Bulky toroidal and vesicular self-assembled nanostructures from fullerene end-capped rod-like polymers

Article abstract of DOI:10.1039/c4cc01477d

In this work, we present novel fullerene (C₆₀) end-capped rod-like polypeptide-polymers, obtained by one-pot thiol-ene chemistry. These systems are able to self-assemble in water creating precise bulky microstructures of toroidal or vesicular shapes. Independent molecular dynamics simulations supported the observed experimental results. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01477d

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Bulky toroidal and vesicular self-assembled
nanostructures from fullerene end-capped
rod-like polymers†
Cite this: Chem. Commun., 2014,
50, 4571
Received 26th February 2014,
Accepted 17th March 2014
D. Mazzier, M. Mba, M. Zerbetto* and A. Moretto*
DOI: 10.1039/c4cc01477d
www.rsc.org/chemcomm
In this work, we present novel fullerene (C60) end-capped rod-like from commercial sources or could be designed and synthesized
polypeptide-polymers, obtained by one-pot thiol–ene chemistry. depending on the finalities. Moreover, polypeptides are fascinating
These systems are able to self-assemble in water creating precise and unique due to their ability to self-assemble into ordered
bulky microstructures of toroidal or vesicular shapes. Independent structures,15 making them attractive in those many applications
molecular dynamics simulations supported the observed experimental where well-organized architectures are essential to accomplish a
results.
function. Substantial progresses have been made in the NCA/
polypeptide field in the past decade14 and today several innovative
One of the novel themes in areas such as materials science, polypeptide-based materials have been produced and studied.16
nanochemistry and biomimetic chemistry is the construction Apart from the ability to assemble in a wide range of different
of supramolecular architectures characterized by well-distinct supramolecular structures of nanoscale dimensions,17 another
shapes and functions.1,2 Undoubtedly, the self-assembly process of unique characteristic of rod-polypeptidic segments is the possibility
polymers through non-covalent interactions, including hydrophilic to endow various functionalities such as photophysical and electro-
and hydrophobic effects, electrostatic contacts, hydrogen bonding, chemical properties to the supramolecular materials. An example of
microphase segregation and shape effects, has great potential for a well-known rod-like polypeptide is poly(g-benzyl-L-glutamate)
creating such supramolecular architectures.3–6 Recently, the (PBLG). PBLG has intramolecular hydrogen bonds that stabilize
improvement in synthetic methodologies associated with the use the secondary a-helical structure and shows mesoscopic liquid
of designed conformational constraints led to the ability to per- crystal order because of its good solubility and rod-like structure.18
form nanometric control, on both size and shape, even at the Inspired by the recent work of Yagci and co-workers,19 we decided to
single polymer molecule level.7 In the literature many examples of functionalize both N- and C-termini of PBLG using thiol–ene
supramolecular vesicles and micelles formed by block copolymers chemistry20 to prepare C60–PBLG (C60, fullerene) conjugates in a
containing at least one high-molecular weight polymeric peptide way that only C or C/N-terminus mono-addition products are
domain,8–10 as well as by small- or medium-sized peptides formed without any contamination of ill-defined derivatives. In
conjugated with molecules of nonpeptidic nature,9,11 can be found. particular, herein we report the one-pot synthesis of two C60–PBLG
Rarely, homo-polypeptides alone were reported as precursors of self- derivatives, namely NH2–PBLG–C60 (3) and C60–PBLG–C60 (6) (Fig. 1)
assembled microstructures that are spherical in shape. Polypeptides and their controlled self-assembly in water.21
prepared by the ring-opening polymerization (ROP) of a-amino acid
N-carboxyanhydrides (NCAs), which have been known since many
decades,12 are nowadays emerging biomaterials13 that are gaining
increasing attention for various applications including drug delivery,
gene therapy, antibiotics and tissue engineering.14 Polypeptides can
offer considerable chemical diversity: beyond the twenty canonical
amino acids alone, non-canonical amino acids are readily accessible
Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131
Padova, Italy. E-mail: alessandro.moretto.1@unipd.it, mirco.zerbetto@unipd.it
† Electronic supplementary information (ESI) available: Synthetic details, SEC
traces, 13C-NMR, 1H-NMR and solid state IR characterization of the polymers (1, 3
Fig. 1 Molecular structures of NH2–PBLG–C60 (3) and C60–PBLG–C60 (6).
Chem. Commun., 2014, 50, 4571--4574 | 4571
and 6). See DOI: 10.1039/c4cc01477d
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region. We assign the observed UV absorptions in 1 and 2 mono and
bis fullerene-conjugates to the fullerene moiety19 considering the
small differences in the resonance frequencies with respect to the
isolated fullerene due to perturbations of the electronic structure
caused by the bonding with the PBLG polymer. The third indication
of the formation of PBLG-conjugated fullerenes comes from the
thermal stability of 1, 2 and 3 that was investigated via thermo-
gravimetric analysis (TGA; 100–700 1C) under nitrogen flow with a
heating rate of 10 1C minÀ1 (Fig. 2B). Accordingly, PBLG polymers
undergo thermal decomposition between 250 and 350 1C, whereas
the thermally stable fullerene component maintains its stability
up to almost 600 1C.19 The weight percentages of the components
(polymer 3, C60/PBLG 4 : 96%; polymer 6, C60/PBLG 7 : 93%)
determined by TGA are in agreement with those of SEC analysis. All
of the experimental results suggested an average number of BLG
amino acids, for the polymer unit, close to 85. The secondary
structure of polymers 1, 3 and 6 was analyzed by ECD (electronic
circular dichroism), which confirmed the preservation of the
a-helical conformation for all the synthesized compounds
(Fig. S1, ESI†), which in turn implies the rod-like shape of the
polymers. Synthetic details, SEC traces, 13C-NMR, 1H-NMR (the
last two characterizations do not allow the detection of C60 due
to the large amount of PBLG with respect to C60) and solid state
IR characterization of the polymers (1, 3 and 6) are reported in
the ESI† and Fig. S2–S5, respectively.
From polymer 3, donut-like morphologies were generated,
in the form of a milk-like suspension, starting from a 20 mg
(3 : 7, v/v) DMF–THF solution (10 mL) dialyzed (membrane
cutoff, 12 KD) against ultrapure water (48 hours) (Fig. 3).
TEM (transmission electron microscopy) images show tor-
oidal shapes with an external diameter ranging between
200 nm and 650 nm (Fig. 3A and B, stained and unstained
samples, respectively), additionally confirmed by SEM (scan-
ning electron microscopy) analysis (Fig. 3C). Moreover, AFM
(atomic force microscopy) details of the selected nanodonut
shown in Fig. 3C reveal a diameter of 200 nm, a thickness of
Scheme 1 Synthesis of polymers NH2–PBLG–C60 (3) and C60–PBLG–C60 (6).
BLG–NCA (4 mmol) was polymerized in dioxane for 24 h at
room temperature, using Trt-cysteamine (Trt, triphenylmethyl)
as the initiator (0.04 mmol) (Scheme 1). Polymer 1 was obtained
after precipitation with methanol in 81% yield (MW = 19100 and
PDI 1.11 by SEC). Subsequently, the Trt-protecting group was
removed yielding polymer 2 (not shown in Scheme 1). Next, 2
(0.01 mmol) was dissolved in DMF and added to toluene. Then,
fullerene (0.05 mmol) and AIBN (0.15 mmol) were added (in this
order) to the previous solution and the subsequent homogeneous
mixture was heated at 90 1C under stirring for 5 h. Subsequently,
the solvent was removed and the resulting solid was suspended in
DMF, passed through a 20 mm filter, and then precipitated by
addition of methanol, giving polymer 3 as a brown solid in 85%
yield (MW = 19 550 and PDI 1.09 by SEC).
Moreover, polymer 3 was successively N-end capped with a
large excess of pre-activated Trt-mercaptopropionic acid, yielding
polymer 4 (not shown in Scheme 1). Polymer 4 was C-terminus
Trt-deprotected (as described before, yielding polymer 5, not
shown in Scheme 1) and placed to react under the same condi-
tions described above for the synthesis of polymer 3, thus to
generate the bisconjugate C60–PBLG–C60, polymer 6 in 78% yield
(MW = 20 300, PDI 1.11 by SEC). Since all the synthesized polymers
possess the same PBLG part (as it comes from polymer 1), the
differences in MWs determined by SEC constitute a first evidence
for the addition of one or two fullerenes.
A second evidence for the covalent attachment of fullerenes arises
from the absorption spectra of 1, 3 and 6, which were recorded in a
THF solvent and are shown in Fig. 2A. The observed patterns are
close to that of fullerene, which shows high UV absorptions at
around l = 287 and 330 nm and tails spanning over the 600 nm
Fig. 3 (A and B) TEM (A, stained and B, unstained) images of the resulting
self-assembled nanostructures obtained from polymer 3. (C) SEM image
from the very same prepared sample used in TEM analysis. (D and E) AFM
micrograph and high-profile of the selected (1) toroidal nanostructure.
Fig. 2 (A) UV/Vis spectra of polymers 1, 3 and 6 in THF solution (concen-
tration: 0.01 mg mLÀ1). (B) Thermogravimetric analysis comparison of
polymers 1, 3 and 6.
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large scale, coil–coil spiral-like, self-assembly process. According to
this hypothesis, all of the fullerenes could be aligned along an
internal axle, thus to result buried by the PBLG hydrophobic helices,
+
while the charged NH3 groups may be exposed to water solution
(a schematic representation is shown in Fig. S7, ESI†) in accordance
with previously reported similar, but much smaller, structures.22
This hypothesis is in agreement with the pH-depending experi-
ments described above. Moreover, the same but shorter functiona-
lized PBLG-polymer series was also synthesized using a BLG–NCA/
initiator ratio of 25 : 1. After the self-assembly assay, the shorter 1
and 3 analogues did not provide any specific microstructure
propensities while the shorter 6 analogue afforded smaller spherical
structures (10–35 nm diameter) compared to the previously
described (Fig. S8, ESI†).
Calculus was used to support experimental observations and
in particular to investigate the atomistic structure of the
vesicles formed by polymer 6. To this purpose we employed a
bottom-up coarse-graining protocol with the intent to set up a
simulation able to independently recover the experimental
observations, i.e. we did not use the experimental results to
bias the parameterization of the model. To limit the computa-
tional weight of the whole procedure we used polymer 6
constituted by 25 BLG units as a model molecule. The coarse-
Fig. 4 (A and B) TEM (unstained) images of the resulting self-assembled
nanostructures obtained from polymer 6. (C and D) AFM and SEM images,
respectively, from the very same sample used for TEM analysis.
70 nm and a hole of 30 nm (Fig. 3D). Additionally, after running graining representation sees each of the BLG units and the two
the same self-assembly process but starting from a more terminal moieties as single beads. Differently from systems recently
diluted solution of 3 (2–5 mg in 10 ml), smaller but uniform reported in the literature,23 in which a balance between attractive
in size (60 nm diameter), toroidal nanostructures were and repulsive forces of the building blocks drives the assembly,
obtained as detected by AFM (Fig. S6, ESI†). Sphere-shaped here the driving force of the self-assembly is the low affinity of the
aggregates were generated from polymer 6 under the same hydrophobic PBLG molecules with water. Thus, in our simulations
conditions described above for polymer 3 (Fig. 4). TEM, we needed to include explicitly the medium to observe aggregation
unstained, images reveal the formation of deep-dark and round of polymer 6 molecules. We employed a coarse-grained 4-water
morphologies (Fig. 4A) with diameters ranging between 250 model for the solvent, in which one bead is used to represent a
and 700 nm. Moreover, these aggregates displayed an internal cluster of 4 water molecules.24,25 Details on the coarse graining
cavity as shown in the TEM image in Fig. 4B; thus, the observed procedure and the parameterization of the bead–bead energy
spherical morphologies can be attributed to hollow vesicle-like function are provided in the ESI.† We simulated a system contain-
microstructures. These results were additionally confirmed by ing 1600 polymer molecules and 1 600 608 water beads (see the
AFM (Fig. 4C) and SEM analyses (Fig. 4D). While polymers 3 ESI† for details). The starting configuration, in which the polymer
and 6 were able to self-assemble into ordered structures, molecules are randomly placed and oriented in space, is shown in
polymer 1, bearing a Trt group instead of fullerene, did not Fig. 5A (water was omitted for clarity).
give any specific nano-structuration under similar conditions.
The situation just after 5 ns of simulation time is shown in
To get polymer 3 from 1, the Trt protecting group was removed Fig. 5B. The simulation box results are populated by a small
under acidic conditions, thus in polymer 3 the N-terminus number of aggregates with different shapes. Most of them are
+
amine is protonated (NH3 ). Importantly, when NaOH 0.2 M spherical aggregates of dimensions ranging from 15 to 20 nm
was added to a solution containing the toroidal objects in diameter. However, a couple of assemblies are present which
obtained by the self-assembly of 3, immediate precipitation of show a bent layer of about 4 nm thickness (very close to the
the polymer was observed. On the other hand, the addition of length of one polymer molecule) that can be thought to be a
0.2 M HCl did not affect the self-assembled structures, since piece of a larger vesicle that could not be formed in this
donut-like structures could still be detected by TEM. This simulation due to an insufficient number of polymer mole-
information suggested that: (i) fullerene is necessary for the cules. A number of important observations can be drawn from
formation of ordered nanostructures and (ii) the NH3+ group is the simulations. The first and most important observation
mandatory for the formation of the toroid-shaped structures.
reflecting the experiments is that in water the polymer mole-
Since, at a fixed volume, a toroidal shape exposes a larger cules aggregate into ordered structures instead of forming an
surface with respect to the spherical shape, our hypothesis is amorphous precipitate. Second, all the aggregates are formed
that to generate self-assembled toroidal forms in water (starting by layers of limited size ranging from 4 to 6 nm, which is in the
from the hydrophobic polymer 1) the single polymer chain may order of the length of the polymers. Third, the layer is not
firstly undergo a tail-to-tail dimerization and successively a structured in a simple way as it happens in phospholipids:
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2 G. M. Whitesides and M. Boncheva, Proc. Natl. Acad. Sci. U. S. A.,
2002, 99, 4769.
3 G. M. Whitesides and B. Grzybowski, Science, 2002, 295, 2418.
4 O. Ikkala and G. ten Brinke, Science, 2002, 295, 2407.
5 J. A. A. W. Elemans, A. E. Rowan and R. J. M. Nolte, J. Mater. Chem.,
2003, 13, 2661.
¨
6 V. Percec, C.-H. Ahn, G. Ungar, D. J. P. Yeardley, M. Moller and
S. S. Sheiko, Nature, 1998, 391, 161.
7 P. J. M. Stals, Y. Li, J. Burdynska, R. Nicola¨y, A. Nese,
´
Fig. 5 The color scheme is: yellow represents BLG beads, red for the N-
terminal group, and blue for the C-terminal group. Beads radii are set to
0.43 nm for the BLG units and 0.53 nm for the terminal moieties. These
values have been determined from the average translational diffusion
tensor of moieties calculated using a hydrodynamic approach.26 Water
molecules have been omitted for clarity. (A) Initial random configuration of
the 1600 PBLG molecules constituted by 25 BLG amino acids. (B) Final
configuration of the system after 5 ns of simulation time. (C) Details of a
selected spherical aggregate with front and rear clipping planes applied to
highlight the hollow part of the spherical aggregate.
A. R. A. Palmans, E. W. Meijer, K. Matyjaszewski and S. S. Sheiko,
J. Am. Chem. Soc., 2013, 135, 11421.
8 Recent reviews: (a) G. Fuks, R. M. Talom and F. Gauffre, Chem. Soc.
Rev., 2011, 40, 2475; (b) S. F. M. van Dongen, H.-P. M. DeHoog,
R. J. R. W. Peters, M. Nallani, R. J. M. Nolte and J. C. M. van Hest,
Chem. Rev., 2009, 109, 6212; (c) E. Soussan, S. Cassel, M. Blanzat and
I. Rico-Lattes, Angew. Chem., 2009, 121, 280; E. Soussan, S. Cassel,
M. Blanzat and I. Rico-Lattes, Angew. Chem., Int. Ed., 2009, 48, 274.
9 Recent reviews: (a) Z. Luo and S. Zhang, Chem. Soc. Rev., 2012,
´
41, 4736; (b) C. Valery, F. Artzner and M. Paternostre, Soft Matter,
2011, 7, 9583; (c) X. Yan, P. Zhua and J. Li, Chem. Soc. Rev., 2010,
39, 1877; (d) I. W. Hamley, Angew. Chem., 2007, 119, 8274–8295;
I. W. Hamley, Angew. Chem., Int. Ed., 2007, 46, 8128–8295.
10 Recent reviews: (a) B. Tian, X. Tao, T. Ren, Y. Weng, X. Lin, Y. Zhang
and X. Tang, J. Mater. Chem., 2012, 22, 17404; (b) C. Cai, L. Wang
and J. Lin, Chem. Commun., 2011, 47, 11189; (c) A. Carlsen and
S. Lecommandoux, Curr. Opin. Colloid Interface Sci., 2009, 14, 329.
11 (a) N. Gour, D. Kedracki, I. Safir, K. X. Ngo and C. Vebert-Nardin,
Chem. Commun., 2012, 48, 5440; (b) H. Wang, C. Yang, L. Wang,
D. Kong, Y. Zhang and Z. Yang, Chem. Commun., 2011, 47, 4439;
(c) Y. Zhou, M. Kogiso and T. Shimizu, J. Am. Chem. Soc., 2009,
131, 2456; (d) S. Ghosh, M. Reches, E. Gazit and S. Verma, Angew.
Chem., 2007, 119, 2048; S. Ghosh, M. Reches, E. Gazit and S. Verma,
Angew. Chem., Int. Ed., 2007, 46, 2002.
couple of polymers form coiled-coil super structures, which in
turn create a network giving rise to the layer, with the C60
termini preferentially exposed to the solvent. We shall also
notice that, while being quite compact, the spherical structures
obtained in the simulation are hollow (Fig. 5C). The obtained
aggregates are in quite good accord with those observed
experimentally for the shorter 6 analogue.
In this work, we reported the one-pot synthesis of two novel
PBLG-polymers, C- and C/N-terminus capped with fullerenes, 12 R. B. Woodward and C. H. Schramm, J. Am. Chem. Soc., 1947,
69, 1551.
by means of thiol–ene chemistry. The self-assembly of such rod-
like systems in water led to the formation of bulky, toroid-like
13 J. Huang and A. Heise, Chem. Soc. Rev., 2013, 42, 7373.
14 (a) H. R. Kricheldorf, Angew. Chem., Int. Ed., 2006, 45, 5752;
or vesicle-like, nanostructures depending on the fullerene
position over the main chain of the polymer. Based on experi-
mental results, we suggested a mechanism for the formation of
(b) H. Lu, J. Wang, Z. Song, L. Yin, Y. Zhang, H. Tang, C. Tu,
Y. Lin and J. Cheng, Chem. Commun., 2014, 50, 139.
15 T. O. Yeates and J. E. Padilla, Curr. Opin. Struct. Biol., 2002, 12, 464.
16 M. Zelzer and R. V. Ulijn, Chem. Soc. Rev., 2010, 39, 3351.
the toroidal nanostructures and we explored vesicle-like systems 17 M. Lee, B.-K. Cho and W.-C. Zin, Chem. Rev., 2001, 101, 3869.
18 P. Doty, J. H. Bradbury and A. M. Holtzer, J. Am. Chem. Soc., 1956,
by means of computational modelling. In conclusion, such
polymer–fullerene conjugates may be interesting systems to
78, 947.
19 B. Iskin, G. Yilmaz and Y. Yagci, Chem. – Eur. J., 2012, 18, 10254.
prepare organized, at a nanoscale level, fullerene-based devices. 20 C. E. Hoyle and C. N. Bowman, Angew. Chem., Int. Ed., 2010,
49, 1540.
Financial support from the University of Padova (PRAT A. M.
CPDA114475 and M. M. CPDA119117) and MIUR, Ministero
21 (a) D. M. Guldi, F. Zerbetto, V. Georgakilas and M. Prato, Acc. Chem.
Res., 2005, 38, 38; (b) V. Georgakilas, F. Pellarini, M. Prato,
`
dell’Istruzione, Universita e Ricerca, (FIRB2012) is gratefully
D. M. Guldi, M. Melle-Franco and F. Zerbetto, Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 5075.
22 Y. Kim, W. Li, S. Shin and M. Lee, Acc. Chem. Res., 2013, 46, 2888.
23 C. Cai, Y. Li, J. Lin, L. Wang, S. Lin, X.-S. Wang and T. Jiang,
Angew. Chem., Int. Ed., 2013, 52, 7732.
acknowledged. A.M. thanks Dr F. Caicci for assistance with
TEM data recording.
24 S. J. Marrink and A. E. Mark, J. Am. Chem. Soc., 2003, 125, 15233.
25 J. Marrink, H. J. Risselada, S. Yefimov, D. P. Tieleman and A. H. de
Vries, J. Phys. Chem. B, 2007, 111, 7812.
Notes and references
1 A. R. Hirst, B. Escuder, J. F. Miravet and D. K. Smith, Angew. Chem., 26 V. Barone, M. Zerbetto and A. Polimeno, J. Comput. Chem., 2009,
Int. Ed., 2008, 47, 8002.
30, 2.
4574 | Chem. Commun., 2014, 50, 4571--4574
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