Dynamically crosslinked materials via recognition of amino acids by cucurbit[8]uril

Article abstract of DOI:10.1039/c3tb20180e

Hydrogels, an increasingly important class of material, have physical properties amenable to many potential uses, particularly in the biomedical area. Utilisation of hydrogels, however, relies not only on their mechanical properties but also on a favourable toxicity profile. Self assembly of polymers through naturally occurring and non-toxic units is therefore a very attractive option. The aromatic amino acids phenylalanine and tryptophan are two such molecular units that form 2 : 1 complexes with cucurbit[8]uril (CB[8]) with high binding equilibrium constants (K_eq up to 10¹² M ^-2). Herein, water soluble styrenic monomers were copolymerised with synthetically derived aromatic amino acid monomers of phenylalanine and tryptophan. The resulting polymers were shown to form dynamic and self-healing physically crosslinked hydrogels via recognition and binding of the amino acids to cucurbit[8]uril.

Full text of DOI:10.1039/c3tb20180e

Journalof
Materials Chemistry B
Materials for biology and medicine
www.rsc.org/MaterialsB
Volume 1 | Number 23 | 21 June 2013 | Pages 2887–2994
ISSN 2050-750X
PAPER
Oren A. Scherman et al.
Dynamically crosslinked materials via recognition of amino acids by cucurbit[8]uril
2050-750X(2013)1:23;1-W

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Dynamically crosslinked materials via recognition of
amino acids by cucurbit[8]uril†
Cite this: J. Mater. Chem. B, 2013, 1,
2904
*
Matthew J. Rowland,‡ Eric A. Appel,‡ Roger J. Coulston and Oren A. Scherman
Hydrogels, an increasingly important class of material, have physical properties amenable to many
potential uses, particularly in the biomedical area. Utilisation of hydrogels, however, relies not only on
their mechanical properties but also on a favourable toxicity profile. Self assembly of polymers through
naturally occurring and non-toxic units is therefore a very attractive option. The aromatic amino acids
phenylalanine and tryptophan are two such molecular units that form 2 : 1 complexes with cucurbit[8]-
uril (CB[8]) with high binding equilibrium constants (Keq up to 1012 Mꢀ2). Herein, water soluble styrenic
monomers were copolymerised with synthetically derived aromatic amino acid monomers of
phenylalanine and tryptophan. The resulting polymers were shown to form dynamic and self-healing
physically crosslinked hydrogels via recognition and binding of the amino acids to cucurbit[8]uril.
Received 6th February 2013
Accepted 19th April 2013
DOI: 10.1039/c3tb20180e
www.rsc.org/MaterialsB
such as methyl viologen (MV), and an electron-rich second guest
such as naphthol, pyrene and dibenzylfuran.18,20–22 In favourable
1 Introduction
Hydrogels are becoming an increasingly important class of
material and can be prepared using either covalent or non-
covalent approaches.1–4 However, covalently crosslinked hydro-
gel materials are oen brittle and lack the ability to self heal once
the network is broken.5 These shortcomings have oen been
addressed by employing dynamic and reversible non-covalent
interactions as the structural crosslinks in hydrogels.6 Cucurbit
[n]uril (n ¼ 5–8 and 10; CB[n]) are a family of macrocyclic host
molecules, which offer attractive supramolecular interactions for
such applications. These macrocyclic hosts are methylene-linked
oligomers of glycoluril that are symmetric and ‘barrel’-like in
shape with two identical portal regions laced by ureido-carbonyl
oxygens. The number of glycoluril units determines the size of
the CB[n] cavity without affecting the height of the molecular
container (approximately 0.9 nm), similar to the cyclodextrin
family, and these materials have recently been screened for
toxicity demonstrating that they are generally not toxic.7,8 Smaller
homologues of the CB[n] family (i.e. CB[5], CB[6] and CB[7]) are
capable of binding single guests (typically cationic amines,
metal, imidazolium ions or small molecule drugs).9–12 In contrast
to the smaller CB[n] homologues, CB[8] has a larger cavity
cases, exceptionally high overall equilibrium binding affinities
(Keq (overall) ¼ Keq (1) ꢁ Keq (2) up to 1014 Mꢀ2) were repor-
ted,13,18,21,23 leading to utilisation in a number of applications
ranging from the formation of diblock copolymers,24–26 sequence-
selective recognition of peptides,27 self-sorting systems,28 surface
modication,29,30 protein conjugation,31 to the formation of
nanocapsules,32 nanocomposites33 and hydrogels.34,35
Our approach involves the use of polymeric materials
coupled with strong, reversible, and stimuli-responsive CB[8]-
based 2 : 1 ‘homo’-ternary binding motifs of naturally occurring
amino acid derived guests. Urbach and coworkers have
demonstrated that N-terminally charged aromatic amino acids,
such as phenylalanine and tryptophan, bind in a 2 : 1 fashion
forming a ternary complex with CB[8] through multiple non-
covalent interactions acting synergistically. This results in
exceptionally high equilibrium binding affinities (Keq up to 1011
Mꢀ2).36 The ternary complex likely forms in a stepwise binding
process whereby one amino acid guest enters rst (Keq1) fol-
lowed by the second amino acid guest (Keq2). These differ from
previous guests as they (1) do not always yield a visible charge-
transfer complex, and (2) the guests should not carry a signi-
cant toxicity prole as they are naturally occurring amino acids.
This 2 : 1 host–guest system has been exploited previously in a
variety of systems including some of biological relevance such
as the dimerisation of proteins which could be observed by
FRET analysis,37–40 and also in insulin sensing.41
3
13,14
˚
volume (479 A )
capable of simultaneously accommodating
two planar and hydrophobic guests in a p–p-stack geom-
etry.11,15–20 This host has been used most prominently in a 1 : 1 : 1
‘hetero’-ternary complex using an electron-decient rst guest,
Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of
Cambridge, Lenseld Road, Cambridge, CB2 1EW, UK. E-mail: oas23@cam.ac.uk;
Fax: +44 (0)1223 334866; Tel: +44 (0)1223 331508
Herein, we utilise this 2 : 1 homoternary binding motif for
the preparation of supramolecular hydrogels. By employing
commercially available and natural amino acids such as
phenylalanine and tryptophan, the hydrogel system should not
only display a reduced toxicity prole but is simultaneously
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3tb20180e
‡ These two authors contributed equally to this work.
2904 | J. Mater. Chem. B, 2013, 1, 2904–2910
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 1 Schematic of hydrogel formation upon CB[8] addition. The amino acids (represented by the shaded cylinders) bind in a 2 : 1 fashion with CB[8]. The R group of
the amino acid is encapsulated within the hydrophobic cavity by non-covalent interactions. Further interactions occur between the protonated N-terminus of the amino
acid unit and the CB[8] portal carbonyl groups.
simplied from a three component to a two component system water was then stirred at room temperature and replaced peri-
(Fig. 1). The well known stimuli-responsive nature of the ternary odically over a 24 h time period (ca. 4–5 times). The dialysed
complex and the ease of synthesis of the various components polymer solution was then transferred into a round bottom
make this system well suited for a variety of important ask, frozen with liquid nitrogen and then lyophilised over-
biomedical and industrial applications.
night to yield uffy white solid materials.
2.2 Synthetic procedures
2 Experimental
2.1 Materials and methods
2.2.1 Synthesis of tert-butyl(1-oxo-3-phenyl-1-((4-vinyl-
benzyl)amino)propan-2-yl)carbamate (StPhe). (4-Vinylbenzyl)-
1H-NMR (500 MHz) spectra was recorded using a Bruker Avance amine (0.500 g, 3.75 mmol) was added dropwise to a solution of
500 Cryo Ultrashield. Chemical shis are measured in ppm (d) Boc–Phe–OSu (1.770 g, 4.88 mmol) in dichloromethane (DCM,
ꢂ
in CDCl3 and D2O with the internal references set to d 7.26 ppm 10 ml) at 0 C. Triethylamine (0.988 ml, 7.50 mmol) was then
and 4.79 ppm, respectively. 13C-NMR (125 MHz) spectra were added dropwise to the cooled solution and the reaction was
recorded in CDCl3 with an internal reference set to d ¼ 77.16 allowed to warm to room temperature and stirred for 24 hours.
ppm. ATR FT-IR spectroscopy was performed using a Perki- The reaction was quenched with saturated sodium carbonate
nElmer Spectrum 100 series FT-IR spectrometer equipped with solution and the product extracted with dichloromethane
a universal ATR sampling accessory. High resolution mass (DCM) (ꢁ3). The combined organic extracts were dried with
spectrometry was recorded using a Waters LCT ESI. Gel magnesium sulfate, ltered and concentrated in vacuo. The
permeation chromatography (GPC) was carried out in water crude residue was redissolved in a 50 : 50 mixture of hexane and
(H2O) on Jordi divinylbenzene columns with a Shimadzu SPD- ethyl acetate and washed through a pad of silica. Removal of the
M20A prominence diode array detector, an Optilab refractive solvent in vacuo yielded the title compound as an amorphous
index detector and a dynamic light scattering detector (both white solid which was dried under a high vacuum overnight
Wyatt). Samples were ltered over 0.2 mm PVDF lters before (1.324 g, 93%). 1H-NMR Spectroscopy (CDCl3, 500 MHz) d
injection using a 0.6 ml minꢀ1 ow rate. Rheological charac- (ppm) ¼ 7.35–6.95 (9H, m, Ar–H), 6.73–6.63 (1H, dd, J ¼ 17.5 Hz,
terisation was performed using a TA Instruments DHR-2 10.7 Hz, alkene–H), 6.10–6.02 (1H, br s, N–H), 5.78–5.68 (1H, dd,
controlled stress rheometer tted with a peltier stage set to J ¼ 17.5 Hz, 0.8 Hz, alkene–H), 5.28–5.20 (1H, dd, J ¼ 10.7 Hz,
20 ꢂC. Dynamic oscillatory strain amplitude sweep measure- 0.8 Hz, alkene–H), 5.10–4.95 (1H, br s, N–H), 4.40–4.37 (3H, m,
ments were conducted at a frequency of 10 rad sꢀ1. Dynamic CH2, CH), 3.16–2.99 (2H, m, CH2), 1.39 (9H, s, CH3). 13C-NMR
oscillatory frequency sweep measurements were conducted at a Spectroscopy (CDCl3, 125 MHz) d (ppm) ¼ 170.98 (CO), 155.37
10–50% strain amplitude depending on the material viscosity. (CO), 137.19 (ArC), 136.87 (ArC), 136.63 (ArC), 136.32 (CH),
All measurements were performed using a 40 mm parallel plate 129.33 (ArCH), 128.72 (ArCH), 127.86 (ArCH), 126.95(ArCH),
geometry with a gap of 0.500 mm and analysed using TA 126.41 (ArCH), 113.95 (CH2), 56.08 (CH), 43.19 (CH2), 38.53
Instruments TRIOS soware. Cucurbit[8]uril was prepared (CH2), 28.24 (CH3). Elemental: found C, 72.39; H, 7.51; N,
according to literature procedures.16 (4-Vinylbenzyl) amine was 7.22%. C23H26O3N2 calculated C, 72.60; H, 7.42; N, 7.36. FT-IR
purchased from TCI, all other materials purchased from Aldrich (ATR) n ¼ 3339 (m), 2983 (m), 1678 (s), 1658 (s), 1517 (s) cmꢀ1
.
and used as received. Dialysis of the functionalised polymers HRMS: found 381.2178 [C23H26O3N2]+ calculated 381.2190.
was carried out by dissolving the polymer (on the order of 2 g) in
2.2.2 Synthesis of poly(2-amino-3-phenyl-N-(4-vinylbenzyl)-
pure water (100 ml) and then placing the solution into a dialysis propanamide-co-(vinylbenzyl)trimethylammonium
chloride)
tube (Spectrum Labs, Spectra/Por, standard grade regenerated (StPhe-StAm). StPhe (0.498 g, 1.31 mmol), (vinylbenzyl)trime-
cellulose dialysis membrane 6, MWCO 2000 Daltons), which thylammonium chloride (2.500 g, 11.81 mmol) and 4,40-azo-
was subsequently submerged into 3 l of water. The external bis(4-cyanopentanoic acid) (ACPA, 36.4 mg) were dissolved in
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 2904–2910 | 2905

View Article Online
Journal of Materials Chemistry B
Paper
methanol (MeOH, 7 ml) and degassed with nitrogen for chloride in dioxane solution (20 ml) added dropwise. The
30 minutes. The reaction was heated to 70 ꢂC and stirred for reaction was stirred for 8 hours and the deprotected polymer
24 hours. The product was precipitated with diethyl ether and was precipitated with diethyl ether. The crude residue was
collected by vacuum ltration. The crude residue was redis- puried by dialysis against water over 24 hours and the product
solved in MeOH (20 ml) and 4 N hydrogen chloride in dioxane lyophilised to yield an amorphous white solid (1.752 g, 60%).
solution (20 ml) added dropwise. The reaction was stirred for 8 1H-NMR Spectroscopy (D2O, 500 MHz) see Fig. S2.† FT-IR (ATR)
hours and the deprotected polymer was precipitated with n ¼ 3373 (br), 3012 (br), 2921 (br), 1679 (m), 1614 (m), 1478 (s)
diethyl ether. The crude residue was puried by dialysis against cmꢀ1. GPC (H2O): Mn ¼ 12.1 kDa, PDI ¼ 2.4.
water over 24 hours and the product lyophilised to yield an
amorphous white solid (1.774 g, 62%) that was 7% StPhe and
93% StAm. 1H-NMR Spectroscopy (D2O, 500 MHz) see Fig. S1.†
FT-IR (ATR) n ¼ 3374 (br), 3014 (m), 2923 (m), 1680 (m), 1614
(m), 1479 (s) cmꢀ1. GPC (H2O): Mn ¼ 10.9 kDa, PDI ¼ 2.2.
2.2.3 Synthesis of tert-butyl(3-(1H-indol-3-yl)-1-oxo-1-((4-
vinylbenzyl)amino)propan-2-yl)carbamate (StTrp). (4-Vinyl-
benzyl) amine (0.500 g, 3.75 mmol) was added dropwise to a
solution of Boc–Phe–OSu (1ꢂ.960 g, 4.88 mmol) in dichloro-
methane (DCM, 10 ml) at 0 C. Triethylamine (0.988 ml, 7.50
mmol) was then added dropwise to the cooled solution and the
reaction was allowed to warm to room temperature and stirred
for 24 hours. The reaction was quenched with saturated sodium
carbonate solution and the product extracted with DCM (ꢁ3).
The combined organic extracts were dried with magnesium
sulfate, ltered and concentrated in vacuo. The crude residue
was redissolved in a 50 : 50 mixture of hexane and ethyl acetate
and washed through a pad of silica. Removal of the solvent in
vacuo yielded the title compound as an amorphous white solid
which was dried under a high vacuum overnight (1.541 g, 98%).
1H-NMR Spectroscopy (CDCl3, 500 MHz) d (ppm) ¼ 8.07 (1H, s,
Ar–H), 7.70–7.62 (1H, d, J ¼ 7.8 Hz, Ar–H), 7.39–7.31 (1H, d, J ¼
8.1 Hz, Ar–H), 7.30–7.20 (1H, m, Ar–H), 7.20–7.15 (1H, ddd, J ¼
8.1, 6.9, 1.1 Hz, Ar–H), 7.15–7.10 (1H, ddd, J ¼ 8.1, 6.9, 1.1 Hz,
Ar–H), 7.00–6.85 (3H, m, Ar–H), 6.71–6.61 (1H, dd, J ¼ 17.7, 10.9
Hz), 6.08–5.90 (1H, br s, N–H), 5.73–5.65 (1H, dd, J ¼ 17.7, 0.8
Hz, alkene–H), 5.27–5.20 (1H, dd, J ¼ 10.9, 0.8 Hz, alkene–H),
5.27–5.10 (1H, br s, N–H), 4.51–4.37 (1H, br s, CH), 4.32–4.18
(2H, m, CH2), 3.39–3.25 (1H, dd, J ¼ 14.3, 5.2 Hz, HC–H), 3.25–
3.10 (1H, dd, J ¼ 14.3, 7.5 Hz, HCH), 1.41 (9H, s). 13C-NMR
Spectroscopy (CDCl3, 125 MHz) d (ppm) ¼ 171.52 (CO), 155.45
(CO), 136.74 (ArC), 136.20 (CH2), 136.05(ArC), 128.02 (ACH),
127.82 (ArC), 127.37 (ArCH), 126.32 (ArCH), 123.19 (ArCH),
123.03 (ArC), 122.35 (ArCH), 119.86 (ArCH), 113.92 (CH), 111.18
(ArCH), 110.67 (ArC), 55.31 (CH), 43.21 (CH2), 28.43 (CH2), 28.27
(CH3). Elemental: found C, 69.27; H, 6.98; N, 9.44%.
2.3 Hydrogel preparation
Polymer solutions (20% w/v) were prepared and diluted with
equivalent volumes of aqueous CB[8] solutions of various
concentrations resulting in a nal polymer concentration of 10%
w/v. The combined solutions were mildly heated and shaken (or
preferably agitated with a vortex) for a few seconds before
allowing to cool to room temperature so the hydrogel could set.
3 Results
3.1 Design and synthesis of functional building blocks
(Vinylbenzyl)trimethylammonium chloride derived polymers
are rigid and highly water soluble on account of their cationic
charge, making this monomer ideal for copolymerisation with
guest-functional monomers. Polymer rigidity is particularly
important for this system in order to enhance hydrogel strength
by limiting intramolecular binding of the amino acid units on
the same polymer chain. Rigid polymers are ideal as they
promote intermolecular complex formation, leading to stronger
materials.
For the purpose of copolymerisation, a compatible amino
acid monomer was also required to ensure random distribution
of the functional units. Therefore, synthesis of a styrene derived
amino acid monomer was undertaken, as shown in Fig. 2.
Coupling of the activated amino acids Boc–L-phenylalanine
N-hydroxysuccinimide ester and Boc–L-tryptophan N-hydroxy-
succinimide ester with (4-vinylbenzyl) amine in the presence of
triethylamine afforded the Boc-protected amino acid monomers
(StPhe, 3a and StTrp, 3b) in good yields.
With the StPhe and StTrp monomers in hand, ‘traditional’
free-radical copolymerisation with (vinylbenzyl)trimethy-
lammonium chloride was performed using 4,40-azobis(4-cya-
nopentanoic acid) (ACPA) (Fig. 3). Following acid treatment for
C
25H29O3N3 calculated C, 71.57; H, 6.97; N, 10.02. FT-IR (ATR)
n ¼ 3310 (br), 2978 (m), 2930 (m), 1693 (s), 1655 (s), 1494 (s)
cmꢀ1 HRMS: found 420.2303 [C25H30O3N3]+ calculated
420.2287.
2.2.4 Synthesis of poly(2-amino-3-(3H-indol-3-yl)-N-(4-
.
vinylbenzyl)propanamide-co-(vinylbenzyl)trimethylammonium
chloride) (StTrp–StAm). StTrp (0.550 g, 1.31 mmol), (vinyl-
benzyl)trimethylammonium chloride (2.500 g, 11.81 mmol) and
ACPA (36.4 mg) were dissolved in MeOH (7 ml) and degassed
with nitrogen for 30 minutes. The reaction was heated to 70 ꢂC
and stirred for 24 hours. The product was precipitated with
diethyl ether and collected by vacuum ltration. The crude
residue was redissolved in MeOH (20 ml) and 4 N hydrogen Fig. 2 Synthesis of StPhe (3a) and StTrp (3b) monomers.
2906 | J. Mater. Chem. B, 2013, 1, 2904–2910
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
Boc-deprotection, the cationic styrenic amino acid random
copolymers (5a, StPhe-StAm and 5b, StTrp-StAm) were afforded
as HCl salts, which were highly water soluble and easily puried
by dialysis. Proton NMR analysis of these copolymers deter-
mined that 7% of the monomeric repeat units were functional
amino acids in both the phenylalanine and tryptophan cases
(see ESI†). By comparing the integration of the aromatic signal
(7.5–6.0 ppm) with the integration of the trimethylammonium
singlet (2.7 ppm), the excess of aromatic protons in the polymer
was determined, which directly correlated to the number of
guest functional monomers incorporated into the nal poly-
mers (Fig. S1 and S2†).
3.2 Hydrogel formation
Phenylalanine and tryptophan bind within the CB[8] cavity in a
2 : 1 fashion.36 Therefore, 0.5 equivalents of CB[8] theoretically
affords 100% crosslinking of the aromatic amino acid units
pendant from the copolymer backbone. Initial CB[8] titrations
into aqueous polymer solutions exemplied this well. Polymer
solutions containing 20% by weight of the functional polymer
were diluted with equivalent volumes of solutions containing
various equivalents of CB[8], mildly warmed and shaken. As
hypothesised, addition of CB[8] resulted in large increases in
viscosity, which was easily visualised by inverted vial tests
(Fig. 4). It was observed that with no CB[8] the polymer
solution remained transparent and the colourless solution
owed readily. With 0.35 equivalents of CB[8] the solution
became much more viscous but retained some ow. Addition
of $0.5 equivalents of CB[8] caused the solution ow to slow
dramatically upon vial inversion. Addition of 1 equivalent
of CB[8] could in theory favour 100% formation of 1 : 1
amino acid3CB[8] complexes. As strong hydrogels are still
formed with 1 equivalent of CB[8] (Fig. 4) this is clearly not
the case.
Fig. 4 Inverted vial test of 10% w/v of 5a (A) or 5b (B) solutions with varying
equivalents of CB[8] and a control experiment wherein addition of CB[7], which
can only accommodate one guest (i.e. crosslinking is impossible), does not cause a
visible increase in viscosity.
(p-StAm), was also synthesised. Upon addition of CB[8] to an
aqueous solution of the control StAm homopolymer at 10% by
weight, no visible change in viscosity was observed (Fig. S3†).
Hydrogel formation, therefore, arises exclusively from the 2 : 1
binding of the charged amino acids inside the CB[8] cavity and
the cationic polymer backbone is not involved in the cross-
linking process.
CB[7] is large enough to bind only one Phe or Trp unit and
unable to promote crosslinking, thus, as a control, 0.5 equiva-
lents of CB[7] were also added to an aqueous polymer solution.
With CB[7] addition no change in viscosity was observed,
therefore 1 : 1 binding of Phe or Trp to a CB[n] molecule is not
constructive for gel formation. As a second control, a homo-
3.3 Rheological characterisation of the supramolecular
hydrogels
Supramolecular hydrogels have been successfully designed and
prepared and rheological characterisation was performed to
quantify their mechanical properties. Rheological analysis was
carried out on supramolecular hydrogels prepared from the
mixture of the 5a and 5b solutions with various molar equiva-
lents of CB[8] (relative to amino acid functionalised units, e.g. 0,
0.05, 0.15, 0.35, 0.50, 0.70 equivalents) corresponding to a
various theoretical percentage crosslinking. Proceeding beyond
the theoretical limit of 100% crosslinking (from 0.5 equivalents
of CB[8] to 0.7 equivalents) are two possible outcomes: (a) the
gel properties, including viscosity, decrease. In the phenylala-
nine case 20% of the amino acid units would be bound in a 1 : 1
fashion and only 80% of the amino acid units in a 2 : 1 fashion
leading to crosslinking. This would be due to the rst binding
constant being equivalent to the second. In the tryptophan
case, the second binding constant is in fact weaker than the
rst and so 40% of the amino acids would be bound in a 1 : 1
fashion and 60% in a 2 : 1 fashion.36 Therefore, at 0.7 equiva-
lents of CB[8] the actual crosslinking would be either 80% or
polymer,
poly[(vinylbenzyl)trimethylammonium
chloride]
Fig. 3 Two stage synthesis of StAm-StPhe (5a) and StAm-StTrp (5b) copolymers. 60% dependant on the amino acids used. (b) As 2 : 1 binding is
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 2904–2910 | 2907

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 5 Frequency-dependent oscillatory rheology of (A) StPhe-StAm, (B) StTrp-
StAm hydrogels with varying equivalents of CB[8]. Open symbols are loss moduli
G00, and closed symbols are storage moduli G0.
Fig. 6 Steady shear rheological measurements of hydrogels formed with (A)
StPhe-StAm (10% w/v), (B) StTrp-StAm (10% w/v) with increasing loading of CB[8].
concentration, the plateau modulus of each material increased,
more favourable than 1 : 1 binding (i.e. higher Keq value for the even when CB[8] loading exceeded 0.5 equivalents relative to the
ternary complex in comparison with the binary complex) the guest moieties. Interestingly, the moduli for the StPhe-StAm are
excess CB[8] suspended in the solution would not prevent distinctly larger than those of the StTrp-StAm hydrogels by an
crosslinking as in case ‘a’ but instead increase viscosity by order of magnitude.
acting as a solid viscosity modulator. It is also possible that
Frequency-dependant oscillatory rheological measurements
excess CB[8] in solution would promote formation of the were also performed on the materials within the linear visco-
ternary complex pushing the system towards 100% crosslinking elastic regime (Fig. 5). As with the strain-dependant oscillatory
and reducing the apparent rate of dissociation of the measurements, the moduli of the StPhe-StAm hydrogels are a
components.
magnitude higher than those of the StTrp-StAm materials. It is
3.3.1 Oscillatory measurements. Strain-dependant oscilla- also worthwhile noting that the StPhe-StAm hydrogels become
tory shear measurements at 20 ꢂC were rst performed in order elastically active at lower angular frequencies than the StTrp-
to determine the linear viscoelastic properties of the material. StAm hydrogels. In both cases, more equivalents of CB[8] added
Throughout the experiment all hydrogels were shown to have a caused an increase in both the storage and loss moduli of the
broad viscoelastic window and no deviation from linearity was hydrogel and also a decrease in the angular frequency at which
observed even at the highest oscillation strain, except for the the storage modulus (G0) becomes dominant over the loss
StTrp-StAm random copolymer hydrogels containing 0.7 modulus (G00). With more CB[8] present at any one time a
equivalents CB[8] (see ESI Fig. S4 and S5†). Both materials, greater number of crosslinks will exist between the polymers,
regardless of CB[8] concentration, retained their broad visco- allowing the material to behave more elastically as the supra-
elastic regions. It was observed that, with increasing CB[8] molecular network undergoes a higher energy penalty to break.
2908 | J. Mater. Chem. B, 2013, 1, 2904–2910
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
As the crossover point occurs at lower angular frequencies distinctly different. The StPhe-StAm copolymer requires only
with greater CB[8] concentration it can be inferred that a greater 0.31 equivalents of CB[8] for gelation to begin whereas the
degree of crosslinking has been achieved and that higher CB[8] StTrp-StAm hydrogel requires 0.5 equivalents. For the StTrp-
concentrations (even beyond 0.5 equivalents) enforce 2 : 1 StAm copolymer, viscosity is not dramatically altered with CB[8]
complexation. This leads to the conclusion that the equilibrium concentration until surpassing the theoretical 100% cross-
of ternary complex formation does not lie completely toward 2 : 1 linking point. This is accountable for by the difference in
homoternary complex. Instead we envisage that in such cases the association constants of the amino acids with CB[8] (vide supra)
equilibrium between the free amino acid units and CB[8] and how this may effect the dynamic equilibrium present.
and their respective 1 : 1 binary complex lies towards the free
units. By increasing the CB[8] concentration, 1 : 1 complexation
is enforced by Le Chatelier's principle, quickly proceeding to
4 Conclusions
ternary complex formation, which is energetically favourable.
Rigid polymer chains with pendant phenylalanine or trypto-
3.3.2 Steady shear measurements. Steady shear rheological phan amino acids have the ability to form hydrogels in the
measurements were performed on both the StPhe-StAm and presence of CB[8]. It has been determined that the phenylala-
StTrp-StAm hydrogels to determine the mechanical effect of nine unit affords much stronger hydrogel materials than its
varying amounts of CB[8] (Fig. 6). In both cases, the zero-shear tryptophan counterpart. This work sheds light on improved
viscosity of the materials increased with added equivalents of biomedical systems for drug delivery as hydrogel formation can
CB[8], even beyond 0.5 equivalents. Initially, the hydrogels now be achieved without the requirement of potentially toxic
behaved as Newtonian materials with no change in viscosity, guest moieties for the CB[8]-based crosslinking. Further devel-
however, at high shear rates some hydrogels with higher CB[8] opment of this system will entail attachment of these amino
loading displayed a slight degree of shear-thickening as shear acids to natural polymers such as cellulose derivatives, that will
rate increased. All materials displayed shear thinning behaviour lead to biomedical applications in 3D cell culture, drug delivery
under high shear conditions.
and regenerative medicine and would bypass limitations of
Considering the zero-shear viscosities alone, upon potentially toxic polymer metabolites. We have demonstrated
increasing addition of CB[8] both the StPhe-StAm and StTrp- that the material properties of hydrogels can be readily altered
StAm materials displayed an increase as would be expected by attenuating the CB[8] concentration as well as by choosing an
(Fig. 7). Whilst initially at low CB[8] concentrations both appropriate amino acid based guest.
materials have very low and similar viscosities, once beyond
0.15 equivalents of CB[8], the StPhe-StAm hydrogel zero-shear
viscosity increases at a much greater rate. This suggests that the
Acknowledgements
homoternary Phe23CB[8] complex is much stronger than the M.J.R. thanks the University of Cambridge Chemical Biology
Trp23CB[8] complex, consistent with previous observations by and Molecular Medicine programme for funding. E.A. thanks
Urbach et al.36 Fig. 7 also exemplies the equivalents of CB[8] Schlumberger for nancial support. R.J.C thanks the ERC for
required to induce gelation. Extrapolating lines of best t for the proof of concept grant ‘SMARTmicroCAPS’. This work was
each material show gelation points for the two materials to be also supported by an ERC Starting Investigator Grant (ASPiRe)
and a Next Generation Fellowship provided by the Walters-
Kundert Foundation.
References
1 M. P. Lutolf, Nat. Mater., 2009, 8, 451–453.
2 M. A. C. Stuart, W. T. S. Huck, J. Genzer, M. Mueller, C. Ober,
M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk,
M. Urban, F. Winnik, S. Zauscher, I. Luzinov and S. Minko,
Nat. Mater., 2010, 9, 101–113.
3 K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869–1879.
4 R. Langer and D. A. Tirrell, Nature, 2004, 428, 487–492.
5 N. A. Peppas, Y. Huang, M. Torres-Lugo, J. H. Ward and
J. Zhang, Annu. Rev. Biomed. Eng., 2000, 2, 9–29.
6 L. A. Estroff and A. D. Hamilton, Chem. Rev., 2004, 104, 1201–
1217.
7 V. D. Uzunova, C. Cullinane, K. Brix, W. M. Nau and A. I. Day,
Org. Biomol. Chem., 2010, 8, 2037–2042.
8 G. Hettiarachchi, D. Nguyen, J. Wu, D. Lucas, D. Ma, L. Isaacs
and V. Briken, PLoS One, 2010, 5, e10514.
Fig. 7 The effect of CB[8] concentration on zero-shear viscosity at increasing
9 N. Zhao, L. Liu, F. Biedermann and O. A. Scherman, Chem.–
Asian J., 2010, 5, 530–537.
shear rate. Note, six data points were obtained for both systems but the first three
are the same.
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 2904–2910 | 2909

View Article Online
Journal of Materials Chemistry B
Paper
10 C. Marquez, R. R. Hudgins and W. M. Nau, J. Am. Chem. Soc., 26 X. J. Loh, J. del Barrio, P. Toh, T.-C. Lee, D. Jiao, U. Rauwald,
2004, 126, 5806–5816.
11 J. Lagona, P. Mukhopadhyay, S. Chakrabarti and L. Isaacs,
Angew. Chem., Int. Ed., 2005, 44, 4844–4870.
E. A. Appel and O. A. Scherman, Biomacromolecules, 2012, 13,
84–91.
27 J. J. Reczek, A. A. Kennedy, B. T. Halbert and A. R. Urbach,
J. Am. Chem. Soc., 2009, 131, 2408–2415.
12 E. A. Appel, M. J. Rowland, X. J. Loh, R. M. Heywood, C. Watts
and O. A. Scherman, Chem. Commun., 2012, 48, 9843– 28 P. Mukhopadhyay, A. Wu and L. Isaacs, J. Org. Chem., 2004,
9845. 69, 6157–6164.
13 F. Biedermann, V. D. Uzunova, O. A. Scherman, W. M. Nau 29 F. Tian, N. Cheng, N. Nouvel, J. Geng and O. A. Scherman,
and A. De Simone, J. Am. Chem. Soc., 2012, 134, 15318–
Langmuir, 2010, 26, 5323–5328.
´
´
15323.
30 A. Gonzalez-Campo, M. Brasch, D. A. Uhlenheuer, A. Gomez-
Casado, L. Yang, L. Brunsveld, J. Huskens and P. Jonkheijm,
Langmuir, 2012, 28, 16364–16371.
14 W. M. Nau, M. Florea and K. I. Assaf, Isr. J. Chem., 2011, 51,
559–577.
15 A. Day, A. P. Arnold, R. J. Blanch and B. Snushall, J. Org. 31 F. Biedermann, U. Rauwald, J. M. Zayed and O. A. Scherman,
Chem., 2001, 66, 8094–8100. Chem. Sci., 2011, 2, 279–286.
16 J. Kim, I. S. Jung, S. Y. Kim, E. Lee, J. K. Kang, S. Sakamoto, 32 J. Zhang, R. J. Coulston, S. T. Jones, J. Geng, O. A. Scherman
K. Yamaguchi and K. Kim, J. Am. Chem. Soc., 2000, 122, 540–
541.
17 E. Masson, X. Ling, R. Joseph, L. Kyeremeh-Mensah and
X. Lu, RSC Adv., 2012, 2, 1213–1247.
18 J. Zhao, H.-J. Kim, J. Oh, S.-Y. Kim, J. W. Lee, S. Sakamoto,
K. Yamaguchi and K. Kim, Angew. Chem., Int. Ed., 2001, 40,
4233–4235.
19 J. Kim, I.-S. Jung, S.-Y. Kim, E. Lee, J.-K. Kang, S. Sakamoto,
K. Yamaguchi and K. Kim, J. Am. Chem. Soc., 2000, 122, 540–
541.
20 J. W. Lee, S. Samal, N. Selvapalam, H.-J. Kim and K. Kim, Acc.
Chem. Res., 2003, 36, 621–630.
21 U. Rauwald, F. Biedermann, S. Deroo, C. V. Robinson and
O. A. Scherman, J. Phys. Chem. B, 2010, 114, 8606–8615.
22 F. Biedermann and O. A. Scherman, J. Phys. Chem. B, 2012,
116, 2842–2849.
23 M. Bush, N. Bouley and A. R. Urbach, J. Am. Chem. Soc., 2005,
127, 14511–14517.
24 U. Rauwald and O. A. Scherman, Angew. Chem., Int. Ed., 2008,
47, 3950–3953.
and C. Abell, Science, 2012, 335, 690–694.
33 R. J. Coulston, S. T. Jones, T.-C. Lee, E. A. Appel and
O. A. Scherman, Chem. Commun., 2011, 47, 164–166.
34 E. A. Appel, F. Biedermann, U. Rauwald, S. T. Jones,
J. M. Zayed and O. A. Scherman, J. Am. Chem. Soc., 2010,
132, 14251–14260.
35 E. A. Appel, X. J. Loh, S. T. Jones, F. Biedermann, C. A. Dreiss
and O. A. Scherman, J. Am. Chem. Soc., 2012, 134, 11767–
11773.
36 L. M. Heitmann, A. B. Taylor, P. J. Hart and A. R. Urbach,
J. Am. Chem. Soc., 2006, 128, 12574–12581.
37 D. T. Dang, H. D. Nguyen, M. Merkx and L. Brunsveld, Angew.
Chem., Int. Ed., 2013, 32, 2915–2919.
38 T. D. Dang, J. Schill and L. Brunsveld, Chem. Sci., 2012, 3,
2679–2684.
39 D. A. Uhlenheuer, J. F. Young, H. D. Nguyen, M. Scheepstra
and L. Brunsveld, Chem. Commun., 2011, 47, 6798–6800.
40 H. Nguyen, D. Dang, J. van Dongen and L. Brunsveld, Angew.
Chem., Int. Ed., 2010, 49, 895–898.
41 J. M. Chinai, A. B. Taylor, L. M. Ryno, N. D. Hargreaves,
C. A. Morris, P. J. Hart and A. R. Urbach, J. Am. Chem. Soc.,
2011, 133, 8810–8813.
25 S. Deroo, U. Rauwald, C. V. Robinson and O. A. Scherman,
Chem. Commun., 2009, 644–646.
2910 | J. Mater. Chem. B, 2013, 1, 2904–2910
This journal is ª The Royal Society of Chemistry 2013