Peptide-based stimuli-responsive biomaterialssafinya/Physics-Materials 135/peptides for...
Transcript of Peptide-based stimuli-responsive biomaterialssafinya/Physics-Materials 135/peptides for...
Peptide-based stimuli-responsive biomaterials
Robert J. Mart,a Rachel D. Osborne,b Molly M. Stevens*b and Rein V. Ulijn*a
Received 31st May 2006, Accepted 31st July 2006
First published as an Advance Article on the web 25th August 2006
DOI: 10.1039/b607706d
This article explores recent advances in the design and engineering of materials wholly or
principally constructed from peptides. We focus on materials that are able to respond to changes
in their environment (pH, ionic strength, temperature, light, oxidation/reduction state, presence of
small molecules or the catalytic activity of enzymes) by altering their macromolecular structure.
Such peptide-based responsive biomaterials have exciting prospects for a variety of biomedical
and bionanotechnology applications in drug delivery, bio-sensing and regenerative medicine.
1. Introduction
Materials that change properties in response to local environ-
mental stimuli are increasingly being studied in the context of
biomedical applications. For example, physical or chemical
hydrogels loaded with drug molecules may release their
payload, only when and where required, in response to
changes in the local environmental conditions, such as pH,
temperature, presence of small molecules or enzymes, and
oxidising/reducing environment, among others.1 Another key
application is injectable gels for minimal invasive surgery.
These materials may be applied through a syringe, and
undergo a solution-to-gel transition when triggered by
temperature, pH, ionic strength, oxidative species or enzymes
at the site of injury to act as a scaffold for tissue regrowth. A
third area is in bio-sensing, where small chemical or physical
aSchool of Materials and Manchester Interdisciplinary Biocentre(MIB), Grosvenor Street, Manchester, UK M1 7HS.E-mail: [email protected]; Fax: +44 161 3068877;Tel: +44 161 3065986bDepartment of Materials and Institute for Biomedical Engineering,Imperial College of Science, Technology and Medicine, Prince ConsortRoad, London, UK SW7 2AZ. E-mail: [email protected];Fax: +44 20 7594 6757; Tel: +44 20 7594 6804
Robert Mart
Robert Mart received aMasters degree from UMIST,before completing a PhD onasymmetric organic catalysis ofthe Morita-Baylis–Hillmanreaction with Dr D. J.Berrisford. He then spent ayear as a postdoctoral researchassociate with Dr S. J. Webb inthe newly created University ofManchester studying vesicle–vesicle interactions before join-ing the Ulijn group where hesynthesises enzyme responsivebiomaterials. Rachel Osborne
Rachel Osborne read for aMasters in Materials,Economics and Managementat Oxford University beforespending a year as aMarketing Co-ordinator forL’Occitane in New York.Despite all the free lunchesshe wanted to pursue scienceand under the direction of DrM. M. Stevens she is currentlyundertaking a PhD looking atthe bio-functionalization ofgold nanoparticles at ImperialCollege, London.
Molly Stevens
Molly Stevens received herPhD from The University ofNottingham and spent 2.5 yearsas a postdoctoral researcher atMIT. She is currently a readerat Imperial College London.She has recently been recog-nised by Technology Review’sTR100 Young InnovatorsAward (2004) and the PhilipLeverhulme Prize forEngineering (2005) for herresearch in regenerative medi-cine and nanotechnology.
Rein Ulijn
Rein Ulijn received his Mastersfrom Wageningen University,PhD from The University ofStrathclyde and spent 2 yearsas a postdoctoral researcher atthe University of Edinburgh.He is currently an advancedresearch fellow and seniorlecturer in biomedicalmaterials at the University ofManchester. His research isinterdisciplinary and focuseson the design, characterisationand application of responsivemolecular biomaterials.
REVIEW www.rsc.org/softmatter | Soft Matter
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changes in the sensing environment trigger macroscopically
observable changes in material properties, thereby reporting
them, for example by gelation or nanoparticle (dis)-assembly.
These responsive biomaterials contain molecular building
blocks that undergo molecular level changes which result in
altered non-covalent interactions that, in turn, translate into
macroscopic responses.
In this Review Article, we focus on recent (since 2000)
reports on responsive biomaterials that use peptides as their
stimuli-responsive elements. Peptides are ideally suited for this
purpose because of the range of distinct physical properties
available from the naturally occurring amino acids (Fig. 1).
This diversity allows for rational incorporation of non-
covalent interactions including electrostatic (acidic and basic
amino acids), hydrophobic, p-stacking (aromatic amino acids),
hydrogen bonding (polar amino acids) as well as covalent
(disulfide) bonds and steric contributions (strand directing
amino acids). While individually these interactions are quite
weak (see Fig. 1), collectively they can give rise to very stable
structures. Crucially, each of these interactions depend in
different ways on environmental conditions such as ionic
strength, pH and temperature. In addition, specific short
peptide sequences can introduce responsiveness via small
molecule recognition. Enzyme responsiveness can be pro-
grammed into these materials by incorporation of peptide
sequences that are known substrates for proteases, kinases, or
phosphatases.1n The dynamic nature of these interactions then
allows the molecular organisation to be altered in response to
Fig. 1 Schematic descriptions of different classes of amino acids and the types of peptide interactions they are involved in.
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changes in the direct environment. Each type of interaction has
different requirements, for example hydrogen bonding requires
precisely positioned and directed residues with the donor and
acceptor approximately 2.8 A apart. p–p stacking interactions
require the overlap of two p systems approximately 3.4 A
apart. In contrast, electrostatic interactions are generally not
directional and tend to be more flexible regarding the distance
between the participating charges, although this depends
strongly on the ionic strength of the solution. Hydrophobic
interactions are even less geometrically constrained. In nature,
responsive peptide based materials, for example, enzymes
and motor-proteins, use a combination of these individually
weak interactions, which work cooperatively to dynamically
organise the secondary, tertiary and quaternary structures of
proteins.
It is a major challenge for scientists and engineers to
incorporate these design concepts into useful peptide based
materials and devices. The following sections examine
strategies which involve the design of a peptide macro-
monomer consisting of a primary sequence that is either
amphiphilic or forms a known secondary structural motif
(a-helix, b-sheet, b-turn, elastin-like sequence), in which
responsive elements are rationally incorporated.
Macroscopically observed transitions in response to external
stimuli are then achieved by further quaternary interactions
between individual peptides. The resulting switchable assem-
blies may take the shape of nanometre sized fibres, spheres
or tubes (consisting of superhelices, coiled-coils, amphiphilic
assemblies such as micelles). In other examples, peptide
motifs are used as responsive elements in multi component
systems, to allow macroscopic transitions such as nanoparticle
(dis-)assembly, switching of surface properties, and even
control the action of bioactive proteins. A number of
peptide-based biomaterials where responsiveness was not a
major design aspect have been excluded from the current
review. We focus mainly on systems composed of relatively
short oligopeptides, thereby excluding a number of studies on
responsive proteins.
2. Systems based on helices and coiled-coils
a-Helices are a key secondary structure of peptides, charac-
terised by a single, spiral chain of amino acids stabilised by
hydrogen bonds. Typically, peptide chains that form a-helices
exhibit amino acids of similar character every three or four
residues. This spacing corresponds to the structural repeat of
3.6 residues per a-helical turn. Dynamic self-assembly of
helical structures has been achieved by rational incorporation
of stimuli-responsive amino acids within these structures.2–15
For example, a range of a-helical peptides (Table 1 , entry 1)
have been modified to undergo dynamic conformational
changes in response to light. Cysteine residues were incorpo-
rated into a de novo hexadecapeptide to allow the bonding of
an azobenzene based cross-linker to the peptide backbone.2a
The extended trans isomer of the cross-linker was synthesised
to match an i, i + 11 substitution pattern and was tested
alongside peptide sequences with cysteine residues placed at
the relative positions i, i + 4, i, i + 7 and i, i + 11, resulting in
their near-vertical alignment in the helical stack. Circular
dichroism shows that, as expected, the trans to cis photo-
isomerism of the azobenzene linker increases the a-helical
content for the i, i + 4 and i, i + 7 peptides as the over-long
cross-linking molecule is effectively shortened. Conversely,
there is a decrease in the a-helical content for the i, i + 11
peptide as the cross-linker becomes too short to permit ready
helix formation.
An important a-helix based quaternary structure of peptides
is the coiled-coil. Characterised by two or more a-helices
organised into a supercoil, each peptide length contains a 3, 4
heptad motif repeat (abcdefg). The interhelical interactions
are captured by pairwise interactions by four key positions;
a, d, e, g (see top panel, Fig. 2). Hydrophobic residues found at
positions a and d form the hydrophobic core of a coiled-coil.
Positions e and g are either side of the hydrophobic core and
can participate in electrostatic interhelical contacts and also
alter core hydrophobicity. Changing the nature of these
contacts by introducing responsive amino acids can alter the
stability of the conformation and provide a mechanism for
control of dynamic materials.
The use of acidic and basic amino acids that can be
protonated or deprotonated by a change in pH allows dynamic
control over the secondary structure of the peptides and can be
used to control the assembly of coiled-coils. For example, a
coiled-coil a-helix with leucine at position d and glutamic
acid residues at positions e and g (Table 1, entry 2) forms
homodimeric coiled-coils which are destabilised in basic
solutions.3 This leucine zipper amino acid sequence was
covalently bonded to a gold-substrate via the formation of a
gold–thiolate bond to form a monolayer. An extended version
of the quartz crystal microbalance (QCM-D) in combination
with surface plasmon resonance was used to probe the
formation of the peptide functionalised surface and its
response to changes in pH. Characteristic shifts in dissipation,
D, consistent with the formation of a rigid layer at low pH
(pH 4.5) which increases in fluidity as the pH is increased
(pH 7.4 then pH 11.2) due to disruption of the coiled-coil
structure and unfolding of the alpha-helices, as monitored
with the QCM-D.
The same acidic leucine-zipper like peptides were also used
in a separate study by Stevens et al. to dynamically assemble
gold nanoparticles functionalised with the peptides (Table 1,
entry 3).4 At pH 11.5 when the coiled-coil structure is relatively
unstable, the gold nanoparticles were dispersed, whereas at
pH 4.5 the nanoparticles were aggregated and stabilised by
the specific biomolecular interactions between peptides on
adjacent nanoparticles forming coiled-coils. The transition to
the dispersed or aggregated state occurred between pH 8.5 and
pH 7, and can be monitored by noting shifts in the UV–visible
spectra and CD spectra (top panel, Fig. 2). The system
also showed dynamic disassembly in response to changes in
temperature due to the thermal unfolding of the a-helices.
A system (Table 1, entry 4) designed by Woolfson et al.5
incorporated glutamic acid and lysine pairs at the e and g
positions to stabilise the coiled-coil. These peptides formed
nanosized fibres that unwound into random coils in response
to an increase in ionic strength as these charges were screened,
destabilising the helix–helix interaction. By designing an amino
acid sequence that resembled both that of a leucine-zipper
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coiled-coil and that of a b-hairpin (Table 1, entry 5), a system
was created that not only changed conformation from a-helix
to b-hairpin when heated, but also consequently formed a gel.6
Long and short charge complimentary coiled-coils were
applied to control the aggregation of gold nanoparticles
(Table 1, entry 6) and adhesion of particles to functionalised
gold surfaces.7 These systems were extremely sensitive to the
pH of the solution. In another case, a non-canonical coiled-coil
sequence (a 3-4-4-3-4-3-4 pattern of hydrophilic residues rather
than 3-4-3-4-3-4-3 heptad repeats) inspired by zinc chelating
proteins and loaded with metal binding histidine residues
(Table 1, entry 7) was synthesised. This peptide was then
shown to convert from a coiled-coil to a b-hairpin conforma-
tion on the addition of zinc salts and back when a stronger
chelating agent was added.8 Despite their use in the formation
of a variety of fibre morphologies,9 first generation structures
proved very sensitive to the presence of salts. Later iterations
(Table 2, entry 8 and Fig. 2, lower panel) have recently been
shown to be more tolerant of the ionic strength of the parent
solution.10 Closely related, shorter peptide sequences were
investigated by Dong and Hartgerink, who showed pH
responsive coiled-coil assembly, a-helix to b-sheet conversion
and heterodimerisation of coils bearing all lysine and all
aspartic acid residues at the e and g positions.11 Histidine has
successfully been incorporated at the d position of a helix in
order to facilitate triggered self-assembly of a trimeric coiled-
coil bundle when the histidine is uncharged above pH 6.5.12
The induction of helicity by a small molecule was achieved
by the substitution of lysine residues variously into the b, e
and f positions in combination with a arylporphorin
molecule bearing free sulfonate groups. Electrostatic interac-
tions between the sulfonate and the lysine residues biased the
conformation of the random coil peptide chain, resulting in
helix formation. Pascal et al. have altered a naturally occurring
protein, Par-4 (Table 1, entries 12, 13 and 14), with a tendency
to form coiled-coils by modifying the residues in the e and g
positions, resulting in ionic strength and pH dependant coil
assembly.14,15
In summary, the design rules for responsive a-helices and
coiled-coils are well understood and a number of recent
examples show that these systems can be used either on their
own or immobilised onto (nanoparticle) surfaces as responsive
biomaterials which provide insight into protein folding and
may have applications in bio-sensing.
Table 1 Responsive systems based on helical and/or coiled-coil motifs
System Stimulus Response Ref. Applications
1 Photo-regulated azobenzene cross linked peptide:EACAREAAAREAACRQ
Light Disruption of a-Helix 2 Probing protein function
2 Leucine zipper peptide. Includes the repeat:SGDLENEVAQLEREVRSLEDEAAEL-EQKVSRLKNEIEDLEAE
pH (4.5–11.2) a-Helix coiled-coil torandom Coil
3 Switchable surfaces
3 Peptide-functionalised nanoparticles. Leucinezipper peptide includes the repeat:SGDLENEVAQLEREVRSLEDEAAELE-QKVSRLKNEIEDLKAE
pH (7–8.5) a-Helix Coiled-coil torandom coil
4 Triggered nanoparticle(dis)-assembly forbio-sensing
Temperature(Tm = 90 uC at pH 4.5)
4 Repeat peptides of: KIAALKQKIASLKQEID-ALEYENDALEQ and KIRALKAKNAHL-KQEIAALEQEIAALEQ
Ionic strength (0.5 M KF) Coiled-coil torandom coil
5 Actin/myosin filamentmimics
5 Ac-YGCVAALETKIAALETKKAALETIA-ALC-NH2
Temperature (.60 uC) Coiled-coil sol tob-hairpin gel
6 Conformational switches
6 Ac-AALEKEIAALEQEIAALEKEIAALEY-ENAALEKEIAALEQE-NH2
pH (,6.5 and .7.5) a-Helix coiled-coilto random
7 Nanoparticle assembly
Ac-CGGIAALKQKIAALKQKIAALKYK-OHH-IAALKQKNAALKQKIAALKYKGGC-NH2
7 ZiCo: YIHALHRKAFAKI Zinc(II) ions (1 eq Zn2+) Coiled-coil to b-hairpin 8 Metal ion sensing, proteinfolding modelsARLERHIRALEHAA
8 KIAALKQKIASLKQEIDALEYENDALEQ-KIAALEQ
Ionic strength(120 mM KCl)
Coiled-coil torandom coil
10 Actin/myosin filamentmimics
KIRRLKQKNARLKQKIAALEQEIAAL-EYEIAALEQ
9 EIAQLEYEISQLEQ pH Random coil to a-helix 11 Protein folding models,amyloid fibre modelsEIAQLEYEISQLEQEIQALES
KIAQLKYKISQLKWKIQSLKQ10 TZ1H pH (.6.5) Random coil to
coiled-coil12 Tissue regeneration
Ac-EIAQHEKEIQAIEKKIAQHEYKIAQHKEKIQAIK-NH2
11 Cp3K–N meso-Tetrakis(4-sulfonato-phenyl)porphine
Random coil to a-helix 13 Electron/excitation transferAc-IQQLKNQIKQLLKQ-NH2
12 Par-4 (11–51) pH (5.5) Random coil to a-helix 14 Neurodegenerative andcancer apoptosis researchIGKLKEEIDKLNRDLDDMEDENEQLKQ-
ENKTLLKVVGKLTRTemperature(0 uC at pH 5.75)Ionic strength(140 mM at pH 8.5)
13 Par-4 (11–51)D24N pH (.6.0) Random coil to a-helix 15 Apoptosis researchPar-4 (11–51)E29Q
14 Par-4 (11–51)D24K Temperature (.65 uC) a-Helix to random coil 15 Apoptosis researchPar-4 (11–51)E29K
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3. Systems based on beta sheets
The secondary structural features known as b-sheets are
another key architectural component in naturally occurring
peptides. They are formed by adjacent parallel or anti-parallel
peptide strands hydrogen bonding together to form a weakly
curved sheet. Much recent research on b-sheets has focused on
their participation in disease states including: Alzheimer’s
disease, Parkinson’s disease and transmissible spongiform
encephalopathies, as well as the formation of spider silk.16
These systems are most frequently static structures and whilst
measures to inhibit their formation have been well studied,
relatively few truly responsive systems have been reported. The
sidechains of alternate residues in the primary sequence are
positioned on opposite sides in a b-sheet, allowing ready facial
discrimination and construction of higher order structures.
For example, a primary sequence may consist of alternating
cationic, hydrophobic and anionic amino acid residues in a
format e.g. arginine-alanine-aspartic acid (RAD), phenyl-
alanine-glutamic acid-lysine (FEK) or glutamic acid-alanine-
lysine (EAK) as pioneered and reviewed by Zhang.1e
A development of the FEK sequence uses the presence of
calcium ions to trigger a structural rearrangement, but supplies
these ions by disrupting vesicles.17 When near infrared light is
shone on a mixture of FEK16 peptides (Table 2, entry 1) and
calcium ions encapsulated in carefully prepared vesicles, the
vesicles become leaky, allowing calcium ion escape and
gelation. This release may also be triggered by heating the
vesicle membranes to their phase transition temperature, and
so is highly tunable. A series of undecapeptides (Table 2,
entry 2) have been tailored to react to acidic or basic
conditions by the incorporation of ornithine or glutamine
residues at key positions.18 Whilst the phase transitions that
resulted were more complex than anticipated, they were fully
reversible until the ionic strength of the solution was com-
promised by the repeated addition of aqueous acids and bases.
Similarly the biomimetic (from Zutoin) KFE12 sequence
(Table 2, entry 3) which gelled at physiological pH as well as
under the influence of salts19 was modified by the incorpora-
tion of glutamine, resulting in the synthesis of KFQ12 (Table 2,
entry 4). This peptide only forms a gel under neutral
conditions upon the addition of salts.20 A number of later
systems based on the so-called EAK16 peptides (Table 2,
entry 5), examined by Hong et al. not only display sensitivity
to pH21a and ionic strength21b but also display different
morphologies, ranging from globules to fibrils, under different
conditions.21c The matrices formed by EAK16-II (Table 2,
entry 5) have previously been shown to support mammalian
cell attachment,21d,21e underlining the potential of this type of
material for regenerative medicine and 3D cell culture.21f–i
Fig. 2 Examples of peptide based responsive biomaterials derived from a-helix coiled-coil motifs.
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Peptides developed by Collier and Messersmith allow two-
stage assembly of a functionalised fibrillar network. The Q11
peptide (Table 2, entry 6) self-assembles very slowly in pure
water, but the process is greatly accelerated by the presence of
salts or the alteration of the solution pH.22 The resultant
structure contains several pendant glutamine residues that
may be cross-linked with lysine containing peptides in the
presence of TGase and calcium ions (lower panel, Fig. 3)
to functionalise the self-assembled matrix. Another system
based on Q11 developed by the same group incorporates a
polyethylenegycol chain at the C-terminus (Table 2, entry 7),
and results in longer, thinner, straighter fibres with a regular
helical twist.23 An entirely different approach to triggered
b-sheet formation is the transformation of a non-aggregating
substrate by enzyme action to a product that is capable of self-
assembly. This approach has been demonstrated in two cases.
The first approach utilises a phosphorylated serine embedded
within a sequence (Table 2, entry 9) derived from arachnid
dragline silk.24 Peptides were successfully phosphorylated and
dephosphorylated and consequent structural changes were
observed, although the influence of altering a single residue
was limited. In contrast, the second case provides absolute
control through a single switching residue. An N-terminal
section of the b-sheet forming sequence (Table 2, entry 8) is
removed and appended to the side chain of a newly N-terminal
threonine, serine or cysteine residue.25 The a-nitrogen of this
‘‘switch’’ residue is blocked by an enzyme cleavable group,
whose removal triggers an O– or S– to N-acyl shift, thus
restoring the b-sheet forming sequence and triggering self-
assembly. A sophisticated drug delivery system consisting of a
b-sheet-based matrix host incorporating a b-sheet containing
therapeutic sequence guest has recently been reported.26 The
matrix is constructed from an enzyme cleavable region
sensitive to urokinase plasminogen activator (UPa), an enzyme
linked to cancer states, flanked by two b-sheet forming
domains (Table 2, entry 11). This matrix is mixed with a
peptide containing a further b-sheet forming domain, a
mitochondrial disruption domain and a cell penetration
domain to create a targeted drug delivery system.
As an alternative to using ‘self-complementary’ peptides,
systems have been studied that consist of two separate
populations of cationic and anionic peptides, that are mutually
attractive but self repulsive. For example, Yu et al.27 studied a
combination of peptides KVW10 and EVW10 that showed
very rapid (seconds) and repeatable sol-to-gel transitions.
Substitution of valine with alanine or serine resulted in
formation of weaker gels, while substitution with proline
prevented gel formation. It was demonstrated that the cationic
and anionic peptides on their own could also form b-sheets,
either by charge neutralisation through exposure to CH3–
COOH or NH4OH vapour or screening of charges in high (M)
salt concentrations. The two-component gel system was found
Table 2 Responsive systems based on b-sheet structures
System Stimulus Response Ref. Applications
1 FEK16 FEFEFKFKFEFEFKFK Ca2+ ions via temperature or lightstimulated vesicle rupture
Soluble to aggregate 17 Drug delivery, wound healing,tissue engineering
2 P11–4 Ac-QQRFEWEFEQQ-NH2 pH .7.0 Nematic to isotropic 18 Hydrogels, organogels, liquidcrystalsP11–5 Ac-QQXFXWXFQQQ-NH2
(Where X denotes Ornithine)pH , 7.5
3 KFE12 FKFEFKFEFKFE Ionic strength (1 mM NaCl) Sol-to-gel 19 Drug delivery, wound healing,tissue engineeringpH (5–10)
4 KFQE12 FKFQFKFQFKFQ Ionic strength (50 mM NaCl) Sol-to-gel 20 Drug delivery, wound healing,tissue engineering
5 EAK16-I AEAKAEAKAEAKAEAK Monovalent cations Morphology change 21 Cell culture, tissue repair,nerve cell regrowthEAK16-II AEAEAKAKAEAEAKAK (Li+, Na+, K+)
EAK16-IV AEAEAEAEAKAKAKAK pHRAD16-I Ac-RADARADARADAR-
ADA-NH2
RAD16-II Ac-RARADADARARAD-ADA-NH2
6 Q11 Ac-QQKFQFQFEQQ-Am Ionic strength Sol-to-gel 22 Drug delivery, tissue engineering(NaCl 1 mM) (CaCl 1 mM)
7 Q11 Ac-QQKFQFQFEQQ-PEGn Ionic strength (y2 mM) Sol-to-gel 23 Drug delivery, tissue engineeringAverage n = 86
8 SGRGYBLGGQGAGAAA Enzymatic Soluble to aggregate 24 Silk assembly studiesAAGGAGQGGYGGLGSQG
9 Switch peptides, from non-linear tolinear
Enzymatic Sol-to-gel 25 Triggered gel formation,prodrug design, biosensors
10 6-u-8 (FITC)-KLDLKL-SGRSANA-DLKLDLKL
Enzymatic Gel-to-sol 26 Drug delivery
11 EVW10 Ac-EWEXEXEXEX-NH2 pH, ionic strength, mixing ofcharge-complementary peptides
Sol-to-gel 27 3D Gel for protein entrapmentKVW10 Ac-WKXKXKXKXK-NH2
KVW15 Ac-KWKVKVKVKVKV-KVK-NH2
Where X = V,A,S,P12 L4K8L4 (all L or all D) pH . 9 Random coil to
b-sheet28 Amyloid model system
13 DDDAAAVVV-NH(CH2)14CH3 pH or Ca2+ Ions Random coil tob-sheet
29 Advanced medicine, cell cultureKKKVVVVVVeD-NH(CH2)14CH3
DDDAAAVVVeD-NH(CH2)14CH3
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to allow entrapment of proteins in their native form. Higashi
et al. demonstrated pH responsive twisted nanofibres with
opposite handedness from D or L tri-block peptides consisting
of an octalysine sequence flanked with two hydrophobic tetra
leucines (Table 2, entry 12). When a racemic mixture of both
peptides was used, globular aggregates were observed.28 Other
peptide amphiphiles (Table 2, entry 13) assemble into b-sheets
from random coils when the pH is altered to neutralise charged
lysine or aspartic acid residues or when calcium ions are
added to shield the charges on the monomeric peptides.29
Amphiphilic peptide systems are discussed in more detail in
section 5.
In summary, the design rules for responsive peptide
materials based on b-sheets are well understood and usually
consist of peptide chains with alternating hydrophilic and
hydrophobic amino acids. These peptides can be used either on
their own (self complementary) or in pairs of opposite charge.
They fold into (twisted) sheets that may further assemble into
super-helices. Responsiveness of these systems can be tuned by
rational incorporation of acidic and basic amino acids, while
the stability towards temperature can be controlled by tuning
hydrophobicity of the uncharged residues. Primary sequences
have been modified with bioactive peptide regions, to render
the system responsive to enzymes. Applications of responsive
b-sheet based biomaterials include a number of examples of
3D cell culture and (enzyme triggered) drug delivery.
4. Systems based on beta hairpins
A further important secondary structure available to peptides
is the b-hairpin, which occurs when an amino acid sequence
contains a pair of turn inducing residues such as proline
followed by glycine or threonine.30 The cyclic structure of
proline (denoted i + 1, see Fig. 4) causes a slight kink in the
a-carbon backbone and in combination with the more
conformationally flexible glycine or threonine residues (i + 2)
that follow allows a complete reversal of the direction of the
a-carbon backbone. The loop is cemented by the two residues
before and after the turn-inducing pair (i and i + 3) and
subsequent residues to hydrogen bond together, holding the
peptide strand into a tight hairpin. The MAX1 (Table 3,
entry 1) peptide consists of alternating polar lysine and apolar
valine residues either side of the turn-inducing residues,
resulting in an amphiphilic hairpin structure able to self-
assemble and form a hydrogel. The lysine residues are
protonated at physiological pH and the resulting charge–
charge repulsion inhibits b-hairpin and hydrogel formation.
Gelation may be triggered either by increasing the pH to 9.0 to
Fig. 3 Fibrillar networks based on b-sheet structures formed from peptide macro-monomers.
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neutralise the lysine residues,31 or at pH 7 by increasing the
ionic strength of the solution to 150 mM to screen their
charges.32 Self-assembly of MAX1 and the closely related
MAX2 and MAX3 sequences may also be controlled by
changing the temperature, hairpin formation and assembly
being triggered by elevated temperatures (Table 3, entries 2
and 3).33 A modified version of MAX1 resulted in photo-
sensitive hydrogelation (Table 3, entry 4). Here a cysteine
residue was incorporated in place of a valine and decorated
with a charged, photo-sensitive molecule. With the charged
a-carboxy-2-nitrobenzyl molecule in place, interaction between
hydrophobic faces is disrupted and self-assembly inhibited.
Photo-cleavage of the polar side chain removed this inhibition,
triggering self-assembly.34
5. Systems based on amphiphiles
Peptide amphiphiles consisting of a polar peptide region and
an apolar aliphatic tail constitute a versatile class of molecules
which undergo dynamic self-assembly to form a variety of
peptide nanofibres. The wedge-shaped monomers align with
the narrower hydrophobic tails inwards and the bulkier polar
region outwards to form fibres.35 The surface of the fibres
displays the peptide sequence, making these materials good
candidates for the construction of responsive biomaterials.
These surface peptides may be further stabilised laterally by
forming b-sheets.29 Studies using a peptide amphiphile
whose polar section includes several cysteine residues and a
well known cell binding epitope (Table 4, entry 1) show that
when an aqueous solution of the peptide amphiphile is
acidified, self-assembly occurs; a process which is reversed at
neutral or basic pH.36 Once the fibres are self-assembled, the
inclusion of cysteine residues allows them to be reversibly
polymerised by oxidative cross-linking (top panel, Fig. 5) to
enhance their stability and diminish their pH sensitivity.
Further studies have shown the self-assembly of supramole-
cular nanofibres can be initiated by electrolyte solutions or
changes in pH, and these reactions determine the bulk
properties of the macroscopic gel that is formed.
Recently, Hartgerink et al. demonstrated a peptide amphi-
phile molecule (Table 4, entry 2) containing a cell binding
epitope and a matrix metalloprotease cleavable sequence,
which mimics the ability of the extracellular matrix to degrade
by the action of cell-mediated enzymes. Gel formation was
triggered by the addition of calcium ions, then the gel was
dissolved by type IV collagenase.37 Using the PA-1 peptide
(Table 4, entry 3), Stupp and co-workers demonstrated peptide
amphiphile self-assembly triggered by screening of charged
aspartate sidechains by di- and tri-valent metal ions or
neutralisation by pH adjustment.38 A molecule in this series
bearing a laminin epitope (Table 4, entry 4) was gelled by
electrolyte action on mixing with murine neural progenitor
Table 3 Responsive systems based on b-hairpin formation
System Stimulus Response Ref. Applications
1 MAX1 VKVKVKVKVDPPTKVKVKVKV-NH2 pH (9) Sol-to-gel 31 Biomedical and tissue engineering2 MAX1 VKVKVKVKVDPPTKVKVKVKV-NH2 Ionic strength
(150 mM NaCl)Sol-to-gel 32 Tissue engineering/regeneration
MAX2 VKVKVKVKVDPPTKVKTKVKV-NH2
MAX4 KVKVKVKVKDPPSVKVKVKVK-NH2
MAX5 VKVKVKVKVDPPSKVKVKVKV-NH2
3 MAX1 VKVKVKVKVDPPTKVKVKVKV-NH2 Heat (y25 uC) Sol-to-gel 33 Stimuli responsive materialsMAX2 VKVKVKVKVDPPTKVKTKVKV-NH2 Heat (y40 uC)MAX3 VKVKVKTKVDPPTKVKTKVKV-NH2 Heat (y60 uC)
4 MAX7 VKVKVKVKVDPPTKVKXKVKV-NH2
(X = Cys or Cys(a-carboxy-2-nitrobenzyl)Light Sol-to-gel 34 Tissue engineering regeneration,
drug delivery
Fig. 4 Self-supporting hydrogels formed from aggregates of b-hairpin molecules.
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cells in physiological fluids.39 Neural progenitor cells encap-
sulated by the gelation process survived and were induced to
rapidly differentiate into neurons. Further molecules synthe-
sised by the same group (Table 4, entry 5) incorporate groups
which strongly chelate gadolinium(III) ions.40 The contrast
enhancing spin properties of the gadolinium species allows the
decay products of implanted gels to be easily traced in three
dimensions by magnetic resonance imaging. A variation of
this type of dynamic self-assembly is exhibited by bola-
amphiphiles; molecules in which two or more hydrophilic
groups are connected by hydrophobic functionalities. One
such molecule (Table 4, entry 6) has been shown to adopt
different structures under differing pH conditions, forming
crystalline tubules at low pH and helical ribbons at higher
values (lower panel, Fig. 5).41 Once formed, these structures
may be directly interconverted. The different structures are
proposed to be determined by the strength of acid–acid and
acid–acetate pairs, depending on the pH, and the curvature
they allow the polymeric tape to adopt. Kogiso et al. describe
the gelation of divaline (Table 4, entry 7) bola-amphiphiles by
a selection of divalent metal ions, to form colloidal suspensions
and hydrogels depending on the pH.42 A natural extension of
this structure leads to three-armed amphiphiles such as
those investigated by van Bommel et al. These C3 symmetric
scaffolds are based on cyclohexane rings modified by three
identical, pendant pairs of amino acids to create molecules
that can form hydrogels in response to either acidic (Table 4,
entry 8) or basic (Table 4, entry 9) conditions.43
The responsiveness of amphiphile based peptide materials
is mainly driven by the hydrophobic effect and therefore
allows great flexibility in the peptide structures that are used.
Hence, these systems are ideally suited for displaying bioactive
peptides for bio-recognition and enzyme responsiveness in gel
scaffolds.
6. Systems based on aromatic interactions
A number of very short peptide motifs containing aromatic
groups have been found to self-assemble in aqueous condi-
tions. These systems are believed to be stabilised, through p–p
interactions, the attractive interactions between p-electrons in
aromatic rings, in addition to hydrogen bonds and ionic
interactions. The use of p-stacking as a driving force for self-
assembly has been practised for decades in supramolecular
chemistry, generally in organic solvent systems.44 This
technique was recently rediscovered for use in aqueous
solutions. A first example of self-assembly of short peptides
through p-stacking has been described by Reches and Gazit,
who observed that aromatic dipeptides (Table 5, entry 1) could
self-assemble into straight nanotubes,45a hollow spherical
structures45b and amyloid-like fibres45c upon dilution from a
fluorinated organic solvent. To rule out electrostatic interac-
tions between terminal carboxylic acids and amines as the
driving force for nano-tube formation in diphenylalanine a
number of N– and/or C– terminal capped analogues
were tested. The formation of tubular structures was still
observed, demonstrating that the self-assembly process
must instead be explained in terms of p–p interactions.
Xu and co-workers reported that certain fluorenylmethoxy-
carbonyl (Fmoc) -protected amino acids and dipeptides
spontaneously formed fibrous scaffolds upon application of
a pH switch. Ulijn et al.46 and Xu et al.47 collectively studied a
small library of Fmoc–dipeptides made up of combinations of
the amino acids serine, threonine, glycine, alanine, leucine,
phenylalanine encompassing a range of hydrophobicities
(Table 5, entries 2 and 3). The pH value at which gelation
took place varied with the amino acid sequence and no gel
formation was observed by Fmoc-glycine-phenylalanine and
Fmoc-glycine-threonine peptides under any of the conditions
tested. Three peptide gels that were stable at neutral pH were
found to support 3D cell culture of chondrocytes for periods of
up to three weeks.46 In addition to being temperature and pH
responsive, gel-to-sol transition upon binding to a small
molecule ligand, vancomycin, was demonstrated. The mole-
cular recognition event dramatically increased the elasticity of
these gels.48 Whilst the L,L diastereoisomers of Fmoc–
dialanine and pyrenyl–dialanine were found to be unrespon-
sive, the D,D diastereoisomers formed a gel.49 Fluorescence
spectroscopy has been used to provide evidence for p–p
interactions within the gels. In Fmoc and pyrenyl systems, the
Table 4 Responsive systems derived from amphiphilic monomers
System Stimulus Response Ref Application
1 12 Peptides, e.g. PA-4: CH3(CH2)14CO-CCCCGGGS(PO4)RGD
pH (Various acidic) Sol-to-nanofibres 36 Cell culture, regenerative medicine,biomineralisationDi- and Tri-valent metal
ions (20 mM)2 (CH3(CH2)14CO- GTAGLIGQRGDS Ionic stength (0.1 M Ca2+) Sol-to-gel 37 Cell culture, regenerative medicine
Type IV collagenase Gel-to-sol3 PA-1: CH3(CH2)14CO-AAAAGGGS-
(PO4)KGEpH (,9) Sol-to-gel 38 Regenerative medicineIonic strength (.30 mM M2+/3+)
4 CH3(CH2)14CO- AAAAGGGIKVAV Cell culture media (DMEM) Sol-to-gel 39 Cell culture, neuroregenerativemedicine
5 KK(DOTA-e-K-e-K) LLCCCK-(CO(CH2)14CH3)
pH (.7) Sol-to-gel 40 Magnetic resonance imaging,metabolic studies
KK(DOTA-e-KGRGDS) LLLAAA-(CO(CH2)14CH3)
6 GG-(CO(CH7)nCO)-GG pH (8) Tubes to ribbons 41 Drug delivery7 VV-(CO(CH2)nCO)-VV Divalent metal ions (10 mM) Sol-to colloid 42 Nanomaterials research
Sol-to-gel8 Cyclohexane-(FG)3 pH (.5) Sol-to-gel 43 Drug delivery9 Cyclohexane-(MH)3 pH (.6) Sol-to-gel 43 Drug delivery
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emissions from monomeric residues were visible and in a
number of cases red shifted emissions were assigned to dimeric
species (excimers). Circular dichroism spectra were used to
further unravel the molecular arrangements, generally thought
to be super-helical in nature. FT-IR spectroscopy analysis of
dried samples of three different diphenylalanine urethane
derivatives—butoxycarbonyl (Boc), carboxybenzyl (CBz) and
Fmoc (Table 5, entry 1)—suggested significantly different
Fig. 5 Responsive systems based on self-assembled peptide amphiphiles.
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structures depending on the N-protecting group, ranging
from a-helical for Boc-diphenylalanine to b-sheets for CBz-
diphenylalanine.45c A more complete picture of the interac-
tions in these short peptide gels is likely to emerge in the future
as research in this area continues.
Enzyme-responsive assembly or disassembly was demon-
strated by Reches and Gazit, who used proteinase K to trigger
the hydrolysis of diphenylalanine nanotubes45a (Table 5,
entry 1). Enzymatic sol-to-gel transitions were demonstrated
by Xu and co-workers in (de)phosphorylation of Fmoc–
tyrosine47b,50 systems and more recently on napthyl–pentapep-
tide (See Fig. 6).51 In this case, a pair of enzymes with
complementary and opposite activities were used assembly and
disassembly. Tyrosine residues were de-phosphorylated by a
phosphatase to induce gelation and a kinase was used to
reverse the process (Table 5, entry 5). Electrostatic repulsion of
Table 5 Responsive systems derived from amphiphilic monomers bearing aromatic groups
System Stimulus Response Ref. Application
1 FF Proteinase K Disassembly ofnanotubes
45 Nanowire templating,micro/nano electronics
2 Fmoc-GG,AA,FG,GF,FF,FF/Ka, FF/GGa pH 4-8 Sol-to-gel 46 3D cell cultureNo gel formed for Fmoc-GF
3 Fmoc-AA (L,L and D,D) , GG, GA, GS pH 3–5 Sol-to-gel 47 SensingNo gel formation for Fmoc-GT
4 Fmoc-AA, pyrenyl-AA (L,L and D,D); Presence of ancomycin Gel-to-sol 49 Sensing5 Naphthalene-FFGEY i. Phosphatase i. Sol-to-gel 51 in vivo Gelation,
regenerative medicineii. Kinase ii. Gel-to-sol6 Fmoc-XFF and Fmoc-LLL where
X = F, A, V, L . No gel formationobserved when X = G or P
Thermolysin (gelation byreverse hydrolysis)
Suspension-to-gel 52 3D Cell culture
a 50:50 (mol/mol) ratios were used.
Fig. 6 Enzyme responsive materials featuring aromatic interactions.
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negatively charged phosphate groups prevented gelation of the
precursors, while a combination of p-stacking interactions
between phenyl and napthyl groups and hydrogen bonds
(b-sheets) triggers hydrogel formation. Since the enzyme
reactions proceed under thermodynamic control, it is thought
that this method results in fewer defects in the resulting self-
assembled structures. Indeed, more uniform nanotubular
structures were obtained when comparing the enzymatically
obtained dephosphorylated peptide gel to that of the gel
triggered by pH switching.51
A recent contribution from our laboratory demonstrated
the use of a protease in reverse, to catalyse peptide synthesis
(condensation) instead of hydrolysis, to produce amphiphilic
Fmoc–peptide hydrogelators that spontaneously form
nano-fibrous gel structures (Table 5, entry 6).52 We have
demonstrated that the thermodynamic stabilisation of
Fmoc–peptides upon self-assembly, relative to non-assembling
Fmoc–amino acid and dipeptide pre-cursors, provides a
sufficient driving force to trigger formation of supramolecular
hydrogels.
In summary, systems in which aromatic interactions play
key roles have been studied in the last few years and include
those that respond to pH, enzymes, and small molecules. In
these systems, much shorter peptide sequences have been used
compared to those in any of the other categories.
7. Elastin-like polymers (ELPs)
A final category of peptide-derived responsive materials is
based on a template derived from the naturally occurring
protein elastin. These structures consist of pentad repeats
where four of the constituent amino acids, the first, second,
third and fifth are conserved, and the fourth is variable. The
conserved residues are valine, proline, glycine and glycine
and the ‘‘guest’’ residue may be any amino acid except
proline. Polypeptides based on this template display inverse
temperature phase transition, becoming less soluble and
forming aggregates as their temperature is elevated above a
transition point. This point is dependant on the guest residues
and the number of repeats in the primary sequence and may be
tailored to the desired application.53
The transition temperature of ELPs has been exploited by
Chilkoti and co-workers in the developments of drug carriers
for hyperthermic cancer treatments. In these regimes, local
heating (y42 uC) is applied to a cancerous tissue. By designing
ELP systems with a transition temperature around 39 uC the
carrier is more soluble in general circulation (37 uC) and less so
at the site of the tumour. Enhanced uptake of a fluorescently-
tagged thermally responsive polypeptide by tumour cells has
been demonstrated both in vitro54a and in vivo.54b,c Further
work resulted in doxorubicin-ELP conjugates with a range of
transition temperatures, which demonstrated equivalent cyto-
toxicity to that of free doxorubicin, but were found to localise
differently in tumour cells.54d,e Thermoresponsive gel swel-
ling,55 the thermally triggered aggregation of protein–ELP
conjugates for expressed protein purification,56 and the
aggregation of ELP-modified gold nanoparticles57 have also
been reported by the same group, as have ELP modified
surfaces for the binding of ELP-tagged proteins58 and silica–
ELP hybrids resulting in temperature dependant permeable
membranes.59 Recently elastin-like polypeptides were com-
bined with leucine-zipper type helices which were entwined
with monomeric kinesin-1 units to yield fused biomotor-
protein assemblies (Fig. 7). Using repeated helical segments,
multiple, cooperative motive units were incorporated into the
same molecule. When these multivalent motor arrays were
used to coat a cover slip increased microtubule gliding
velocities relative to monovalent systems were observed.60
Elastin-like polypeptides are designed using a well under-
stood template and through their predictable temperature
responsive behaviour have found utility in drug delivery,
protein sensing and purification and biomotor alignment.
Fig. 7 Elastin-like polypeptides and coiled-coils create a backbone to form multivalent kinesin macromolecules.
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8. Conclusion
In this article we report advances in the design and engineering
of materials wholly or principally constructed from peptide
chains. The principles governing the design of systems based
on a-helices and coiled-coils, b-sheets, b-turns, elastin-like
peptides and amphiphiles are well understood, and those
governing aromatic interactions increasingly so. Rational
incorporation of design elements that are responsive to
environmental changes such as pH, ionic strength, oxidation
state, temperature and the catalytic action of enzymes is
possible by observing these principles. The growing under-
standing of peptide design principles enables a shift in
emphasis from the structure to the function of new materials.
Many exciting existing and potential future applications of pep-
tide based biomaterials in biomedicine have been highlighted.
These include drug delivery,17,19,20,22,21f,26,34,41,43,54 injectable
scaffolds for tissue engineering,15,19,21,29,31,32,34,36–38,51 3D cell
culture,21,46,52 sensing,4,25,33,47,49,54b,54c,59 smart surfaces,3,12,57
and general nano-engineering.13,23,45,55,60 Further noteworthy
applications of responsive peptide sequences include their use
in hybrid materials where peptides play key roles whilst fused
to non-peptide backbones, particularly polyethyleneglycol and
N-isopropylacrylamideacrylamides.61
Limitations of current peptide biomaterials technology
include the high cost of custom chemically synthesised or
fermented peptides. Relatively few topographies are currently
available to peptide based biomaterials; future work will
include the development of motifs for the creation of larger
and more complex architectures. Future materials will also
become more subtle through the incorporation of multiple
responsive elements or glycoprotein-like saccharide. It is surely
a matter of time before synthetic, responsive peptide-based
biomaterials are a clinical reality.
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