Cerium oxide nanoparticle-mediated self-assembly of hybrid supramolecular hydrogels

Article abstract of DOI:10.1039/c2cc33351a

Hybrid supramolecular hydrogels are prepared by non-enzymatic dephosphorylation of N-fluorenylmethyloxycarbonyl tyrosine-(O)-phosphate (FMOC-Tyr-P) using catalytic cerium oxide nanoparticles. The organic-inorganic hydrogels exhibit enhanced viscoelastic p

Full text of DOI:10.1039/c2cc33351a

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Cerium oxide nanoparticle-mediated self-assembly of hybrid
supramolecular hydrogelsw
Avinash J. Patil,* Ravinash Krishna Kumar, Nicholas J. Barron and Stephen Mann*
Received 9th May 2012, Accepted 14th June 2012
DOI: 10.1039/c2cc33351a
Hybrid supramolecular hydrogels are prepared by non-
enzymatic dephosphorylation of N-fluorenylmethyloxycarbonyl
tyrosine-(O)-phosphate (FMOC-Tyr-P) using catalytic cerium
oxide nanoparticles. The organic–inorganic hydrogels exhibit
enhanced viscoelastic properties compared with analogous
materials prepared using alkaline phosphatase.
long-term stability under processing conditions, and difficul-
ties in recovery and/or recycling. On the other hand, metal and
metal oxide nanoparticles display size-dependant catalytic prop-
erties, chemical robustness and extended stability, suggesting that
under certain conditions inorganic nanoparticles could be used to
mimic aspects of biological functions related to specific enzymes.
For instance, there are a growing number of examples in which
Fe3O4, Co3O4, FeS, Au, Au@Pt, CoFe2O4 and V2O5 nano-
particles have been shown to exhibit peroxidase-like activity.8
Specifically, cerium oxide nanoparticles and various lanthanide
ions and/or their complexes have been employed to mimic
alkaline phosphatase functions involved in the dephosphoryla-
tion of small organic molecules, nucleotides and peptides.9
However, application of metal oxide nanoparticles to trigger
the self-assembly of small molecules into supramolecular hydro-
gels remains unexplored. Herein, we demonstrate for the first
time to our knowledge a facile method in which the intrinsic
alkaline phosphatase-like activity of cerium oxide nanoparticles
is exploited for the dephosphorylation of N- fluorenylmethyl-
oxycarbonyl tyrosine-(O)-phosphate (FMOC-Tyr-P) (Scheme 1
ESIw). As a consequence, the hydrolysis reaction converts a
charged amino acid derivative into a hydrogelator species
(FMOC-Tyr) of lower charge, which subsequently self-associates
to form a viscoelastic hydrogel comprising supramolecular
nanofilaments and entrapped cerium oxide nanoparticles.
Spontaneous molecular self-assembly into higher-ordered
structures has been central to the design of novel organic
and hybrid (organic/inorganic) nanostructured materials.1 In
particular, the design of bioactive building blocks, such as
peptides with appropriate intermolecular recognition sites, has
been exploited for the non-covalent self-assembly of 1-D bio-
functionalized nanostructures.2,3 Moreover, amphiphilicity of
the building blocks can be tailored to facilitate entanglement
of the 1-D nanostructures along with solvent entrapment to
produce a wide range of viscoelastic hydrogels.4,5 Although
covalent cross-linking has been used to produce stabilized
hydrogels, this method requires judicious control over the
coupling reactions,6 and alternative approaches based on
supramolecular assembly have been explored.4,5 Recent
studies have shown that the concept of enzyme-directed
assembly of small molecules can be extended to produce
supramolecular hydrogels, for example based on the dephos-
phorylation of derivatized amino acids or peptides with
fluorenyl- or naphthalene-functionalized end groups.5 In many
cases, the enzyme-mediated reaction results in an increase in
the hydrophobicity of the gelator molecules, with the con-
sequence that non-covalent intermolecular interactions such as
hydrogen bonding and p stacking give rise to the reversible
self-assembly of 3-D networks of entangled supramolecular
nanofilaments. In a recent study, this approach was coupled
with calcium phosphate mineralization to produce supra-
molecular hydrogels with integrated hybrid components.7
Although enzymes demonstrate excellent substrate specifi-
city, their potential as agents in materials science is often
hindered by their structural and functional fragility, lack of
Nanoparticle-containing supramolecular hydrogels were pre-
pared by adding 100 mL of aqueous FMOC-Tyr-P in alkaline
buffer (50 mM Tris-HCl, 50 mM Na2CO3, 1 mM MgCl2,
pH 10.2) to 100 mL of an aqueous dispersion of well-crystalline
cerium oxide nanoparticles (0.3 g mLꢀ1, pH 8.5, fluorite, Rhodia,
Fig. S1, ESIw), and leaving the mixture (with final substrate
concentrations of 50, 25, 12.5 or 8.3 mM) to stand at room
temperature in the dark for 24 h. The resulting yellow-coloured
hydrogels (pH 7.8) were semi-transparent and showed no
gravity-induced flow upon inversion of the sample vial
(Fig. 1a). Transmission electron microscopy (TEM) images of
unstained samples of the hydrogels (Fig. 1b and c, and Fig. S2,
ESIw) and tapping mode atomic force microscopy (AFM) studies
(Fig. S3, ESIw) showed a continuum of cerium oxide nano-
particles that were penetrated by a semi-rigid and entangled
matrix of flat ribbon-like helical nanofilaments, which were
several micrometers long and 10–20 nm in width. High resolution
TEM and energy dispersive elemental analysis further confirmed
that the cerium nanoparticles were present along the seam of the
Centre for Organized Matter Chemistry, School of Chemistry,
University of Bristol, Bristol, BS8 1TS, UK.
E-mail: avinash.patil@bristol.ac.uk, s.mann@bristol.ac.uk;
Fax: 0044-117-9290509; Tel: 0044-117-3317215
w Electronic supplementary information (ESI): Structural data for cerium
oxide nanoparticles, TEM, AFM images of hydrogel nanofilaments, and
NMR spectroscopy data are provided. DOI: 10.1039/c2cc33351a
c
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Fig. 1 (a) Photograph showing self-supported supramolecular
hydrogel produced by cerium oxide nanoparticle-mediated dephos-
phorylation of FMOC-Tyr-P (50 mM). (b,c) Unstained TEM micro-
graphs of corresponding FMOC-Tyr hydrogel network of semi-rigid
nanofilaments (b), and high magnification image showing ribbon-like
helical morphology (arrows) of individual nanofilaments (c). Scale
bars; (b) 500 nm, and (c) 100 nm.
Fig. 2 (a) UV-vis spectra of aqueous FMOC-Tyr-P (i), and corres-
ponding FMOC-Tyr hydrogel (ii) produced in the presence of cerium
oxide nanoparticles, (Note: The broad absorption spectrum of the
hydrogel masks the peak at 290 nm corresponding to cerium oxide
nanoparticles) (Fig. S8, ESIw) (ii). (b) DSC thermogram of hydrogel
prepared from 50 mM FMOC-Tyr-P showing broad gel to sol transition
centred at 80 1C. (c) Oscillatory amplitude sweeps at a constant frequency
of 1 Hz for hydrogel prepared at 50 mM FMOC-Tyr-P showing linear
viscoelastic region for the storage (G0, ’) and loss (G0 0, K) moduli (d)
Viscosity profiles of cerium oxide nanoparticle-catalysed hydrogels
prepared at 50 mM (’) or 25 mM (K) FMOC-Tyr-P concentrations.
nanofilaments (Fig. S4, ESIw). Control solutions involving mixtures
of buffer and aqueous dispersions of cerium oxide nanoparticles
remained fluid, confirming that self-assembly of the small-molecule
amino acid derivative was responsible for hydrogelation.
Cerium oxide-mediated dephosphorylation of FMOC-Tyr-P
was confirmed using 13C and 31P NMR spectroscopy. The
31P NMR spectrum of FMOC-Tyr-P gave a resonance at
ꢀ11.64 ppm corresponding to the phosphate ester group that
was absent in the hydrogel samples, which showed a new peak
at 0.17 ppm assigned to inorganic phosphate (Fig. S5, ESIw).
Similarly, the resonance at 142.4 ppm associated with
C–O–P(O)(OH)2 in the 13C NMR spectrum of FMOC-Tyr-P
disappeared and was replaced in the hydrogel spectra by a
peak corresponding to C–OH at 152.1 ppm (Fig. S6,S7, ESIw).
These observations indicated that cerium oxide was an
effective catalyst for phosphate ester bond hydrolysis of
FMOC-Tyr-P, and that supramolecular self-assembly of
dephosphorylated FMOC-Tyr molecules occurred readily in
the presence of the inorganic nanoparticles.
aged for 1 day showed typical supramolecular hydrogel behav-
iour within the linear viscoelastic region with almost parallel
storage (elastic) G0 and loss (viscous) G0 0 moduli up to a shear
stress of 570 Pa (Fig. 2c). The two cross-over points observed in
the oscillatory amplitude sweep could be attributed to structural
deformation resulting in a severely dehydrated nanofilamentous
network. The three-fold increase seen in the elastic modulus
compared to the viscous modulus was consistent with a persis-
tent semi-rigid gel network, and indicative of solid-like visco-
elastic behaviour. Cerium oxide-containing hydrogels prepared
at lower FMOC-Tyr-P concentrations (25 mM) showed similar
solid-like viscoelastic properties but exhibited structural
deformation at a much lower shear stress value of 63 Pa
(Fig. S9, ESIw). These observations were consistent with the
viscometry measurements, which showed an increase in the
viscosity values with increasing FMOC-Tyr-P concentration
(Fig. 2d). Typically, at very low shear rates, the viscosities were
252 and 60 Pa.s for nanoparticle-containing hydrogels prepared
from 50 and 25 mM FMOC-Tyr-P, respectively.
The structural and physical properties of the as-prepared
FMOC-Tyr/nanoparticle hydrogels were investigated by
several methods. UV-vis spectra of the hydrogels showed three
characteristic absorption peaks corresponding to p–p*, n-p*
and n-p* transitions, at 265, 288 and 299 nm, respectively.
Significantly, the hydrogel samples exhibited characteristic
hypochromicity, as evidenced by changes in the absorption
intensity ratios (A265/A299) from a value of 3.5 : 1 for aqueous
FMOC-Tyr-P to 3.2 : 1 for the hydrogels (Fig. 2a). This was
consistent with the presence of stacking interactions between
fluorenyl moieties associated with fibril formation.5d DSC
thermograms of the nanoparticle-containing hydrogels
showed a broad endothermic peak in the region of 75–80 1C,
corresponding to a reversible gel–sol melting transition.
Oscillatory amplitude sweeps at a constant frequency of 1 Hz
of hydrogels prepared using 50 mM FMOC-Tyr-P solutions and
Cross-polarized optical microscopy images of cerium oxide-
containing hydrogels prepared at a FMOC-Tyr-P concen-
tration of 50 mM revealed 100–150 mm-sized domains displaying
Maltese cross patterns (Fig. 3a), whereas at 25 mM anisotropic
domains with nematic birefringence (Fig. 3b) were observed,
both of which were dispersed in an isotropic phase. The
Maltese cross patterns exhibited a strong blue fluorescence
in the presence of the dye Hoechst 3325 (Fig. S10a, ESIw),
indicative of spherulites comprising radially ordered nanofilaments
with a well-defined, p-stacked FMOC-Tyr superstructure.10,11
This was consistent with environmental scanning electron
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(75–80 1C) approximately 40 1C higher than that observed for
enzyme-mediated FMOC-Tyr hydrogels.5d Entrapment of the
cerium oxide nanoparticles within the gel network could also
contribute towards the increased stiffness and viscosity.
Finally, the use of cerium oxide and other inorganic
nanoparticles9 in the catalytic dephosphorylation of bio-
logically active amino acids and peptides could have general
relevance in developing supramolecular hydrogels with hybrid
structures and functions. In this regard, we note that several
recent studies have demonstrated promising biomedical appli-
cations based on cerium oxide nanoparticles,12 suggesting that
embedding inorganic nano-catalysts of this type within small-
biomolecule hydrogels could represent an important step
towards novel forms of soft, bioactive materials.
Fig. 3 Cross-polarized optical microscopy images of hydrogels
prepared by cerium oxide nanoparticle-mediated dephosphorylation
of FMOC-Tyr-P at concentrations of 50 mM (a) and 25 mM (b),
showing the presence of Maltese cross patterns (spherulites) or nematic
birefringence, respectively. Scale bar = 100 mm.
The authors thank the University of Bristol, UK for
financial support, Dr Andrew Collins, Jonathan A. Jones,
Judith Mantell (Wolfson Bioimaging Facility, University of
Bristol), Prof. Tony Miles and Dr Phil Pollintine (University
of Bath) for assistance with AFM, HRTEM, ESEM and
rheology measurements respectively.
microscopy (ESEM) micrographs, which indicated that the
Maltese cross structures corresponded to discrete spheroidal
100–120 mm-sized particle-like domains (Fig. S10b, ESIw).
Although the differences in texture were attributed primarily to
changes in FMOC-Tyr-P concentration, the possibility that nuclea-
tion of the FMOC-Tyr nanofilaments occurred specifically on the
surface of the cerium oxide nanoparticles could not be ruled out.
In summary, we have shown that cerium oxide nanoparticles
are capable of alkaline phosphatase-like activity, with the
consequence that viscoelastic, amino acid-based supramole-
cular hydrogels comprising an entangled network of helical
ribbon-like nanofilaments and inorganic nanoparticles can be
produced by abiogenic catalysis-mediated self-assembly. The
nanoparticle-containing FMOC-Tyr hydrogels were responsive
to external stimuli such as temperature or mechanical forces,
with the consequence that they could be reversibly assembled
and disassembled by melting or shear thinning of the supra-
molecular structure. The catalytic dephosphorylation behaviour
of the cerium oxide nanoparticles was attributed to the Lewis
acidity associated with Ce3+/Ce4+ surface sites that are capable
of not only coordinating phosphoryl oxygens but also reducing
the activation energy of P–O bond scission by facilitating
nucleophilic attack from neighbouring surface-bound hydroxyl
groups.9 Although mechanistically different, the results indicate
that cerium oxide nanoparticles can be used as plausible mimics
of enzymatic transformations involving peptide dephosphoryla-
tion and coupled supramolecular self-assembly.6 However, we
noted several differences between the physical properties of
the nanoparticle-containing hydrogels and their FMOC-Tyr
counterparts prepared by enzyme-mediated self-assembly under
comparable conditions. For example, use of the cerium oxide
nanoparticle catalyst (0.3 g mLꢀ1) produced hydrogels from
FMOC-Tyr-P concentrations as low as 8.3 mM (Fig. S2, ESIw),
while a limiting amino acid concentration of ca. 50 mM was
required in the presence of alkaline phosphatase. Moreover,
alkaline phosphatase-mediated hydrogels were softer, less viscous,
and displayed a considerable degree of structural deformation
at much lower shear stress values (10 Pa),5d compared with
hydrogels prepared in the presence of cerium oxide nano-
particles. The increase in mechanical strength was attributed
primarily to the nanoparticle-mediated self-assembly of a semi-
rigid matrix of supramolecular filaments, which become
entangled to produce a robust hydrogel comprising spherulitic/
nematic domains, and exhibiting a gel–sol transition temperature
Notes and references
1 (a) S. Mann, Nat. Mater., 2009, 8, 781; (b) S. Stupp, Chem. Rev.,
2005, 105, 1023.
2 R. V. Ulijn and D. N. Woolfson, Chem. Soc. Rev., 2010, 39, 3349.
3 (a) J. B. Matson and S. I. Stupp, Chem. Commun., 2012, 48, 26;
(b) M. Zezler and R. V. Ulijn, Chem. Soc. Rev., 2010, 39, 3351.
4 (a) R. V. Ulijn, J. Mater. Chem., 2006, 16, 2217; (b) Z. Yang, G. Liang
and B. Xu, Acc. Chem. Res., 2008, 41, 315; (c) M. E. Hahn and
N. C. Gianneschi, Chem. Commun., 2011, 47, 11814; (d) D. J. Adams,
Macromol. Biosci., 2011, 11, 160; (e) J. Dash, A. J. Patil, R. N. Das,
F. L. Dowdall and S. Mann, Soft Matter, 2011, 7, 8120.
5 (a) Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam and B. Xu, Adv.
Mater., 2004, 16, 1440; (b) S. Toledano, R. J. Williams,
V. Jayawarna and R. V. Uljin, J. Am. Chem. Soc., 2006,
128, 1070; (c) A. K. Das, R. Collins and R. V. Ulijn, Small,
2008, 4, 279; (d) R. Krishna Kumar, X. Yu, A. J. Patil, M. Li and
S. Mann, Angew. Chem., Int. Ed., 2011, 50, 9343.
6 (a) T. Vermonden, R. Censi and W. E. Hennink, Chem. Rev., 2012,
DOI: 10.1021/cr200157d; (b) K. Y. Lee and D. J. Mooney, Chem.
Rev., 2001, 101, 1869.
7 Z. A. C. Schnepp, R. Gonzalez-McQuire and S. Mann, Adv.
Mater., 2006, 18, 1869.
8 (a) L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang,
J. Feng, D. Yang, S. Perrett and X. Yan, Nat. Nanotechnol., 2007,
2, 577; (b) Z. Dai, S. Liu, J. Bao and H. Ju, Chem.–Eur. J., 2009,
15, 4321; (c) W. He, Y. Liu, J. Yuan, J. J. Yin, X. Wu, X. Hu,
K. Zhang, J. Liu, C. Chen, Y. Ji and Y. Guo, Biomaterials, 2011,
32, 1139; (d) Y. Jv, B. Li and R. Cao, Chem. Commun., 2010, 46, 8017;
(e) R. Andre, F. Natalio, M. Humanes, J. Leppin, K. Heinze,
R. Wever, H. C. Schroder, W. E. G. Muller and W. Tremel, Adv.
Funct. Mater., 2010, 21, 501; (f) W. Shi, X. Zhang, S. He and
Y. Huang, Chem. Commun., 2011, 47, 10785; (g) J. Mu, Y. Wang,
M. Zhao and L. Zhang, Chem. Commun., 2012, 48, 2540.
9 (a) F. Tan, Y. Zhang, J. Wang, J. Wei, Y. Cai and X. Qian, J. Mass
Spectrom., 2008, 43, 628; (b) M. H. Kuchma, C. B. Komanski,
J. Colon, A. Teblum, A. E. Masunov, B. Alvarado, S. Babu,
S. Seal, J. Summy and C. H. Baker, Nanomed.: Nanotechnol., Biol.
Med., 2010, 6, 738; (c) G. Cheng, J.-L. Zhang, Y.-L. Liu,
D.-H. Sun and J.-Z. Ni, Chem. Commun., 2011, 47, 5732;
(d) N. W. Luedtke and A. Schpartz, Chem. Commun., 2005, 5426.
10 A. Aggeli, M. Bell, L. M. Carric, C. W. G. Fishwick, R. Harding,
P. J. Mawer, S. E. Radford, A. E. Strong and N. Boden, J. Am.
Chem. Soc., 2003, 125, 9619.
11 M. R. H. Krebs, C. E. MacPhee, A. F. Miller, I. E. Dunlop,
C. M. Dobson and A. M. Donald, Proc. Natl. Acad. Sci. U. S. A.,
2004, 101, 14420.
12 I. Cleardo, J. Z. Pedersen, E. Traversa and L. Ghibelli, Nanoscale,
2011, 3, 1411.
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