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: R.Ulijn@manchester.ac.uk; 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: M.Stevens@imperial.ac.uk;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

822 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online / Journal Homepage / Table of Contents for this issue

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.

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 823

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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

824 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 825

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

826 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 827

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

828 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 829

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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

830 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 831

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

832 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 833

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

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.

References

1 (a) C. de las Heras Alarcon, S. Pennadam and C. Alexander, Chem.Soc. Rev., 2005, 34, 276–285; (b) B. Jeong and A. Gutowska,Trends Biotechnol., 2002, 20, 305–311; (c) B. D. Ratner andS. J. Bryat, Annu. Rev. Biomed. Eng., 2004, 6, 41–75; (d) K. Pagel,T. Vagt and B. Koksch, Org. Biomol. Chem., 2005, 3, 3843–3850;(e) S. G. Zhang, Biotechnol. Adv., 2002, 20, 321–339; (f) H.-W. Jun,S. E. Paramonov and J. D. Hartgerink, Soft Matter, 2006, 2,177–181; (g) R. Fairman and K. S. Akerfeldt, Curr. Opin. Struct.Biol., 2005, 15, 453–463; (h) S. A. Maskarinec and D. A. Tirrell,Curr. Opin. Biotechnol., 2005, 16, 422–426; (i) N. Nath andA. Chilkoti, Adv. Mater., 2002, 14, 1243–1247; (j) A. Chilkoti,M. R. Dreher and D. E. Meyer, Adv. Drug Delivery Rev., 2002, 54,1093–1111; (k) A. Chilkoti, M. R. Dreher, D. E. Meyer andD. Raucher, Adv. Drug Delivery Rev., 2002, 54, 613–630; (l)G. A. Woolley, Acc. Chem. Res., 2005, 38, 486–493; (m)M. M. Stevens and J. George, Science, 2005, 310, 1135–1138; (n)R. V. Ulijn, J. Mater. Chem., 2006, 16, 2217–2225.

2 (a) J. R. Kumita, O. S. Smart and G. A. Woolley, Proc. Natl. Acad.Sci. U. S. A., 2000, 97, 3803–3808; (b) D. G. Flint, J. R. Kumita,O. S. Smart and G. A. Woolley, Chem. Biol., 2002, 9, 391–397; (c)J. R. Kumita, D. G. Flint, O. S. Smart and G. A. Woolley, ProteinEng., 2002, 15, 561–569; (d) Z. H. Zhang, D. C. Burns,J. R. Kumita, O. S. Smart and G. A. Woolley, BioconjugateChem., 2003, 14, 824–829; (e) J. R. Kumita, D. G. Flint,G. A. Woolley and O. S. Smart, Faraday Discuss., 2003, 122,89–103; (f) D. C. Burns, D. G. Flint, J. R. Kumita, H. J. Feldman,L. Serrano, Z. H. Zhang, O. S. Smart and G. A. Wooley,Biochemistry, 2004, 43, 15329–15338; (g) V. Borisenko andG. A. Woolley, J. Photochem. Photobiol., A, 2005, 173, 21–28;

(h) N. Pozhidaeva, M.-E. Cormier, A. Chaudhari andG. A. Woolley, Bioconjugate Chem., 2004, 15, 1297–1303.

3 M. M. Stevens, S. Allen, J. K. Sakata, M. C. Davies, C. J. Roberts,S. J. B. Tendler, D. A. Tirrell and P. M. Williams, Langmuir, 2004,20, 7747–7752.

4 M. M. Stevens, N. T. Flynn, C. Wang, D. A. Tirrell and R. Langer,Adv. Mater., 2004, 16, 915–918.

5 M. J. Pandya, G. M. Spooner, M. Sunde, J. R. Thorpe, A. Rodgerand D. N. Woolfson, Biochemistry, 2000, 39, 8728–8734.

6 B. Ciani, E. G. Hutchinson, R. B. Sessions and D. N. Woolfson,J. Biol. Chem., 2002, 277, 10150–10155.

7 M. G. Ryadnov, B. Ceyhan, C. M. Niemeyer and D. N. Woolfson,J. Am. Chem. Soc., 2003, 125, 9388–9394.

8 (a) M. G. Ryadnov and D. N. Woolfson, Nat. Mater., 2003, 2,329–332; (b) M. Ryadnov and D. N. Woolfson, Angew. Chem., Int.Ed., 2003, 42, 3021–3023; (c) M. G. Ryadnov and D. N. Woolfson,J. Am. Chem. Soc., 2004, 126, 7454–7455; (d) A. M. Smith,S. F. A. Acquah, N. Bone, H. W. Kroto, M. G. Ryadnov,M. S. P. Stevens, D. R. M. Walton and D. N. Woolfson, Angew.Chem., Int. Ed., 2005, 44, 325–328; (e) M. G. Ryadnov andD. N. Woolfson, J. Am. Chem. Soc., 2005, 127, 12407–12415.

9 E. Cerasoli, B. K. Sharpe and D. N. Woolfson, J. Am. Chem. Soc.,2005, 127, 15008–15009.

10 A. M. Smith, E. F. Banwell, W. R. Edwards, M. J. Pandya andD. N. Woolfson, Adv. Funct. Mater., 2006, 16, 1022–1030.

11 H. Dong and J. D. Hartgerink, Biomacromolecules, 2006, 7,691–695.

12 Y. Zimenkov, S. N. Dublin, R. Ni, R. S. Tu, V. Breedveld,R. P. Apkarian and V. P. Conticello, J. Am. Chem. Soc., 2006, 128,6770–6771.

13 B. C. Kovaric, B. Kokona, A. D. Schwab, M. A. Twomey, J. C. dePaula and R. Fairman, J. Am. Chem. Soc., 2006, 128, 4166–4167.

14 K. Dutta, A. Alexandrov, H. Huang and S. M. Pascal, Protein Sci.,2001, 10, 2531–2540.

15 K. Dutta, F. A. Engler, L. Cotton, A. Alexandrov, G. S. Bedi,J. Colquhoun and S. M. Pascal, Protein Sci., 2003, 12, 257–265.

16 (a) J. R. Silveira, G. J. Raymond, A. G. Hughson, R. E. Race,V. L. Sim, S. F. Hayes and B. Caughey, Nature, 2005, 437,257–261; (b) J. P. Taylor, J. Hardy and K. H. Fischbeck, Science,296, 1991–1995; (c) J. M. Kenney, D. Knight, M. J. Wise andF. Vollrath, Eur. J. Biochem., 2002, 269, 4159–4163.

17 J. H. Collier, B.-H. Hu, J. W. Ruberti, J. Zhang, P. Shum,D. H. Thompson and P. B. Messersmith, J. Am. Chem. Soc., 2001,123, 9463–9464.

18 (a) A. Aggeli, M. Bell, L. M. Carrick, C. W. G. Fishwick,R. Harding, P. J. Mawer, S. E. Radford, A. E. Strong andN. Boden, J. Am. Chem. Soc., 2003, 125, 9619–9628; (b) A. Aggeli,M. Bell, N. Boden, L. M. Carrick and A. E. Strong, Angew. Chem.,Int. Ed., 2003, 42, 5603–5606; (c) V. Kayser, D. A. Turton,A. Aggeli, A. Beevers, G. D. Reid and G. S. Beddard, J. Am.Chem. Soc., 2004, 126, 336–343.

19 M. R. Caplan, P. N. Moore, S. Zhang, R. D. Kamm andD. A. Lauffenburger, Biomacromolecules, 2000, 1, 627–631.

20 M. R. Caplan, E. M. Schwartzfarb, S. Zhang, R. D. Kamm andD. A. Lauffenburger, Biomaterials, 2002, 23, 219–227.

21 (a) Y. Hong, R. L. Legge, S. Zhang and P. Chen, Biomacro-molecules, 2003, 4, 1433–1442; (b) Y. Hong, M. D. Pritzker,R. L. Legge and P. Chen, Colloids Surf., B, 2005, 46, 152–161; (c)Y. Hong, L. S. Lau, R. L. Legge and P. Chen, J. Adhes., 2004, 80,913–931; (d) S. Zhang, T. Holmes, C. M. DiPersio, R. O. Hynes,X. Su and A. Rich, Biomaterials, 1995, 16, 1385–1393; (e)T. C. Holmes, S. de Lacalle, X. Su, G. Liu, A. Rich andS. Zhang, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 6728–6733; (f)D. A. Narmoneva, O. Oni, A. L. Sieminski, S. Zhang, J. P. Gertler,R. D. Kamm and R. T. Lee, Biomaterials, 2005, 26, 4837–4846; (g)M. E. Davis, J. P. M. Motion, D. A. Narmoneva, T. Takahashi,D. Hakuno, R. D. Kamm, S. Zhang and R. T. Lee, Circulation,2005, 111, 442–450; (h) R. G. Ellis-Behnke, Y.-X. Liangm, S.-W.You, D. K. C. Tay, S. Zhang, K.-F. So and G. E. Schneider, Proc.Natl. Acad. Sci. U. S. A., 2006, 103, 5054–5059; (i) T. C. Holmes,S. de Lacalle, X. Su, A. Rich and S. Zhang, Proc. Natl. Acad. Sci.U. S. A., 2000, 97, 6728–6733.

22 J. H. Collier and P. B. Messersmith, Bioconjugate Chem., 2003, 14,748–755.

834 | Soft Matter, 2006, 2, 822–835 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online

23 J. H. Collier and P. B. Messersmith, Adv. Mater., 2004, 16,907–910.

24 S. Winkler, D. Wilson and D. L. Kaplan, Biochemistry, 2000, 39,12739–12746.

25 S. Dos Santos, A. Chandravarkar, B. Mandal, R. Mimna,K. Murat, L. Saucede, P. Tella, G. Tuchscherer and M. Mutter,J. Am. Chem. Soc., 2005, 127, 11888–11889.

26 (a) J. P. Schneider, D. J. Pochan, B. Ozbas, K. Rajagopal,L. Pakstis and J. Kretsinger, J. Am. Chem. Soc., 2002, 124,15030–15037; (b) M. S. Lamm, K. Rajagopal, J. P. Schneider andD. J. Pochan, J. Am. Chem. Soc., 2005, 127, 16692–16700.

27 (a) S. Ramachandran, Y. Tseng and Y. B. Yu, Biomacromolecules,2005, 6, 1316–1321; (b) S. Ramachandran, P. Flynn, Y. Tseng andY. B. Yu, Chem. Mater., 2005, 17, 6583–6588.

28 T. Koga, M. Matsuoka and N. Higashi, J. Am. Chem. Soc., 2005,127, 17596–17597.

29 H. A. Behanna, J. J. J. M. Donners, A. C. Gordon and S. I. Stupp,J. Am. Chem. Soc., 2005, 127, 1193–1200.

30 C. M. Wilmot and J. M. Thornton, J. Mol. Biol., 1988, 203,221–232.

31 (a) J. P. Schneider, D. J. Pochan, B. Ozbas, K. Rajagopal,L. Pakstis and J. Kretsinger, J. Am. Chem. Soc., 2002, 124,15030–15037; (b) M. S. Lamm, K. Rajagopal, J. P. Schneider andD. J. Pochan, J. Am. Chem. Soc., 2005, 127, 16692–16700.

32 (a) B. Ozbas, J. Kretsinger, K. Rajagopal, J. P. Schneider andD. J. Pochan, Macromolecules, 2004, 37, 7331–7337; (b)J. K. Kretsinger, L. A. Haines, B. Ozbas, D. J. Pochan andJ. P. Schneider, Biomaterials, 2005, 26, 5177–5186; (c)K. Rajagopal, B. Ozbas, D. J. Pochan and J. P. Schneider, Eur.Biophys. J., 2006, 35, 162–169.

33 D. J. Pochan, J. P. Schneider, J. Kretsinger, B. Ozbas, K. Rajagopaland L. Haines, J. Am. Chem. Soc., 2003, 125, 11802–11803.

34 L. A. Haines, K. Rajagopal, B. Ozbas, D. A. Salick, D. J.Pochan and J. P. Schneider, J. Am. Chem. Soc., 2005, 127,17025–17029.

35 (a) S. Tsonchev, G. C. Schatz and M. A. Ratner, Nano Lett., 2003,3, 623–626; (b) S. Tsonchev, A. Troisi, G. C. Schatz andM. A. Ratner, Nano Lett., 2004, 4, 427–431; (c) S. E. Paramonov,H.-W. Jun and J. D. Hartgerink, J. Am. Chem. Soc., 2006, 128,7291–7298.

36 (a) J. D. Hartgerink, E. Beniash and S. I. Stupp, Proc. Natl. Acad.Sci. U. S. A., 2002, 99, 5133–5138; (b) E. Beniash, J. D. Hartgerink,H. Storrie, J. C. Stendahl and S. I. Stupp, Acta Biomater., 2005, 1,387–397; (c) J. D. Hartgerink, E. Beniash and S. I. Stupp, Science,2001, 294, 1684–1688; (d) E. D. Sone and S. I. Stupp, J. Am. Chem.Soc., 2004, 126, 12756–12757.

37 H.-W. Jun, V. Yuwono, S. E. Paramonov and J. D. Hartgerink,Adv. Mater., 2005, 17, 2612–2617.

38 J. C. Stendahl, M. S. Rao, M. O. Guler and S. I. Stupp, Adv. Funct.Mater., 2006, 16, 499–508.

39 (a) G. A. Silva, C. Czeisler, K. L. Niece, E. Bensiash,D. A. Harrington, J. A. Kessler and S. I. Stupp, Science, 2004,303, 1352–1355; (b) K. L. Niece, J. D. Hartgerink, J. J. J. M. Donnersand S. I. Stupp, J. Am. Chem. Soc., 2003, 125, 7146–7147.

40 S. R. Bull, M. O. Guler, R. E. Bras, T. J. Meade and S. I. Stupp,Nano Lett., 2005, 5, 1–4.

41 H. Matsui and B. Gologan, J. Phys. Chem. B, 2000, 104,3383–3386.

42 (a) M. Kogiso, Y. Okada, T. Hanada, K. Yase and T. Shimizu,Biochim. Biophys. Acta, 2000, 1475, 346–352; (b) M. Kogiso,Y. Okada, K. Yase and T. Shimizu, J. Colloid Interface Sci., 2004,273, 394–399.

43 K. J. C. van Bommel, C. van der Pol, I. Muizebelt, A. Friggeri,A. Heeres, A. Meetsma, B. L. Feringa and J. van Esch, Angew.Chem., Int. Ed., 2004, 43, 1663–1667.

44 (a) A. P. H. J. Schenning and E. W. Meijer, Chem. Commun., 2005,3245–3258; (b) R. L. E. Furlan, S. Otto and J. K. M. Sanders, Proc.Natl. Acad. Sci. U. S. A., 2002, 99, 4801–4804.

45 (a) M. Reches and E. Gazit, Science, 2003, 300, 625–627; (b)M. Reches and E. Gazit, Nano Lett., 2004, 4, 581–585; (c)M. Reches and E. Gazit, Isr. J. Chem., 2005, 45, 363–371.

46 V. Jayawarna, M. Ali, T. A. Jowitt, A. F. Miller, A. Saiani,J. E. Gough and R. V. Ulijn, Adv. Mater., 2006, 18, 611–614.

47 (a) Z. Yang and B. Xu, Chem. Commun., 2004, 21, 2424–2425; (b)Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam and B. Xu, Adv. Mater.,2004, 16, 1440–1444; (c) Z. Yang, G. Liang, L. Wang and B. Xu,J. Am. Chem. Soc., 2006, 128, 3038–3043.

48 Y. Zhang, Z. Yang, F. Yuan, H. Gu, P. Gao and B. Xu, J. Am.Chem. Soc., 2004, 126, 15028–15029.

49 Y. Zhang, H. Gu, Z. Yang and B. Xu, J. Am. Chem. Soc., 2003,125, 13680–13681.

50 Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam and B. Xu, Adv. Mater.,2004, 16, 1440–1444.

51 Z. Yang, G. Liang, L. Wang and B. Xu, J. Am. Chem. Soc., 2006,128, 3038–3043.

52 S. Toledano, R. J. Williams, V. Jayawarna and R. V. Ulijn, J. Am.Chem. Soc., 2006, 128, 1070–1071.

53 (a) D. W. Urry, C.-H. Luan, T. M. Parker, D. C. Gowda,K. U. Prasad, M. C. Reid and A. Safavy, J. Am. Chem. Soc., 1991,113, 4346–4348; (b) D. E. Meyer and A. Chilkoti, Biomacro-molecules, 2004, 5, 846–851.

54 (a) D. Raucher and A. Chilkoti, Cancer Res., 2001, 61, 7163–7170;(b) D. E. Meyer, G. A. Kong, M. W. Dewhirst, M. R. Zalutsky andA. Chilkoti, Cancer Res., 2001, 61, 1548–1554; (c) D. E. Meyer,B. C. Shin, G. A. Kong, M. W. Dewhirst and A. Chilkoti,J. Controlled Release, 2001, 74, 213–224; (d) D. Y. Furgeson,M. R. Dreher and A. Chilkoti, J. Controlled Release, 2006, 110,362–369; (e) M. R. Dreher, D. Raucher, N. Balu, O. M. Colvin,S. M. Ludeman and A. Chilkoti, J. Controlled Release, 2003, 91,31–43.

55 K. Trabbic-Carlson, L. A. Setton and A. Chilkoti,Biomacromolecules, 2003, 4, 572–580.

56 D. E. Meyer, K. Trabbic-Carlson and A. Chilkoti, Biotechnol.Prog., 2001, 17, 720–728.

57 N. Nath and A. Chilkoti, J. Am. Chem. Soc., 2001, 123, 8197–8202.58 (a) W. Frey, D. E. Meyer and A. Chilikoti, Langmuir, 2003, 19,

1641–1653; (b) N. Nath and A. Chilkoti, Anal. Chem., 2003, 75,709–715.

59 G. V. R. Rao, S. Balamurugan, D. E. Meyer, A. Chilkoti andG. P. Lopez, Langmuir, 2002, 18, 1819–1824.

60 M. R. Diehl, K. Zhang, H. J. Lee and D. A. Tirrell, Science, 2006,311, 1468–1471.

61 (a) P. D. Thornton, G. McConnell and R. V. Ulijn, Chem.Commun., 2005, 5913–5915; (b) M. P. Lutolf, G. P. Raeber,A. H. Zisch, N. Tirelli and J. A. Hubbell, Adv. Mater., 2003, 15,888–892; (c) J. Groll, J. Fiedler, E. Engelhard, T. Ameringer,S. Tugulu, H.-A. Klok, R. E. Brenner and M. Moller, J. Biomed.Mater. Res. A., 2005, 74a, 607–617; (d) T. J. Sanborn,P. B. Messersmith and A. E. Barron, Biomaterials, 2002, 23,2703–2710; (e) B. Jeong and A. Gutowska, Trends Biotechnol.,2002, 7, 305–311; (f) S. Kim and K. E. Healy, Biomacromolecules,2003, 4, 1214–1223; (g) K. N. Plunkett, K. L. Berkowski andJ. S. Moore, Biomacromolecules, 2005, 6, 632–637; (h) E. Smith,J. Bai, C. Oxenford, J. Yang, R. Somayaji and H. Uludag,J. Polym. Sci., Part A: Polym. Chem., 2003, 41, 3989–4000.

This journal is � The Royal Society of Chemistry 2006 Soft Matter, 2006, 2, 822–835 | 835

Publ

ishe

d on

25

Aug

ust 2

006.

Dow

nloa

ded

by U

nive

rsity

of

Cal

ifor

nia

- Sa

nta

Bar

bara

on

03/0

2/20

16 2

2:03

:39.

View Article Online