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Clay Gels For the Delivery of Regenerative
Microenvironments
Jonathan I. Dawson, Janos M. Kanczler, Xuebin B. Yang, George S. Attard,
and Richard O.C. Oreffo*
Hydrogels offer excellent biocompatibility and permeability for
water soluble metabolites with the potential for self-organization
in situ to allow minimally invasive delivery of stem-cells and/or
growth factors. However, the essential hydrophilicity of hydro-
gels presents challenges for the retention, in space and time, of
bioactive molecules encapsulated within the gel network. Cer-
tain clays are known for their ability to adsorb biological mole-
cules due to the large and highly charged specific surface area
of the nano/micro-sized particles. Here we show the potential
to harness the sorptive capacity of clay in regenerative medi-
cine. Critically, we demonstrate the ability of self-assembling
clay gels to rapidly adsorb VEGF165 to promote an angiogenic
response in local endothelial populations in vitro and in vivo.
Such an approach provides significant potential for the gen-
eration of developmentally potent microenvironments able to
direct the growth and differentiation of stem/progenitor cell
populations for tissue regeneration.
Regenerative strategies seek to harness the developmental
potential of stem cells to replace tissue lost or damaged through
injury and disease. Critical to this objective, 3D matrices serve
to deliver and maintain at the site of damage the requisite extra-
cellular niche for stem cell-mediated tissue regeneration.[1]
Important to this function is the ability to retain, in space and
time, requisite bioactive molecules involved in the growth and
differentiation of stem and progenitor populations. While the
high water content (>90%) of hydrogels facilitates diffusion of
nutrients and contributes to the biocompatibility of the mate-
rial,[2,3] the open polymer networks that characterize most
hydrogels typically result in the rapid release of incorporated
Dr. J. I. Dawson, Dr. J. M. Kanczler, Prof. R. O. C. Oreffo
Bone and Joint Research
Centre for Human Development
Stem Cells and Regeneration
Institute of Developmental Sciences
University of Southampton
Southampton, SO16 6YD, UK
E-mail: Richard.Oreffo@soton.ac.uk
Dr. X. B. Yang
Division of Oral Biology
University of Leeds
Leeds, LS2 9LU, UK
Prof. G. S. Attard
School of Chemistry
University of Southampton
Southampton, SO17 1BJ, UK
DOI: 10.1002/adma.201100968
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proteins necessitating elaborate modifications for extended pro-
tein retention.[4–6]
Clays have been widely used in the pharmaceutical industry
both as excipients and active substances.[7] Early studies
observed decreased systemic bioavailability of drugs co-
administered with clays, prompting investigation into their
utility as drug-release modifiers.[8] Clay-protein interaction, via
cation exchange, hydrophobic and interlamellar mechanisms,
have been harnessed in drug delivery formulations to delay
(extended release applications) or localize (site-specific appli-
cations) therapeutic molecules. Typically, clay particles are dis-
persed in drug solutions, recovered as a solid phase upon equi-
libration and dried to allow delivery of the drug-clay complex
in tablet form.[8] Alternative approaches utilizing clay particle
aggregations in slurries to further enhance sorptive potential,[9]
or polymer-clay nanocomposites[10] have also been developed.
In the current study we describe a clay gel based strategy for
the delivery and application of cells, proteins and niche devel-
opment in regenerative medicine.
Laponite, a synthetic smectite,[11] was chosen for its ability to
generate high purity nano-particle dispersions of large surface
area (>350 m2 g−1). Laponite consists of disk-shaped particles of
approximately 25 nm diameter and 1 nm thickness, possessing
a negative face charge and a weak positive rim charge. These
particles form delaminated dispersions in water which self-
organize via face-edge aggregation forming an open, macro-
porous and reversible (thixotropic) gel network.[12,13] The thix-
otropy of Laponite suspensions was observed in the form of a
hysteretic viscous response to a cyclic regime of shear rates via a
rate-controlled rotational viscometer. Following extended expo-
sure to high shear (5 min, 190 l s−1) Laponite gels recovered
to a pre-shear state after 80 min at 21 °C and 20 min at 37 °C
(Figure 1a). Such thixotropic properties allowed the release of
Laponite through a 21G needle and, depending on the solids
content and resting time post-shear (Supporting Information,
Figure 1), re-establishment of a gel network sufficiently cohe-
sive to bridge gaps of 1cm (Figure 1b).
Previous studies recorded faster network formation with
increasing electrolyte concentration to a critical point where-
upon flocculation occurs.[14,15] We therefore investigated the
possibility of generating gel capsules by adding pre-dispersed
Laponite into electrolyte solutions. Drop-wise addition of
Laponite resulted in morphologies progressing from amor-
phous residues at low ionic strength to discrete spherical cap-
sules as physiological ionic strength (equivalent to 100–150 mM
NaCl) was reached, reflecting a decrease in surface area with
increased rate of gelation at higher electrolyte concentrations
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COMMUNICATION
a
21 ° C 37 °C
C d e

Thixotropy
Thixotropy

200 200
Viscosity (Dynes /cm2)

Time (min) Time (min)

150 150

100 100

Pre-shear
50 5 mins 50 Pre-shear
20 mins 5 mins
40 mins 10 mins
80 mins 20 mins
0
0.01 1 100 0.01 1 100 f g
Shear Rate (1/s) Shear Rate (1/s)

b c

h i

Figure 1. Self-organization of Laponite suspensions via thixotropy and in response to an ionic medium. (a) The recovery of a thixotropic response
by Laponite suspensions. Thixotropy was defined by a hysteretic shear-stress response to a cyclic program of changing shear-rate over time which
was measured before (dotted line), and at intervals after (blue lines), exposure to shear. Inset shows thixotropy with time post-shear. (b) Laponite
thixotropy allows discharge through a hypodermic needle and the subsequent re-establishment of a gel network sufficiently cohesive to bridge gaps
between bone fragments. Scale bar = 1 cm. (c) Laponite capsule formation in response to drop-wise addition to media of varying ionic strength (NaCl
concentration). (d) HBMSCs (pre-labeled with 5-chloromethylfluorescein diacetate) suspended in low viscosity Laponite formulations encapsulated
by drop-wise, addition to cell culture media. (e) Single cell encapsulation in Laponite microcapsules. (f) Ring forms generated from a surface impact
retarded by rapid gelation. (g) String forms generated from low velocity addition through an immersed hypodermic needle. Laponite micro-capsules
could be recovered from suspension (h) and sub-encapsulated within a larger host capsule (i). Scale bars = 250 μm.

(Figure 1c). Cells suspended in low viscosity Laponite formu- indicating the importance of cell matrix interaction for chon-
lations could therefore be encapsulated by direct addition to drogenesis,[16] co-encapsulation with the adhesion molecule,
cell culture media (Figure 1d). Atomized droplets of Laponite fibronectin enhanced both matrix synthesis and the number
suspensions generated microcapsules with a mean diameter of Sox-9 expressing cells (Supporting Figure 3). These studies
of 170 μm (+/− 54.9 s.d.; Supporting Figure 2) allowing encap- indicate the cytocompatibility of Laponite encapsulation for cell
sulation of single cells (Figure 1e). Further morphologies were delivery and culture together with the potential for facile incor-
generated by altering the velocity at which Laponite suspen- poration of extra-cellular matrix molecules able to modulate
sions were introduced to an ionic medium. At higher velocity, the behavior of encapsulated populations (see also Supporting
ring-forms were generated due to a surface impact response Figure 4). Interestingly, the osteogenic response of encapsulated
retarded by rapid gelation (Figure 1f). Low velocity (0.01– cells was modest with limited matrix synthesis and staining for
0.1 mL min−1) addition via an immersed needle resulted in the collagens after 28 days (Supporting Figure 3).
generation of long string morphologies (Figure 1g). In addition, Following demonstration of the potential to encapsulate skel-
discrete Laponite microcapsules were recovered via centrifuga- etal populations without loss of viability, the ability of Laponite
tion (Figure 1h) enabling sub-encapsulation of microcapsules gels for growth factor delivery and protein retention was exam-
within a larger host capsule (Figure 1i). ined using two model proteins, albumin (bovine serum; Mw
The viability and function of encapsulated cells was assessed 66 400 Da; PI, 4.7) and lysozyme (egg white; Mw 14 400 Da;
by encapsulating human bone marrow stromal cells (HBMSCs, PI, 11) in comparison with alginate hydrogel capsules. Pro-
passage 1) and subsequent culture for osteogenic and chondro- tein solutions were mixed with Laponite and capsules gener-
genic differentiation. Histological examination after 28 days, ated via drop-wise addition to 1× phosphate-buffered saline
revealed extensive staining for proteoglycan and type II col- (PBS) solution. Release profiles of both proteins from alginate
lagen indicating the potential of Laponite capsules to host the capsules followed exponential-order kinetics, with lysozyme
chondrogenic differentiation of HBMSCs in a chondrogenic showing faster initial diffusion consistent with the protein’s
medium (Supporting Figure 3). Consistent with studies smaller size, but with sub-equilibrium slowing of diffusion

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a Albumin Lysozyme d Albumin Lysozyme

1.6 1.6
Protein in Media (mg/ml)

Protein Release
1.2 r2=0.95 1.2
r2=0.95
0.8 Control 0.8 Control Adsorbed Adsorbed
800 800

Protein Concentration

Protein Concentration
Alginate Alginate Protein Protein
0.4 Laponite 0.4 Laponite Ambient Ambient
600 600

(mg /ml)

(mg /ml)
Protein Protein

0 20 40 60 0 20 40 60 400 400
b 1.6 1.6
Protein in Media (mg/ml)

200 200

1.2 1.2
0 mm 1 0 mm 1
0.8 Control 0.8 Control
Alginate Alginate
Laponite 0.4 Laponite
e
0.4
r2=0.97

Protein Uptake
r2=0.98
0 20 40 60 0 20 40 60
Time (hrs)
c 2.5 2.5
Protein Adsorbed (mg/ml)

Alginate Alginate
Laponite Laponite
2 2

1.5 1.5 f

1 1

0.5 0.5

0 0.5 1 1.5 2 0 0.5 1 1.5 2

Final Protein Concentration (mg/ml)

Figure 2. Protein localization by Laponite capsules. (a) Retention of two model proteins by Laponite. Protein solutions were mixed with Laponite or
alginate sols prior to gelation and protein release measured as a function of protein concentration in media over time. (b-c) Uptake of two model
proteins by Laponite capsules. Unloaded capsules were added drop-wise into solutions of protein and uptake measured as a function of protein con-
centration in media over time (b) and input protein concentration (c). Error bars = S.D. (d) Distribution of model proteins within Laponite capsules
after 2 h incubation in solutions of FITC-labeled protein followed by three washes over 18 h. Concentrations around the periphery of the capsules were
observed to be 4× that of the initial ambient concentration of the media. (e) Distribution of varying concentrations of FITC-labeled albumin premixed
with Laponite prior to encapsulation. (f) Dual sub-encapsulation of microcapsules pre-incubated in the presence of DAPI-labeled DNA or FITC-labeled
albumin. Scale bars = 500 μm.

suggesting a degree of interaction between positively charged at a fixed z-plane. In all capsules added to FITC-labeled pro-
protein and negatively charged alginate network. In contrast, tein, fluorescence was observed as a thin band of approximately
negligible release of either protein was observed from Laponite 50 μm radius at the periphery of the capsules. Protein concen-
capsules over 72 h (Figure 2a). In addition, unloaded Laponite tration at the periphery was approximately four times that of
and alginate capsules were added to protein-PBS solutions and the ambient concentration (Figure 2d). Pre-mixed FITC-labeled
the protein concentration measured over time. In the presence albumin displayed a homogeneous distribution at concentra-
of alginate, albumin remained at the starting concentration tions below 50 μg mL−1, but at higher concentrations regions of
throughout and the lysozyme concentration dropped to a level high fluorescent intensity were observed (Figure 2e).
consistent with the degree of binding implied by the release In addition to bio-molecule localization via the two modes
experiments. Surprisingly, upon addition of Laponite, an described above, further biological conformations of nested
exponential decrease in the concentration of each protein was capsules were achieved via the sub-encapsulation of micro-
observed and by 72 h negligible amounts of either protein were capsules within a host capsule (see Figure 1h–i). As proof of
detected (Figure 2b). Increasing protein concentration revealed concept, unloaded Laponite was sprayed into separate solu-
continued protein removal even at concentrations double the tions of albumin, labeled with FITC, and DNA, labeled with 4’,
solid concentration (1.2 mg mL−1) of Laponite (Figure 2c). This 6-diamidino-2-phenylindole (DAPI). Dual sub-encapsulation of
high sorptive potential of Laponite capsules is consistent with microcapsules pre-incubated in the presence of DAPI-labeled
previous observations that flocculation of clay particles in saline DNA or FITC-labeled albumin were observed (Figure 2f) fur-
solutions enhanced adsorption of active molecules.[9] ther indicating the versatility of the china clay for protein and
Following observation of significant protein uptake, the cell encapsulation.
distribution of protein within Laponite capsules was assessed Critical to many tissue regeneration strategies as well as
using albumin and lysozyme labeled with fluorescein isothiocy- the treatment of ischemic diseases is the induction of local
anate (FITC). After two hours in the presence of FITC-labeled neo-angiogenesis.[17,18] Sustained delivery of angiogenic
protein, capsules were washed three times in PBS over 18 h. growth factors is a key strategy for inducing angiogenesis
Capsules were imaged and compared via confocal microscopy via stimulation of the invasion and organization of local

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a 1.2 c
Mean branch-points per cell
** b 200

Residual VEGF
1
**

(ng/ ml)
0.8 100
**
0.6 0 ***
*** Control Collagen Collagen
0.4
alone + laponite

0.2

0
VEGF VEGF Unbound Bound d e
positive negative VEGF VEGF

Phase contrast Cell identification Skeletonised network

positive
VEGF

Vessel number (per mm)

3 **

Vessel volume (%)

0.3 *
2
negative

0.2
VEGF

1 0.1

0 0
Collagen Collagen Collagen Collagen Collagen Collagen Collagen Collagen
alone + laponite + factors + laponite alone + laponite + factors + laponite
Unbound
VEGF

+ factors + factors

Collagen Collagen Collagen Collagen

alone + laponite + factors + laponite
+ factors
Bound
VEGF

Figure 3. Angiogenesis via Laponite localization of VEGF. (a) In vitro tubule-formation by HUVECs in response to Laponite-bound VEGF. Image
analysis demonstrated equivalent network organization by HUVECs cultured on Laponite pre-incubated with VEGF for 2 h before washing, as HUVECs
cultured in the presence of VEGF throughout. Scale bars = 200 μm. (b-f) In vivo angiogenesis in response to Laponite pre-incubated with VEGF.
(b) Collagen scaffolds impregnated with Laponite were exposed to media containing VEGF for 2 h followed by 3 washes over 18 h. (c) Following 2 h
incubation with Laponite, negligible VEGF remained in the media. (d) Constructs were implanted in a murine critical sized femoral defect (5 mm) for
28 days (X-ray at day 1). (e) The murine vascular network was perfused with microfil to enhance μCT analysis. (f) μCT analysis revealed greater vessel
volume and number only in collagen scaffolds perfused with Laponite and pre-incubated with VEGF. Area = 14 mm3. Error bars = S.D.

endothelial progenitors.[18,5] Following our ability to gen-
erate a variety of clay structures and spatial encapsulation
of biomolecules we set out to demonstrate the efficacy of
this material in a regenerative setting. To test the bioactivity
of a Laponite-bound protein, the effect of Laponite-bound
vascular endothelial growth factor-165 (VEGF) on the in
vitro tubule-formation of human umbilical vein endothelial
cells (HUVECs) was assayed. Medium containing VEGF
was incubated upon a Laponite gel film for two hours, then
removed (and retained to assay unbound VEGF). The Laponite
film was washed twice with PBS before HUVECs were seeded
onto the gel surface. Laponite exposed to VEGF for two hours
before washing (bound) yielded an equivalent degree of tubule
network organization to that observed when VEGF was added
concurrently with the cells and present throughout the culture
period (VEGF positive). Critically, we observed significantly
greater network organization with Laponite-bound VEGF both
to Laponite in the absence of VEGF (VEGF negative; P < 0.001)
and to Laponite in combination with the residual (unbound; P =
0.004) fraction of VEGF (Figure 3a). Robust HUVEC tubule for-
mation was dependent on the presence of a matrix molecule
such as, in this study, fibronectin or type I collagen mixed with
Laponite prior to gelation (Supporting Information, Figure 4).
These results demonstrate the ability of Laponite to bind VEGF
and induce an angiogenic response as well as suggesting poten-
tial for clay-based gels as biologically neutral platforms for in
vitro assays of angiogenesis.
To further examine the utility of this approach for the locali-
zation of growth factors for regenerative medicine, Laponite-
bound VEGF in combination with bone morphogenetic
protein-2 (BMP2) was applied to a murine bone-defect model
to assess neo-angiogenesis, a critical precursor to any new bone
formation. Laponite-bound VEGF was found to enhance neo-
vascularization at the defect site. In combination with a com-
posite collagen scaffold (BDTM) substrate, Laponite was applied
to media containing VEGF and BMP2 for 2 h (Figure 3b)
before being washed 3× in PBS over 18 h. The residual VEGF
in the media following incubation and washing was quantified
revealing almost complete removal of VEGF in the Laponite-
containing samples (Figure 3c). The constructs were implanted
in a 5-mm mouse femur segmental defect (Figure 3d) and, after
4 weeks, assessed for new vessel formation following perfusion
of the murine vascular network with a radio-opaque polymer
(Figure 3e) and analysis via micro-computed tomography
(μCT).[19,20] Three-dimensional μCT reconstructions revealed
significantly higher vessel volume (P = 0.007) and vessel
number (P = 0.011) in defects containing Laponite samples
pre-incubated in the presence of VEGF compared to controls

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(Figure 3f). These results are consistent with additional in vivo
studies that show the induction of angiogenesis by Laponite-
bound VEGF when implanted subcutaneously (Supporting
Figure 5). Further studies are proposed to examine the efficacy
of bone formation over an extended time frame in conjunc-
tion with a mechanically competent substrate as sub-optimal
mechanical environment and time frame utilized were not
conducive to significant bone formation in the femoral defect
model.
In the current study we have demonstrated the potential to
harness the sorptive capacity of clay in regenerative medicine.
Combining in situ self-assembly in response to physiological
saline and a remarkable capacity for protein retention, clay
gels can provide a one-step procedure for the encapsulation
and delivery of agents without the need for complex chem-
ical/physical approaches and methods. The judicious use of
appropriate factors incorporated at the mixing stage or sub-
sequently via uptake from an external medium, together with
the facility to recover gel capsules from suspension, provides
a versatile mode of generating spatially-controlled biological
microenvironments. We have provided proof of concept
of the ability of a smectite clay-gel to stimulate angiogen-
esis in vitro and in vivo following brief exposure to soluble
VEGF. The application of such a material to generate multi-
functional scaffolds and innovative routes for protein and
cell delivery offers significant potential for the modulation of
stem and progenitor cell growth and differentiation for tissue
regeneration.
Experimental Section
Suspensions of Laponite XLG (Rockwood Additives Ltd, UK) were
prepared by adding Laponite powder to d-H2O under rapid agitation
sufficient to produce a vortex. Suspensions were stirred for 6 h at room
temperature, sterilized via standard autoclave protocols and stored at
4 °C for up to 6 months. Laponite solids concentrations (2.5%) were
used throughout unless stated otherwise. Capsules were formed by
applying Laponite suspensions drop-wise into cell culture media. Cell
suspensions were washed twice with sucrose (0.25 M) and mixed as a
10% fraction to 2.5% Laponite suspensions. Fibronectin (50 μg mL−1)
was added to capsules at a concentration of by adding solubilized
Fibronectin (1 mg mL−1) 1:1 to sucrose (0.5 M) before mixing with cells
and Laponite and addition to cell culture media. Microcapsules were
generated by pumping Laponite suspensions (3.5% solids concentration)
through a domestic hydraulic spray nozzle and collecting the atomized
suspension in Petri-dishes containing cell culture media. Microcapsules
were sub-encapsulated by repeated centrifugation and washing in
d-H2O followed by resuspension in Laponite suspensions (2.5%
solids) and drop-wise addition to cell culture media. Ring-forms and
string-forms were generated via application of Laponite suspensions to
cell culture media drop-wise from a height of 5 cm or though an
immersed hypodermic needle (23G) respectively. Laponite localization
of exogenous protein/growth factors was achieved by addition of
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Laponite capsules to media containing protein and incubation for 2 h
before washing.
Additional experimental details are provided in the supporting
information.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors would like to thank Esther Ralph, Joanna Greenhough
and Michelle Bryson of the University of Southampton Bone and Joint
group for technical support, Dr. David A. Johnston of the University of
Southampton Biomedical Imaging Unit for assistance with confocal
microscopy, Dr. Patrick Duriez of the University of Southampton Protein
Core Facility for assistance with protein labeling and Dr. Marcus Dymond
for assistance with rheological studies. This project was funded by
Wessex Medical Research, BBSRC (BB/GO10579/1) and PhD funding to
J.I.D from the BBSRC.
Received: March 15, 2011
Published online: June 10, 2011
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