Supramolecular diversity through click chemistry: Switching from nanomicelles to 1D-nanotubes and tridimensional hydrogels

Article abstract of DOI:10.1021/cm4022613

The size and shape of nanoparticles are of prominent importance for their biological activities and for their application as smart drug delivery systems. Thus, synthetic designs allowing divergent synthesis of nanoscale materials with controlled size, morphology, and surface chemistry are currently highly desirable, but they remain a major challenge. Herein, we report a simple method for the creation of supramolecular diversity from structurally related diacetylenic-based glycolipids. We have found that neoglycolipids with an amide function between the hydrophilic and hydrophobic part of the amphiphile afford tridimensional micelles, while those having a triazole self-organize into 1D-nanotubes. Additionally, at higher concentrations, the clicked amphiphiles form hydrogels through three-dimensional networks of bundled nanotubes. Photopolymerization of the obtained nanomaterials leads to the formation of conjugated polydiacetylene backbone of alternating enyne groups, which rigidify glyconanomaterial structures enhancing their physical stability. The obtained nanostructures were extensively characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) techniques, enabling the confirmation of the formation of tubular structures in water for all triazolo-substituted neoglycolipids and micellar structures for the glycolipid containing an amide group. This fact refutes the so-called isosteric character of 1,2,3-triazole and amide groups, at least, at the supramolecular level and point out to the possibility of using the CuAAC between azides and alkynes to create supramolecular diversity at the nanoscale. The functionality of the gel was, moreover, evaluated as a nanocontainer for the incarceration and controlled release of the antitumoral topotecan.

Full text of DOI:10.1021/cm4022613

Article
pubs.acs.org/cm
Supramolecular Diversity through Click Chemistry: Switching from
Nanomicelles to 1D-Nanotubes and Tridimensional Hydrogels
Mohyeddin Assali,† Juan-Jose Cid,† Inmaculada Fernandez,‡ and Noureddine Khiar†,*
́
́
†Asymmetric Synthesis and Functional Nanosystems Group, Instituto de Investigaciones Químicas (IIQ), CSIC and Universidad de
Sevilla, C/Americo Vepucio 49, 41092 Seville, Spain
‡Departamento de Química Organ
ica y Farmaceutica, Universidad de Sevilla, C/García Gonzal
́
́
́
́
ez 2, 41012 Seville, Spain
S
* Supporting Information
ABSTRACT: The size and shape of nanoparticles are of prominent
importance for their biological activities and for their application as
smart drug delivery systems. Thus, synthetic designs allowing
divergent synthesis of nanoscale materials with controlled size,
morphology, and surface chemistry are currently highly desirable, but
they remain a major challenge. Herein, we report a simple method for
the creation of supramolecular diversity from structurally related
diacetylenic-based glycolipids. We have found that neoglycolipids
with an amide function between the hydrophilic and hydrophobic
part of the amphiphile afford tridimensional micelles, while those
having a triazole self-organize into 1D-nanotubes. Additionally, at
higher concentrations, the clicked amphiphiles form hydrogels
through three-dimensional networks of bundled nanotubes. Photo-
polymerization of the obtained nanomaterials leads to the formation of conjugated polydiacetylene backbone of alternating enyne
groups, which rigidify glyconanomaterial structures enhancing their physical stability. The obtained nanostructures were
extensively characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force
microscopy (AFM) techniques, enabling the confirmation of the formation of tubular structures in water for all triazolo-
substituted neoglycolipids and micellar structures for the glycolipid containing an amide group. This fact refutes the so-called
isosteric character of 1,2,3-triazole and amide groups, at least, at the supramolecular level and point out to the possibility of using
the CuAAC between azides and alkynes to create supramolecular diversity at the nanoscale. The functionality of the gel was,
moreover, evaluated as a nanocontainer for the incarceration and controlled release of the antitumoral topotecan.
KEYWORDS: click chemistry, supramolecular self-assembly, carbohydrate-coated robust nanomaterials, PDA-based hydrogels,
PDA-based micelles, PDA-based lipid nanotubes
their application as smart drug delivery systems.25−27 In this
INTRODUCTION
■
sense, while spherical nanomaterials, preferentially with 50 nm
size, are well suited for processes proceeding through a rapid
cellular uptake, 1D-rodlike structure are more appropriate for
those requiring improved circulation time. On the other hand,
it has recently been reported that whereas spherical mannose-
coated micelles are potent inhibitors of a globular lectin,
concanavalin A, the mannose-coated 1D-cylindrical micelles are
more suited for the inhibition of bacterial motility.28,29 Thus,
synthetic designs allowing the divergent synthesis of function-
alized nanoscale materials with varied morphologies preferen-
tially from a common intermediate, with no additional synthetic
cost are highly desirable. Yet, designing molecular materials in
which the self-assembled nanostructures are predetermined by
molecular architecture remains a major challenge, especially if
self-assembly is used to control the size, the morphology, and
The advent of nanotechnology has increased the need for
bottom-up approaches that can generate complex nanoscale
systems from simple molecules or architectures.1−6 In this
sense, chemical self-assembly, the spontaneous association of
molecules into well-defined structures held together by
noncovalent interactions, is one of the most attractive
approaches for constructing complex supramolecular nano-
structures.7−10 Hierarchical processes typical of chemical self-
assembly, spontaneously produce ordered multivalent sys-
tems,11−14 valuable tools in biology and nanotechnology,
starting from prefunctionalized monomers.15−20 As a result of
the deep impact of nanoscale materials in the nascent field of
nanomedicine, self-assembled nanostructures taking place in
water are receiving special interest, as they can be directly
applied in important biological processes.21−24 Structure−
activity relationship studies have shown that, beside the surface
chemistry, the topology and size of nanoscale molecules are key
factors for their biological activities, including their cellular
uptake, circulation time, interaction with specific receptors, and
Received: July 8, 2013
Revised: September 30, 2013
Published: October 2, 2013
© 2013 American Chemical Society
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Figure 1. Supramolecular self-organization of sugar-coated PDA-amphiphiles, showing the crucial role of the ligation group used to link the
diacetylenic hydrophobic and hydrophilic parts: Starting from a common intermediate a simple change of the amide function to a triazole group in I
allows the formation of 1D-nanotubes (B) which evolve to tridimensional gels (C) instead of forming tridimensional micelles (A).
the surface chemistry of the final nano-objects.30 For example, a
large variety of low molecular mass organogelators (LMOGs)
have been reported to form gels through three-dimensional
networks of bundled nanofibers and nanoribons.31−35 The
development of this nanoengineering field is exemplified by
recent applications of molecular gels in the fields of tissue
engineering,36−38 biomineralization,39,40 molecular elec-
tronics,41,42 and drug delivery.43−46 However, the lack of
knowledge of the fundamental parameters behind self-assembly
into gels difficults the design of systems in which molecular
modification would control nanoscale assembly that ultimately
endows specific materials function.47 A special concern about
the nanoscale materials formed by chemical self-organization,
and which limits their practical applications, is their limited
stability. The utilization of micelles for instance in biomedical
processes is limited by its sensitivity to dilution,48 while
hydrogels are easily collapsed or changed by small perturba-
tions in the environmental conditions.49 Therefore, there is
great interest in the synthesis of monomers that can be further
manipulated in the supramolecular state in order to endow the
nanoassemblies with physicochemical stability. Diacetylenic
amphiphiles are well suited for such a proposal, as they can
undergo topochemical reactions upon UV-irradiation or
thermal stimuli, leading to the anisotropic formation of
poly(diacetylene) (PDA) in the crystalline state.50,51 Closely
packed and properly ordered diacetylenes amphiphiles undergo
a smooth and clean photopolymerization via 1,4-addition
reaction, affording functional PDA-nanomaterials with en-
hanced stability and interesting chromatic properties.52−56
Indeed, PDAs exhibit an intense blue color due to the
absorption of the enyne cross-linked framework in the visible
region. Furthermore, phase transitions of the conjugated
backbone of the polymer can be induced, resulting in
modification of the delocalized conjugated electronic networks,
thereby resulting in dramatic blue-to-red transformations. The
structural/colorimetric transformations of PDA assemblies can
take place in response to heat (thermochromism),57−59 organic
solvents (solvatochromism),60,61 mechanical stress (mechano-
chromism),62,63 magnetic fields (magnetochromism),64 and
ligand−receptor interactions (affinochromism or biochrom-
ism).65−69 These exceptional physicochemical properties have
boosted the utilization of diacetylenic amphiphiles as self-
organizing monomers for the synthesis of various nano-
structured materials, including liposomes,70 micelles,71 films,72
and nanotubes,73 mainly for sensing proposals.
Based on these premises, in the present work, we report a
new design allowing the divergent synthesis of PDA-based
nanomaterials with different morphologies. We have found that,
in the case of amphiphilic neoglycolipid I (Figure 1, center), the
ligation group used to link the diacetylenic hydrophobic part
and the hydrophilic part formed by an oligoethyleneoxide-
tethered sugar play a crucial role in their self-organization.
While diacetylenic-based glycolipids with an amide function
between the hydrophilic and hydrophobic part afford micellar
structures (Figure 1A), those with an additional triazole ring
obtained by click ligation of relatively similar partners
afforded 1D-tubular microstructures (Figure 1B). Hierarchical
aggregation and entanglement of the nanotubes in water allow
us to develop novel hydrogels (Figure 1C), implementing the
attractive functions of the hollow cylinders. Subsequent
photopolymerization turns out stable and blue colored
nanomaterials responsive to external stimuli such as heat and
solvents. The structure, aggregation and self-assembly of the
formed nanomaterials have been investigated by nuclear
magnetic resonance (NMR), infrared spectroscopy (IR),
transmission electron microscopy (TEM), scanning electron
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Figure 2. Procedure for SWCNT/1 nanoassembly formation and synthesis of glyconanosomes (GNS). (A) (i) Formation of micelles by mixing
neoglycolipid 1 above its critical micelle concentration (CMC) in water. (ii) Sonication-promoted supramolecular self-assembly of neoglycolipid 1 in
concentric hemimicelles around SWCNTs. (iii) Intermolecular photopolymerization of neoglycolipid 1 hemimicelles into homogeneous
glyconanorings (GNRs). Above, TEM micrograph of SWCNT/1 nanoassemblies and their idealized representative figures. (B) Structural and
chemical composition of a sought glyconanosome in the present work.
microscopy (SEM), and atomic force microscopy (AFM). One
of the resulting gels was then assayed as a nanocontainer for the
inclusion and release of cytotoxic topotecan drug as a first step
toward their use as smart drug delivery system. As far as we
know, this is the first example of supramolecular nanotube
hydrogel, based on amphiphilic diacetylene building blocks,74
although some bolaamphiphilic diacetylenes are known to gel
organic solvents or a mixture of water and polar organic
solvents.75−78
Preliminary mechanistic studies have shown that neo-
glycolipid 1 first self-organizes into micelles in aqueous
solution, then undergoing a hemimicellar arrangement on the
solid surface of the CNTs. Indeed, a TEM analysis of a water
solution containing 1 over the critical micellar concentration
(CMC) shows the formation of spherical micelles with 12 nm
diameter (Figure 2A, step i). The micellar solution of
neoglycolipid 1 was capable of facilitating and promoting the
deaggregation of SWCNTs bundles, giving rise to stable
dispersions of assembled SWCNTs/1 nanoconstructs. Interest-
ingly, TEM, SEM, and AFM analyses showed that compound 1
self-assembled on the SWCNTs surfaces in a supramolecular
fashion, resulting in rings made of rolled-up half cylinders
(Figure 2A, step ii). The photopolymerization of the
diacetylene function upon ultraviolet irradiation (254 nm)
afforded a conjugated polydiacetylene backbone of alternating
enyne groups (Figure 2A, step iii), which rigidified the inner
core of each hemimicelle, resulting in robust polymerized
glyconanorings (GNRs) around the nanotube in an abacus-like
geometry. Subsequently, ultrasonication of the nanoconstructs
allowed the slid of neutral glyconanorings out of their
assemblies with SWCNTs. These new disc-shaped nanoma-
terials, called glyconanosomes (GNSs), with a 35−55 nm
length diameter have a hydrophobic inner core surrounded by a
RESULTS AND DISCUSSION
■
Synthesis of “Clicked” Diacetylene-Based Glycolipid
Monomers. We have recently reported a bottom-up approach
for the water-solubilization and biofuncionalization of CNTs
(Figure 2)79,80 based on the self-organization of amphiphilic
molecules on the CNTs sidewalls.81−84 In the case of
neoglycolipid 1, the design include a tetraethyleneglycol-
tethered lactose moiety responsible of the water solubilization.
Opposite to the external exposition of the functional
glycoligand, a 25 carbon-based hydrophobic tail was included
for efficient van der Waals interactions with the CNT sidewalls,
bearing furthermore, a photopolymerizable diacetylenic func-
tion (Figure 2A).
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Figure 3. (A) Convergent synthesis of neoglycolipid 2 by CuCAAC: (a) CuI, DIPEA, CH2Cl2, rt, overnight; (88%). (b) (i) NaOMe, MeOH, rt; 1 h.
(ii) Amberlyst Ir-120; (67%). (c) (i) H2O, 70 °C, (ii) hv (254 nm), 24 h. (B) A large-scale height (I) and enlarge phase (II) TEM images of
spherical nanotubes formed by self-organization of neoglycolipid 2 in water.
Figure 4. Chemical structures of neoglycolipids 6−8 with increasing length of the spacer in between the diacetylenic tail and the sugar headgroup.
hydrophilic tetraethyleneglycol chain capped by a biodetectable
lactose moiety are well-suited candidates for active drug
delivery.85 In order to fine-tune the hydrophobicity and
functionality of the inner core of the GNSs, we thought in
including an aromatic ring able to establish further interactions
with guest molecules through π−π interactions (Figure 2B). As
an aromatic fragment, we choose to introduce a triazole ring
connecting the hydrophilic and hydrophobic parts of the
amphiphile by using the unrivalled copper(I)-catalyzed azide−
alkyne cycloaddition (CuAAC), the paradigmatic example of
“click chemistry”.86−88 The synthesis of the required
diacetylenic lipid 2 (Figure 3A), functionalized with thiolactose
has been performed in a convergent manner using a similar
approach than that for compound 1. The click reaction of the
advanced azido intermediate 3 used in the synthesis of 1 and N-
(2-propynyl)pentacosa-10,12-diynamide 4 using Cu(I) in the
presence of Hunig base in methylene chloride, afforded
regioselectively the 1,2,3−triazole derivative 5 in excellent
88% yield, (Figure 3A, step a). Finally, a Zemplen deacetylation
in methanol afforded the desired thiolactose-based neo-
glycolipid 2, as depicted in Figure 3A, step ii.
Surprisingly, despite its structural similarity with compound
1, neoglycolipid 2 was unable to solubilize CNTs in water,
consequence of its incapacity to form micelles. Indeed, a study
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of water solutions of neoglycolipid 2 shows that it self-organizes
forming 1D-tubular nanostructure, able to gel water at
concentration of 1% w/w. TEM analysis of a negatively stained
diluted solution (0.1%) of 2 showed the formation of
nanotubes with several micrometer lengths, an outer diameter
of 70 nm, an inner diameter of 14 nm, and a membrane
thickness of 28 nm (Figure 3A, step iii).
In order to decipher the exact role of the triazole ring on the
self-organization of 2 into 1D tubular nanostructure, we
designed various related neoglycolipids and studied their self-
organizations. To fine-tune the hydrophilic/hydrophobic
balance of the designed amphiphiles and generalize the
observed effect to other sugar coated nanomaterials, we used
as sugar headgroup the monosaccharide mannose, instead of
the disaccharide lactose. The neoglycolipids designed are
depicted in Figure 4 and include neoglycolipid 6 with no
hydrophilic spacer between the hydrophobic moiety and the
sugar headgroup, the neoglycolipid with a hydrophilic spacer
derived from a tetraethyleneglycol 7, and, finally, the neo-
glycolipid 8 with a hydrophilic spacer 2-fold larger than in
compound 7. These triazolo-substituted neoglycolipids were
synthesized by means of a convergent synthetic routes similar
to that used for the synthesis of 2, employing as a key reaction
the regioselective CuAAC reaction between the corresponding
azido-substituted sugars and the trialkyne N-(2-propynyl)-
pentacosa-10,12-diynamide 4.
Study of the Self-Association of the “Clicked”
Diacetylene-Based Glycolipid Monomers in Water.
Once in hand, each neoglycolipid was suspended in Milli-Q
water, affording diluted solutions (0.1%) that were sub-
sequently heated beyond the melting temperature of pure
solids (up to 50 °C in all cases), so ensuring the complete
deaggregation and homogeneity of the mixtures (qualitatively
showing as noncloudy solutions). Then, they were slowly
cooled to room temperature, observing the apparition of
homogeneous white fibrillar suspensions for all the neo-
glycolipids 6−8 studied. Once all the basic supramolecular
assemblies were attained, subsequent photopolymerization
reactions comprising the diynic fragment into poly(diacetylene)
polymer (PDA) derivatives were carried out by irradiation at
254 nm using a UV lamp. A series of covalently fixed PDA-
derived structures with a dark-blue color were obtained,
accompanied by the appearance of an absorption band in the
visible region with a maximum at 605 nm (Figure 5).
PDA-based nanomaterials are responsive to various external
stimuli and as such, are excellent sensors able to detect and
report the change occurring in their surrounding medium. In
this sense, heating the clicked glyconantubes 6 and 7 at 100 °C
for 5 min induces a drastic chromatic change from blue to red
(Figure 5C, spectra I and II) with the concomitant
disappearance of the band at 605 and the appearance of a
new band at 505 in the UV−vis spectra. Beside their
characteristic thermochromism, glyconanotubes are also
sensitive to the solvent present in the medium (solvatochrom-
ism) as shown by the change to red of the fibrillar
nanostructures upon adding methanol.
Next, the resulting structures and morphologies of the
diluted mixtures of clicked nanomaterials were analyzed by
TEM and AFM microscopies (Figure 6). In all the series,
micrometer length glyconanotubes were formed (Figure 6A)
having relatively similar internal diameters (around 15 nm) and
external diameters varying from 46 nm for 6, 54 nm for 7 to 75
nm for 8, (Figure 6B). These results, demonstrate that the self-
Figure 5. (A) Photographs of vials showing the formation of blue
colored PDA-based nanotube suspensions after photopolymerization
of neoglycolipids 2 (I), 6 (II), 7 (III), and 8(IV). (B) Photographs of
vials highlighting the thermochromatic changes from blue to red of the
nanotube suspensions derived from 6 (I) and 7 (II) upon heating at
100 °C for 5 min, characterized by disappearance of the band at 605
and the appearance of a new band at 505 in the UV−vis spectra (C).
organization of clicked diacetylenic glycolipids is indeed
independent of the hydrophobic/hydrophilic balance of the
amphiphile and consistently lead to 1D-hollow glyconanotubes
with relatively similar structures in form, size, and thickness.
Moreover, AFM (specially the AFM-3D image) of
glyconanotube 7 (Figure 6C and D) corroborated the tubular
nature of the nanoconstructs, reflecting consecutive striations
on the tube surfaces, as a consequence of the regular folding,
and curl of rolled tapes composed from the superposition of
neoglycolipidic bilayers (see also Figures S7 and S8 in the SI
and the tubular model depicted in Figure 8C vide infra).
Gelation Study. All the clicked neoglycolipids with the
triazole ring led to the formation of gel-state at 1%
concentration, and in the following section, we will detail the
synthesis, characterization, and use as nanocontainer for the
stabilization and controlled delivery of antitumoral drug of the
gel formed from neoglycolipid 7. An aqueous solution of
neoglycolipid 7 (1%) was heated at 50 °C for 30 min, affording
upon cooling to room temperature a white waxy material whose
jelly nature was proved by the method of being “stable to
inversion of the container”, highlighting a complete absence of
flow (Figure 7).89 The stability of the nonpolymerized gel at
room temperature and in absence of light was about three
weeks, after which solid depletion and precipitation was
observed. Photopolymerization of the gel 7 by irradiation at
254 nm with a 6 W UV lamp at a distance of 10 cm for 24 h led
to a remarkable color change to intense blue (Figure 7A),
accompanied by the appearance of an absorption band in the
visible region with a maximum at 605 nm (Figure 7B). These
results reflect the formation of poly(diacetylenes) as the result
of 1,4-addition reaction of the diyne groups in the gel phase.
Although it is well-known that the photopolymerization
reaction is really efficient only in highly organized systems,
the intense blue color observed after irradiation implies the
extension of the π-delocalization along the polymerized
nanotube.90 The formation of conjugated poly(diacetylene)
backbone of alternating enyne groups rigidifies the glyco-
nanotube wall, resulting in a robust gel with temporal stability
beyond one year. The structure of the polymerized hydrogel
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Figure 6. Large-scale height (A) and enlarged phase (B) TEM images of spherical nanotubes formed by self-organization of neoglycolipid 6 (I), 7
(II), and 8 (III). Bidimensional (C) and tridimensional (D) AFM images obtained by the tapping mode of a water-solution drop of helical nanotubes
formed by self-organization of 8 in water, after deposition and drying on a just exfoliated mica plate.
formed from amphiphile 7 was further characterized by TEM,
SEM, and AFM microscopies (Figure 7C−E). Meshes of
agglomerated tubule with uniform diameters (50−70 nm) and
lengths in the micrometer scale were consistently found in the
photopolymerized hydrogel 7 under TEM (Figure 7C).
However, in comparison with nonpolymerized gel no
significant morphological differences were found for the
polymerized xerogel 7. This observation has been ascribed to
the tube-like preorganization of glycolipid 7 molecules by
intermolecular hydrogen-bonding and π-stacking forces of the
amido and triazole groups respectively in nonpolymerized gel,
in such a way that the subsequent photopolymerization
reaction just fixed covalently the structure without altering
the primary structure. The observation of the photopolymer-
ized xerogel 7 under SEM at the hundreds of micrometer scale
showed interesting intertwined and twisted structures similar to
posies in a bush-form (Figure 7D). Moreover HR-SEM,
allowed observing the intimate fibrillar aggregates composing
the posies shown on the standard SEM images (Figure 7D).
Going into an intermediate scale (1 μm), AFM microscopy
showed a dense material composed by intercrossed and well-
packed bundles of smaller and overlapped fibers, mimicking the
seams in a cloth patch (Figure 7E). On the other hand, HR-
AFM corroborated the aforementioned TEM data turning on
fiber widths at around 52−67 nm (z-axis in the tapping mode).
Fourier transform infrared (FT-IR) measurements were
performed to understand the nature of the interactions that
participate in the assemblies (Figure 8). It is known that both
amide I and amide II bands become weaker and shift into lower
wavenumber when passing from free to H-bonded amide
groups.91 Something similar happens with the vibration of
1,2,3-triazole rings in π−π stacked aggregates. As such, the
starting neoglycolipid 7 exhibits the amide I and II vibration
modes at 1636 and 1550 cm−1, respectively, while in the
condensed phase the same peaks appeared at around ∼1628
and ∼1547 cm−1 values, together with a decreased intensity in
both the dilute solutions and photopolymerized hydrogel
(Figure 8A). In a similar way, the peak at 1480 cm−1 assigned
to a stretching mode of the 1,2,3-triazole ring,92 weaken and
shifted at ∼1475 cm−1 in both examples. On the other hand,
the symmetric and antisymmetric CH2 stretching bands of the
alkyl chains are found without modification at ∼2850 and
∼2920 cm−1, respectively, thus indicating a well packing and
arrangement in all-trans conformation and light disorder in the
different structures. These values highlight a cooperative self-
assembly involving hydrogen bonding, π−π stacking and
hydrophobic interactions simultaneously.
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Figure 7. Photographs of vials showing the formation of hydrogel from neoglycolipid 7 and the chromatic change from white to blue upon
irradiation at 254 nm with a 6 W UV lamp, highlighting the polymerization within the gel phase and the formation of PDA, accompanied by the
appearance of an absorption band in the visible region with a maximum at 605 nm (B). TEM (C), SEM (D), and AFM (E) micrographs of xerogel
derived from 7 on Cu grids (C and D) and on just-exfoliated mica (E).
Complementary small-angle X-ray scattering (SAXS) studies
allowed stating the intimate structure of the hydrogel derived
from 7. Maximum peaks corresponding to averaged repeating
distances of ca 9.8 nm was obtained (Figure 8B). Taking into
account that the estimated size of a bilayer comprising two
molecules of 7 with the alkyl-diyne chains in the center is 10
nm long (Figure 8D), the values match with a structure formed
up by two concentric bilayers of glycolipids arranged in a
tubular fashion in agreement with TEM results (∼ 20 nm), as
depicted in the sketch in Figure 8C. Based on these results, we
suggest that the 1D-tubular supramolecular structures formed
by the clicked glycolipids are stabilized by hydrogen bonds
between the amide, the solvophobic interactions of the alkyl
chains, and the π−π interactions between the triazole rings
(Figure S12 in the SI).
However, compound 1, which self-organizes into micelles has
all the aforementioned interactions with the exception of the
claimed π−π interactions. This observation raises the question
if the driving force in the self-organization into 1D-nanoma-
terials is consequence of the structural restriction imposed by
the π−π interaction or if it is simply a result of the higher
number of interactions involved in clicked nanotubes 2, 6−8
with regard to glycolipid 1. In other words: What structure
would result if we change the triazole ring by a different
functional group able to establish further interaction such as
hydrogen bonding for instance? At this point, it is worthy of
mention that one reason at the basis of the great success of
CuAAC in different research fields is the claim that the triazole
ring act as a robust nonhydrolyzable amide mimic.93
Structurally, the 1,4-disustituted 1,3 triazole can indeed mimic
the E amide bond, with the N-3 electrons acting as the carbonyl
oxygen of the amide, the polarization of the C5−H bond allows
it to act as a hydrogen bond donor equivalent to the N−H
bond of the amide, while the electrophilic and polarizable
nature of the C-4 is electronically similar to the carbonyl carbon
of the amide. Additionally, taking into account that the dipole
moment of the triazole ring is higher than that of the amide
bond, its hydrogen donor−acceptor properties are higher than
those of the amide.
Based on these premises, we synthesized glycolipid 9, similar
to 7 with the exception that an amide function has been
included instead of the triazole ring (Figure 9). The synthesis of
the sought glycolipid was done starting from the amine 10
obtained in 80% yield from the common azide derivative used
previously in the synthesis of 7 by catalytic hydrogenation.
Condensation of the amine with N-Boc glycine 11 using DIPC
as activator in the presence of DMAP afforded the Boc
derivative 12 in 63% yield (Figure 9, step a). Boc-deprotection
was accomplished by using trifluoroacetic acid in methylene
chloride, leading to the free amine 13 in good yield (Figure 9,
step b). Subsequently, condensation of the amine with 10,12-
pentacosadiinoic acid (PCDA) 14 in the presence of TBTU
and DMAP afforded the fully protected glycolipid 15 in 52%
yield (Figure 9, step c). Finally, Zemplen deacetylation
followed by purification of the product with G-20 sephadex
column turned out pure glycolipid 9 (Figure 9, step d).
Once in hand, neoglycolipid 9 was submitted to the same
conditions than those used for the formation of nanotubes and
gel from 7 (Figure 9, step e). In this case, a clear solution was
obtained with no apparent formation of nanotubes or gel
neither at 0.1% nor at 1% concentration. The TEM analysis of
the clear solution obtained, reveals the formation of spherical
micelles with 8 nm diameter.
This result refutes the generalized assumption that the
triazole ring is a good amide bond mimic and indicate that, at
the supramolecular level, the claimed analogy should be used
with extreme precaution as they can, and indeed do, generate
completely different supramolecular assemblies. We ascribe this
behavior to the ability of the triazole ring to establish effective
π−π interactions, and also to the larger distance between the
triazole substituents, connected by three atoms, compared with
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Figure 8. (A) FT-IR spectra of free neoglycolipid 7 (black) and self-assembled hydrogel (blue). (B) Small angle X-ray scattering of hydrogel derived
from neoglycolpid 7. (C) Model of the molecular bilayer nanotube derived from SAXS study. (D) Hyperchem schematic illustration of the molecular
packing in the self-assembled state.
those of the amide connected only with two atoms, with a
global increase of 1.1 Å.
administration or even by the direct implantation into the
body.98 Within the tested gels, polymeric hydrogels exhibit a
similar range of physical, chemical, and biological properties for
biocompatible drug delivery systems than discrete molecules-
based hydrogels, but offering the possibility to give controlled,
pulsed, and triggered drug release profiles in a variety of tissues
while maintaining their mechanical integrity.99 Surprisingly, as
far as we know, no preliminary studies on controlled release of
drugs have been yet reported for robust PDA-based hydrogels.
With this aim in mind, we conducted some preliminary
studies directed toward the utilization of polymerized PDA
hydrogel 7 as a drug delivery depot system and stabilizer of the
chemo-therapeutic drug topotecan (TPT) in order to
determine its controlled-release properties (Figure 10).100,101
TPT is a hemisynthetic analogue of the alkaloid camptothecin,
a natural cytotoxic compound with high anticarcinogenic
activity against breast, colon, ovary, and lung tumors. In
contrast to camptothecin, TPT is soluble in water thanks to the
tertiary amine at C-9 position, but similar to other CPT-family
drugs, suffer from poor stability and from the preponderance of
Study of Topotecan Release from Prepared PDA
Hydrogels. It has been recently demonstrated that the
controlled and constant supply of targeted anticarcinogenic
drugs results in a better prognostic and cure of certain cancers
than the administration of nonspecific medicines at tolerated
maximum doses in reiterative cycles.94 In this regard, a slow but
constant and controlled release of anticarcinogen quantities has
been achieved, for instance, by means of implants of
antitumoral-swollen gels, resulting in an increased global
efficacy.95
Despite the wide use of DMSO-based organogels for this and
other pathologies,96,97 hydrogels are the more desirable
biocompatible materials for controlled release and for
bioadhesive and targetable devices of therapeutic agents. This
fact is ascribed to the specific control of drug release rate that
can be achieved, while maintaining the pharmacologically
effective drug levels for extended periods of time in blood,
through oral, rectal, ocular, epidermal, or subcutaneous
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Figure 9. Synthesis of neoglycolipid 9: (a) DIPC, DMAP, CH2Cl2, rt, 16 h; (63%), (b) TFA, CH2Cl2, rt, 5 h; (99%.), (c) (i) PCDA (14), TBTU,
DIPEA, DMF, rt, 5 min. (ii) 13, DIPEA, DMF, rt, 14 h; (52%), (d) (i) NaOMe, MeOH, rt; 1 h. (ii) Amberlyst Ir-120; (82%), (e) H2O, 50 °C;
together with a TEM image showing the resulting self-organization in water in a micellar fashion.
Figure 10. (A) Hydrolysis reaction of TPT from the active lactone form (red) to the inactive carboxylate form (blue) at physiologic pH. (B)
Preliminary study by absorption spectrophotometry of the TPT release in water from TPT-containing hydrogel 7 vs time.
a less active carboxylate form over the active lactone form at
physiologic pH (Figure 10A).
67% from hydrogel TPT/7 was attained at t = 30 h (Figure
10B). In comparison with the few examples in the literature
including cross-linked TPT-containing hydrogels, we obtained
a minor release percentage than that of a poly(ethyleneglycol)
copolymer with vinylsulphone cross-linker.100 To the contrary,
the TPT/gel-7 system resulted in a 3-fold more prolonged
release time than the cited example.
The synthesis of TPT/gel complex was carried out by
dissolving 100 μg of TPT in an aqueous solution of 7 (1 mg/
100 μL) followed by gel formation, as described before. Upon
irradiation at 254 nm for 24 h, the color of the gel-TPT
assembly changed from white to deep blue, indicating that the
polymerization takes place ensuring the complete entrapping of
the chemo-medicine within the polymeric framework in the
form of the composite TPT/gel-7.
Freshly prepared hydrogel TPT/gel-7 was disposed in a test
vial containing Milli-Q water (1.00 mL) at 37.0 °C. Afterward,
aliquots of identical volume of the supernatant were taken at
different intervals of time, filling up to 1.00 mL and the
absorptions at 381 nm recorded in a spectrophotometer. The
absorbance data were corrected and converted into concen-
tration values according to the previously stated Lambert−Beer
law and the calculated molar extinction coefficient from
standards of TPT at 37 °C (for more details, see SI). As
deduced from sigmoid curve of Figure 10 and the reach of a
plateau, a maximum percentage value of TPT release at around
CONCLUSIONS
■
In conclusion, we have synthesized a series of glycolipids by
means of convergent synthetic pathways that include as key
ligation reaction either CuAAC or amidation, resulting in
amphiphiles with 1,2,3-trizolo or amido groups, respectively.
From the comparison of the supramolecular self-organization in
water, we have observed that the neoglycolipids with an amide
ligating the hydrophilic and hydrophobic part of the amphiphile
afford tridimensional micelles, while those having a triazole self-
organize into 1D-nanotubes. Additionally, at higher concen-
tration (1%), the clicked amphiphiles form hydrogels through
three-dimensional networks of bundled nanotubes. Photo-
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Chemistry of Materials
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nanotubes, as demonstrated by the remarkable color change from
white to intense blue.
polymerization of obtained nanomaterials by irradiation at 254
nm leads to the formation of conjugated poly(diacetylene)
backbones of alternating enyne groups, which rigidify the
glyconanomaterials, thus enhancing their physical stability, a
critical issue for their future medical uses. The 1D-based
nanomaterials have an intense blue color, indicative of the long-
range π-delocalization along the polymerized nanotube, and are
excellent sensors of the surrounding media able to change their
color to red as a consequence of heat (thermochromism) or
solvent (solvatochromism). The obtained nanostructures were
extensively characterized using TEM and AFM techniques,
enabling the confirmation of the formation of tubular structures
in water for all triazolo-substituted neoglycolipids (2, 6−8) and
micellar structures for the glycolipid containing an amide group
(1 and 9). This fact refutes the so-called isosteric character of
1,2,3-triazole and amide groups, at least at the supramolecular
level, and points to the possibility of using the CuAAC between
azides and alkynes to create supramolecular diversity at the
nanoscale. According to SAXS, FT-IR, and molecular modeling,
the resulting structures are explained in terms of glycolipidic
bilayers associated in concentric tubules, with all the association
motifs participating in the assembly. The functionality of the
latter has been, moreover, evaluated as a drug releaser of
topotecan, giving rise to a maximum release value of ca. 67%
after 30 h.
Preparation of Gel-7/TPT Inclusion Complex. TPT (100 μg)
was dissolved in an aqueous solution of neoglycolipid 7 (1 mg/100
μL). After heating to 50 °C during 30 min, the solution was allowed to
cool to room temperature inducing the formation of the hydrogel-7/
TPT inclusion complex. Next, the photopolymerization of the
diacetylenic tails as described before afforded the polymerized blue-
colored hydrogel-7/TPT complex with high stability.
ASSOCIATED CONTENT
* Supporting Information
Detailed methods and additional figures. This material is
■
S
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +34 95 448 9559. Fax: +34 954460565. E-mail: khiar@
iiq.csic.es.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the Ministerio de Economia y
■
́
Competitividad (Grant No. CTQ2010-21755-CO2-01) and the
́
Junta de Andalucia (Grant Nos. P07-FQM-2774 and P08-CTS-
A salient feature of our designed functional monomers is the
formation of biologically active hydro-soluble nanomaterials
exposing a dense layer of carbohydrates to the water phase
similar to the glycocalyx at the cell surface. In this sense, recent
investigations have confirmed the prime importance of
carbohydrates in important biological events including cell−
cell communication, cell adhesion, fertilization, differentiation,
development, inflammation, tumor cell metastasis, and
pathogen infections.102,103 Therefore, the polymerized nano-
micelles, which are covered by a dense carbohydrate array can
be used as synthetic multivalent systems to treat pathologies
mediated by carbohydrate−lectin interactions. Additionally, the
presence of a poly(ethyleneglycol) (PEG) fragmentknown to
reduce the rapid uptake and clearance in vivo by the cell
mononuclear phagocytic systemmakes the formed nano-
micelles excellent nanovectors for the active targeting of
hydrophobic antitumoral molecules. On the other hand, the
formed hydrogels mimic the extracellular matrix and, as such,
are excellent materials for tissue engineering. Moreover, the
nanotube-based 3D-nanogels can also encapsulate biologically
active molecules including proteins in the inner hollow cavity of
the tubules, preventing their fast denaturation behavior and
therefore, keeping their biological activity. Based on our
findings and on the previously reported works, the applications
of both polymerized micelles and LNT-based hydrogels as
smart nanocontainers for active drug delivery and as 3D-matrix
for tissue engineering are being actively investigated in our
laboratories, and the results will be reported in due courses.
04297). We acknowledge CITIUS for TEM, AFM, and NMR
facilities. Dr. M. Assali thanks the MAEC-AECID for PhD
scholarship. Dr. J.-J. Cid thanks CSIC for the postdoctoral JAE
DOC scholarship.
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