface
of the protein a-chymotrypsin (Cht).[24] Activity assays were
conducted (for details, see the Supporting Information) using
N-succinyl-l-phenylalanine-para-nitroanilide[24]
(SPNA;
Figure 4) as a chromogenic substance that can be hydrolyzed
Figure 4. a) Time versus concentration plot of para-nitroaniline gener-
ated from the hydrolysis of SPNA in the presence of Cht (3.2m) after
incubation with NDI-1 or NDI-2 (100m) or without addition of an
amphiphile at pH 9.0. b) Cht activity in the absence or presence of an
NDI vesicle.
Figure 3. a) Solvent-dependent FT-IR spectra of NDI-1 and NDI-2.
b) UV/Vis spectra of NDI-1 (solid line) and NDI-2 (dotted line) in THF
(black) and H2O (before urea addition: light-gray line; after urea
addition: dark-gray line). [NDI]=0.5 mm, [urea]=11.3m and 9.9m for
NDI-1 and NDI-2, respectively. c) UV/Vis spectra (CT band) of NDI-1
and pyrene butyric acid (1:1; black line) and NDI-2 and pyrene butyric
acid (1:1; light-gray line) before (solid line) and after (dotted line) urea
addition. [NDI]=10.0 mm, [pyrene butyric acid]=10.0 mm,
[urea]=9.1m.
in the presence of Cht generating a new absorption peak at
405 nm, which is due to the formation of para-nitroaniline.
The activity of Cht in the absence of NDI was taken as 100%.
When Cht was incubated with NDI-1, a remarkable decrease
in activity by approximately 80% was observed (Figure 4). In
striking contrast, the enzymatic activity was mostly retained
in the presence of the NDI-2 vesicles as their surfaces consist
of nonionic OE chains. A mere 20% reduction in activity
could be a consequence of the non-specific adsorption of the
protein on NDI-2 vesicles. Unchanged emission (Fig-
ure S15a) and CD (Figure S15b) spectra during the activity
assay confirmed the lack of protein denaturation.[25–27] DLS
studies (Figure S16) indicated no disassembly of vesicles in
the presence of the protein.
Therefore, the pronounced drop in enzyme activity
selectively in the presence of NDI-1 is indeed an outcome
of the display of negatively charged phenoxides on the
surface, enabling effective electrostatic recognition of Cht. It
is noteworthy that the extent of activity inhibition (80%) can
be ranked among the best reported values except for the
recent report on Cht surface recognition by graphene
which did not occur in similar NDI amphiphiles lacking the
hydrazide groups,[7b] and thus the current observation indi-
rectly corroborates the FT-IR results confirming the presence
of hydrogen bonding. In fact, zipping the donor–acceptor
complex with a hydrogen bond led to unusually high
association constants (Ka) of 1.1 ꢂ 103 mÀ1 and 4.9 ꢂ 103 mÀ1
(Figure S12)[21] for the CT complexes of NDI-1 and NDI-2,
respectively, with pyrene butyric acid. Comparatively lower
Ka values of the NDI-1/pyrene butyrate complex can be
attributed to the electrostatic repulsion among the carboxyl-
ate and phenoxide ions as in this case both are located on the
same side of the membrane wall. Most importantly, the
intercalation of pyrene butyrate does not alter the vesicle
morphology other than a small enlargement in particle size,
which was observed by DLS (Figure S13) and TEM (Fig-
4
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