Peptide-Based Stimuli-Responsive Biomaterials

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REVIEW www.rsc.org/softmatter | Soft Matter

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 biomedical applications. For example, physical or chemical hydrogels loaded with drug molecules may release their Materials that change properties in response to local environ- payload, only when and where required, in response to mental stimuli are increasingly being studied in the context of 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 aSchool of Materials and Manchester Interdisciplinary Biocentre (MIB), Grosvenor Street, Manchester, UK M1 7HS. application is injectable gels for minimal invasive surgery. E-mail: [email protected]; Fax: +44 161 3068877; These materials may be applied through a syringe, and Tel: +44 161 3065986 undergo a solution-to-gel transition when triggered by b Department of Materials and Institute for Biomedical Engineering, temperature, pH, ionic strength, oxidative species or enzymes Imperial College of Science, Technology and Medicine, Prince Consort Road, London, UK SW7 2AZ. E-mail: [email protected]; at the site of injury to act as a scaffold for tissue regrowth. A Fax: +44 20 7594 6757; Tel: +44 20 7594 6804 third area is in bio-sensing, where small chemical or physical

Robert Mart received a Rachel Osborne read for a Masters degree from UMIST, Masters in Materials, before completing a PhD on Economics and Management asymmetric organic catalysis of at Oxford University before the Morita-Baylis–Hillman spending a year as a reaction with Dr D. J. Marketing Co-ordinator for Berrisford. He then spent a L’Occitane in New York.

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. year as a postdoctoral research Despite all the free lunches associate with Dr S. J. Webb in she wanted to pursue science the newly created University of and under the direction of Dr Manchester studying vesicle– M. M. Stevens she is currently vesicle interactions before join- undertaking a PhD looking at ing the Ulijn group where he the bio-functionalization of synthesises enzyme responsive gold nanoparticles at Imperial Robert Mart biomaterials. Rachel Osborne College, London.

Molly Stevens received her Rein Ulijn received his Masters PhD from The University of from Wageningen University, Nottingham and spent 2.5 years PhD from The University of as a postdoctoral researcher at Strathclyde and spent 2 years MIT. She is currently a reader as a postdoctoral researcher at at Imperial College London. the University of Edinburgh. She has recently been recog- He is currently an advanced nised by Technology Review’s research fellow and senior TR100 Young Innovators lecturer in biomedical Award (2004) and the Philip materials at the University of Leverhulme Prize for Manchester. His research is Engineering (2005) for her interdisciplinary and focuses research in regenerative medi- on the design, characterisation cine and nanotechnology. and application of responsive Molly Stevens Rein Ulijn molecular biomaterials.

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changes in the sensing environment trigger macroscopically amino acids), hydrophobic, p-stacking (aromatic amino acids), observable changes in material properties, thereby reporting hydrogen bonding (polar amino acids) as well as covalent them, for example by gelation or nanoparticle (dis)-assembly. (disulfide) bonds and steric contributions (strand directing These responsive biomaterials contain molecular building amino acids). While individually these interactions are quite blocks that undergo molecular level changes which result in weak (see Fig. 1), collectively they can give rise to very stable altered non-covalent interactions that, in turn, translate into structures. Crucially, each of these interactions depend in macroscopic responses. different ways on environmental conditions such as ionic In this Review Article, we focus on recent (since 2000) strength, pH and temperature. In addition, specific short reports on responsive biomaterials that use peptides as their peptide sequences can introduce responsiveness via small stimuli-responsive elements. Peptides are ideally suited for this molecule recognition. Enzyme responsiveness can be pro- purpose because of the range of distinct physical properties grammed into these materials by incorporation of peptide available from the naturally occurring amino acids (Fig. 1). sequences that are known substrates for proteases, kinases, or This diversity allows for rational incorporation of non- phosphatases.1n The dynamic nature of these interactions then covalent interactions including electrostatic (acidic and basic allows the molecular organisation to be altered in response to Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39.

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 dichroism shows that, as expected, the trans to cis photo- different requirements, for example hydrogen bonding requires isomerism of the azobenzene linker increases the a-helical precisely positioned and directed residues with the donor and content for the i, i + 4 and i, i + 7 peptides as the over-long acceptor approximately 2.8 A˚ apart. p–p stacking interactions cross-linking molecule is effectively shortened. Conversely, require the overlap of two p systems approximately 3.4 A˚ there is a decrease in the a-helical content for the i, i +11 apart. In contrast, electrostatic interactions are generally not peptide as the cross-linker becomes too short to permit ready directional and tend to be more flexible regarding the distance helix formation. between the participating charges, although this depends An important a-helix based quaternary structure of peptides strongly on the ionic strength of the solution. Hydrophobic is the coiled-coil. Characterised by two or more a-helices interactions are even less geometrically constrained. In nature, organised into a supercoil, each peptide length contains a 3, 4 responsive peptide based materials, for example, enzymes heptad motif repeat (abcdefg). The interhelical interactions and motor-proteins, use a combination of these individually are captured by pairwise interactions by four key positions; weak interactions, which work cooperatively to dynamically a, d, e, g (see top panel, Fig. 2). Hydrophobic residues found at organise the secondary, tertiary and quaternary structures of positions a and d form the hydrophobic core of a coiled-coil. proteins. Positions e and g are either side of the hydrophobic core and It is a major challenge for scientists and engineers to can participate in electrostatic interhelical contacts and also incorporate these design concepts into useful peptide based alter core hydrophobicity. Changing the nature of these materials and devices. The following sections examine contacts by introducing responsive amino acids can alter the strategies which involve the design of a peptide macro- stability of the conformation and provide a mechanism for monomer consisting of a primary sequence that is either control of dynamic materials. amphiphilic or forms a known secondary structural motif The use of acidic and basic amino acids that can be (a-helix, b-sheet, b-turn, elastin-like sequence), in which protonated or deprotonated by a change in pH allows dynamic responsive elements are rationally incorporated. control over the secondary structure of the peptides and can be Macroscopically observed transitions in response to external used to control the assembly of coiled-coils. For example, a stimuli are then achieved by further quaternary interactions coiled-coil a-helix with leucine at position d and glutamic between individual peptides. The resulting switchable assem- acid residues at positions e and g (Table 1, entry 2) forms blies may take the shape of nanometre sized fibres, spheres homodimeric coiled-coils which are destabilised in basic or tubes (consisting of superhelices, coiled-coils, amphiphilic solutions.3 This leucine zipper amino acid sequence was assemblies such as micelles). In other examples, peptide covalently bonded to a gold-substrate via the formation of a motifs are used as responsive elements in multi component gold–thiolate bond to form a monolayer. An extended version systems, to allow macroscopic transitions such as nanoparticle of the quartz crystal microbalance (QCM-D) in combination (dis-)assembly, switching of surface properties, and even with surface plasmon resonance was used to probe the control the action of bioactive proteins. A number of formation of the peptide functionalised surface and its peptide-based biomaterials where responsiveness was not a response to changes in pH. Characteristic shifts in dissipation, major design aspect have been excluded from the current D, consistent with the formation of a rigid layer at low pH review. We focus mainly on systems composed of relatively (pH 4.5) which increases in fluidity as the pH is increased

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. short oligopeptides, thereby excluding a number of studies on (pH 7.4 then pH 11.2) due to disruption of the coiled-coil responsive proteins. structure and unfolding of the alpha-helices, as monitored with the QCM-D. 2. Systems based on helices and coiled-coils The same acidic leucine-zipper like peptides were also used in a separate study by Stevens et al. to dynamically assemble a-Helices are a key secondary structure of peptides, charac- gold nanoparticles functionalised with the peptides (Table 1, terised by a single, spiral chain of amino acids stabilised by entry 3).4 At pH 11.5 when the coiled-coil structure is relatively hydrogen bonds. Typically, peptide chains that form a-helices unstable, the gold nanoparticles were dispersed, whereas at exhibit amino acids of similar character every three or four pH 4.5 the nanoparticles were aggregated and stabilised by residues. This spacing corresponds to the structural repeat of the specific biomolecular interactions between peptides on 3.6 residues per a-helical turn. Dynamic self-assembly of adjacent nanoparticles forming coiled-coils. The transition to helical structures has been achieved by rational incorporation the dispersed or aggregated state occurred between pH 8.5 and of stimuli-responsive amino acids within these structures.2–15 pH 7, and can be monitored by noting shifts in the UV–visible For example, a range of a-helical peptides (Table 1 , entry 1) spectra and CD spectra (top panel, Fig. 2). The system have been modified to undergo dynamic conformational also showed dynamic disassembly in response to changes in changes in response to light. Cysteine residues were incorpo- temperature due to the thermal unfolding of the a-helices. rated into a de novo hexadecapeptide to allow the bonding of A system (Table 1, entry 4) designed by Woolfson et al.5 an azobenzene based cross-linker to the peptide backbone.2a incorporated glutamic acid and lysine pairs at the e and g The extended trans isomer of the cross-linker was synthesised positions to stabilise the coiled-coil. These peptides formed to match an i, i + 11 substitution pattern and was tested nanosized fibres that unwound into random coils in response alongside peptide sequences with cysteine residues placed at to an increase in ionic strength as these charges were screened, the relative positions i, i +4,i, i + 7 and i, i + 11, resulting in destabilising the helix–helix interaction. By designing an amino their near-vertical alignment in the helical stack. Circular acid sequence that resembled both that of a leucine-zipper

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Table 1 Responsive systems based on helical and/or coiled-coil motifs

System Stimulus Response Ref. Applications 1 Photo-regulated azobenzene cross linked peptide: Light Disruption of a-Helix 2 Probing protein function EACAREAAAREAACRQ 2 Leucine zipper peptide. Includes the repeat: pH (4.5–11.2) a-Helix coiled-coil to 3 Switchable surfaces SGDLENEVAQLEREVRSLEDEAAEL- random Coil EQKVSRLKNEIEDLEAE 3 Peptide-functionalised nanoparticles. Leucine pH (7–8.5) a-Helix Coiled-coil to 4 Triggered nanoparticle zipper peptide includes the repeat: Temperature random coil (dis)-assembly for SGDLENEVAQLEREVRSLEDEAAELE- (Tm =90uC at pH 4.5) bio-sensing QKVSRLKNEIEDLKAE 4 Repeat peptides of: KIAALKQKIASLKQEID- Ionic strength (0.5 M KF) Coiled-coil to 5 Actin/myosin filament ALEYENDALEQ and KIRALKAKNAHL- random coil mimics KQEIAALEQEIAALEQ 5 Ac-YGCVAALETKIAALETKKAALETIA- Temperature (.60 uC) Coiled-coil sol to 6 Conformational switches ALC-NH2 b-hairpin gel 6 Ac-AALEKEIAALEQEIAALEKEIAALEY- pH (,6.5 and .7.5) a-Helix coiled-coil 7 Nanoparticle assembly ENAALEKEIAALEQE-NH2 to random Ac-CGGIAALKQKIAALKQKIAALKYK-OH H-IAALKQKNAALKQKIAALKYKGGC-NH2 7 ZiCo: YIHALHRKAFAKI Zinc(II) ions (1 eq Zn2+) Coiled-coil to b-hairpin 8 Metal ion sensing, protein ARLERHIRALEHAA folding models 8 KIAALKQKIASLKQEIDALEYENDALEQ- Ionic strength Coiled-coil to 10 Actin/myosin filament KIAALEQ (120 mM KCl) random coil mimics KIRRLKQKNARLKQKIAALEQEIAAL- EYEIAALEQ 9 EIAQLEYEISQLEQ pH Random coil to a-helix 11 Protein folding models, EIAQLEYEISQLEQEIQALES amyloid fibre models KIAQLKYKISQLKWKIQSLKQ 10 TZ1H pH (.6.5) Random coil to 12 Tissue regeneration Ac-EIAQHEKEIQAIEKKIA coiled-coil QHEYKIAQHKEKIQAIK-NH2 11 Cp3K–N meso-Tetrakis(4-sulfonato- Random coil to a-helix 13 Electron/excitation transfer Ac-IQQLKNQIKQLLKQ-NH2 phenyl)porphine 12 Par-4 (11–51) pH (5.5) Random coil to a-helix 14 Neurodegenerative and IGKLKEEIDKLNRDLDDMEDENEQLKQ- Temperature cancer apoptosis research ENKTLLKVVGKLTR (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 research Par-4 (11–51)E29Q 14 Par-4 (11–51)D24K Temperature (.65 uC) a-Helix to random coil 15 Apoptosis research Par-4 (11–51)E29K Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. coiled-coil and that of a b-hairpin (Table 1, entry 5), a system and heterodimerisation of coils bearing all lysine and all was created that not only changed conformation from a-helix aspartic acid residues at the e and g positions.11 Histidine has to b-hairpin when heated, but also consequently formed a gel.6 successfully been incorporated at the d position of a helix in Long and short charge complimentary coiled-coils were order to facilitate triggered self-assembly of a trimeric coiled- applied to control the aggregation of gold nanoparticles coil bundle when the histidine is uncharged above pH 6.5.12 (Table 1, entry 6) and adhesion of particles to functionalised The induction of helicity by a small molecule was achieved gold surfaces.7 These systems were extremely sensitive to the by the substitution of lysine residues variously into the b, e pH of the solution. In another case, a non-canonical coiled-coil and f positions in combination with a arylporphorin sequence (a 3-4-4-3-4-3-4 pattern of hydrophilic residues rather molecule bearing free sulfonate groups. Electrostatic interac- than 3-4-3-4-3-4-3 heptad repeats) inspired by zinc chelating tions between the sulfonate and the lysine residues biased the proteins and loaded with metal binding histidine residues conformation of the random coil peptide chain, resulting in (Table 1, entry 7) was synthesised. This peptide was then helix formation. Pascal et al. have altered a naturally occurring shown to convert from a coiled-coil to a b-hairpin conforma- protein, Par-4 (Table 1, entries 12, 13 and 14), with a tendency tion on the addition of zinc salts and back when a stronger to form coiled-coils by modifying the residues in the e and g chelating agent was added.8 Despite their use in the formation positions, resulting in ionic strength and pH dependant coil of a variety of fibre morphologies,9 first generation structures assembly.14,15 proved very sensitive to the presence of salts. Later iterations In summary, the design rules for responsive a-helices and (Table 2, entry 8 and Fig. 2, lower panel) have recently been coiled-coils are well understood and a number of recent shown to be more tolerant of the ionic strength of the parent examples show that these systems can be used either on their solution.10 Closely related, shorter peptide sequences were own or immobilised onto (nanoparticle) surfaces as responsive investigated by Dong and Hartgerink, who showed pH biomaterials which provide insight into protein folding and responsive coiled-coil assembly, a-helix to b-sheet conversion may have applications in bio-sensing.

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Fig. 2 Examples of peptide based responsive biomaterials derived from a-helix coiled-coil motifs.

3. Systems based on beta sheets calcium ions encapsulated in carefully prepared vesicles, the vesicles become leaky, allowing calcium ion escape and

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. The secondary structural features known as b-sheets are gelation. This release may also be triggered by heating the another key architectural component in naturally occurring vesicle membranes to their phase transition temperature, and peptides. They are formed by adjacent parallel or anti-parallel so is highly tunable. A series of undecapeptides (Table 2, peptide strands hydrogen bonding together to form a weakly entry 2) have been tailored to react to acidic or basic curved sheet. Much recent research on b-sheets has focused on conditions by the incorporation of ornithine or glutamine their participation in disease states including: Alzheimer’s residues at key positions.18 Whilst the phase transitions that disease, Parkinson’s disease and transmissible spongiform resulted were more complex than anticipated, they were fully 16 encephalopathies, as well as the formation of spider silk. reversible until the ionic strength of the solution was com- These systems are most frequently static structures and whilst promised by the repeated addition of aqueous acids and bases. measures to inhibit their formation have been well studied, Similarly the biomimetic (from Zutoin) KFE12 sequence relatively few truly responsive systems have been reported. The (Table 2, entry 3) which gelled at physiological pH as well as sidechains of alternate residues in the primary sequence are under the influence of salts19 was modified by the incorpora- positioned on opposite sides in a b-sheet, allowing ready facial tion of glutamine, resulting in the synthesis of KFQ12 (Table 2, discrimination and construction of higher order structures. entry 4). This peptide only forms a gel under neutral For example, a primary sequence may consist of alternating conditions upon the addition of salts.20 A number of later cationic, hydrophobic and anionic amino acid residues in a systems based on the so-called EAK16 peptides (Table 2, format e.g. arginine-alanine-aspartic acid (RAD), phenyl- entry 5), examined by Hong et al. not only display sensitivity alanine-glutamic acid-lysine (FEK) or glutamic acid-alanine- to pH21a and ionic strength21b but also display different lysine (EAK) as pioneered and reviewed by Zhang.1e morphologies, ranging from globules to fibrils, under different A development of the FEK sequence uses the presence of conditions.21c The matrices formed by EAK16-II (Table 2, calcium ions to trigger a structural rearrangement, but supplies entry 5) have previously been shown to support mammalian these ions by disrupting vesicles.17 When near infrared light is cell attachment,21d,21e underlining the potential of this type of shone on a mixture of FEK16 peptides (Table 2, entry 1) and material for regenerative medicine and 3D cell culture.21f–i

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Table 2 Responsive systems based on b-sheet structures System Stimulus Response Ref. Applications

1 FEK16 FEFEFKFKFEFEFKFK Ca2+ ions via temperature or light Soluble to aggregate 17 Drug delivery, wound healing, stimulated vesicle rupture tissue engineering 2P11–4 Ac-QQRFEWEFEQQ-NH2 pH .7.0 Nematic to isotropic 18 Hydrogels, organogels, liquid P11–5 Ac-QQXFXWXFQQQ-NH2 pH , 7.5 crystals (Where X denotes Ornithine) 3 KFE12 FKFEFKFEFKFE Ionic strength (1 mM NaCl) Sol-to-gel 19 Drug delivery, wound healing, pH (5–10) tissue engineering 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, EAK16-II AEAEAKAKAEAEAKAK (Li+,Na+,K+) nerve cell regrowth EAK16-IV AEAEAEAEAKAKAKAK pH RAD16-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 engineering Average n = 86 8 SGRGYBLGGQGAGAAA Enzymatic Soluble to aggregate 24 Silk assembly studies AAGGAGQGGYGGLGSQG 9 Switch peptides, from non-linear to Enzymatic Sol-to-gel 25 Triggered gel formation, linear prodrug design, biosensors 10 6-u-8 (FITC)-KLDLKL-SGRSANA- Enzymatic Gel-to-sol 26 Drug delivery DLKLDLKL 11 EVW10 Ac-EWEXEXEXEX-NH2 pH, ionic strength, mixing of Sol-to-gel 27 3D Gel for protein entrapment KVW10 Ac-WKXKXKXKXK-NH2 charge-complementary peptides KVW15 Ac-KWKVKVKVKVKV- KVK-NH2 Where X = V,A,S,P 12 L4K8L4 (all L or all D) pH . 9 Random coil to 28 Amyloid model system b-sheet 2+ 13 DDDAAAVVV-NH(CH2)14CH3 pH or Ca Ions Random coil to 29 Advanced medicine, cell culture e KKKVVVVVV D-NH(CH2)14CH3 b-sheet e DDDAAAVVV D-NH(CH2)14CH3

Peptides developed by Collier and Messersmith allow two- ‘‘switch’’ residue is blocked by an enzyme cleavable group, stage assembly of a functionalised fibrillar network. The Q11 whose removal triggers an O– or S– to N-acyl shift, thus peptide (Table 2, entry 6) self-assembles very slowly in pure restoring the b-sheet forming sequence and triggering self-

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. water, but the process is greatly accelerated by the presence of assembly. A sophisticated drug delivery system consisting of a salts or the alteration of the solution pH.22 The resultant b-sheet-based matrix host incorporating a b-sheet containing structure contains several pendant glutamine residues that therapeutic sequence guest has recently been reported.26 The may be cross-linked with lysine containing peptides in the matrix is constructed from an enzyme cleavable region presence of TGase and calcium ions (lower panel, Fig. 3) sensitive to urokinase plasminogen activator (UPa), an enzyme to functionalise the self-assembled matrix. Another system linked to cancer states, flanked by two b-sheet forming based on Q11 developed by the same group incorporates a domains (Table 2, entry 11). This matrix is mixed with a polyethylenegycol chain at the C-terminus (Table 2, entry 7), peptide containing a further b-sheet forming domain, a and results in longer, thinner, straighter fibres with a regular mitochondrial disruption domain and a cell penetration helical twist.23 An entirely different approach to triggered domain to create a targeted drug delivery system. b-sheet formation is the transformation of a non-aggregating As an alternative to using ‘self-complementary’ peptides, substrate by enzyme action to a product that is capable of self- systems have been studied that consist of two separate assembly. This approach has been demonstrated in two cases. populations of cationic and anionic peptides, that are mutually The first approach utilises a phosphorylated serine embedded attractive but self repulsive. For example, Yu et al.27 studied a within a sequence (Table 2, entry 9) derived from arachnid combination of peptides KVW10 and EVW10 that showed dragline silk.24 Peptides were successfully phosphorylated and very rapid (seconds) and repeatable sol-to-gel transitions. dephosphorylated and consequent structural changes were Substitution of valine with alanine or serine resulted in observed, although the influence of altering a single residue formation of weaker gels, while substitution with proline was limited. In contrast, the second case provides absolute prevented gel formation. It was demonstrated that the cationic control through a single switching residue. An N-terminal and anionic peptides on their own could also form b-sheets,

section of the b-sheet forming sequence (Table 2, entry 8) is either by charge neutralisation through exposure to CH3– removed and appended to the side chain of a newly N-terminal COOH or NH4OH vapour or screening of charges in high (M) threonine, serine or cysteine residue.25 The a-nitrogen of this salt concentrations. The two-component gel system was found

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Fig. 3 Fibrillar networks based on b-sheet structures formed from peptide macro-monomers.

to allow entrapment of proteins in their native form. Higashi b-sheet based biomaterials include a number of examples of

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. et al. demonstrated pH responsive twisted nanofibres with 3D cell culture and (enzyme triggered) drug delivery. opposite handedness from D or L tri-block peptides consisting of an octalysine sequence flanked with two hydrophobic tetra 4. Systems based on beta hairpins leucines (Table 2, entry 12). When a racemic mixture of both peptides was used, globular aggregates were observed.28 Other A further important secondary structure available to peptides peptide amphiphiles (Table 2, entry 13) assemble into b-sheets is the b-hairpin, which occurs when an amino acid sequence from random coils when the pH is altered to neutralise charged contains a pair of turn inducing residues such as proline lysine or aspartic acid residues or when calcium ions are followed by glycine or threonine.30 The cyclic structure of added to shield the charges on the monomeric peptides.29 proline (denoted i + 1, see Fig. 4) causes a slight kink in the Amphiphilic peptide systems are discussed in more detail in a-carbon backbone and in combination with the more section 5. conformationally flexible glycine or threonine residues (i +2) In summary, the design rules for responsive peptide that follow allows a complete reversal of the direction of the materials based on b-sheets are well understood and usually a-carbon backbone. The loop is cemented by the two residues consist of peptide chains with alternating hydrophilic and before and after the turn-inducing pair (i and i + 3) and hydrophobic amino acids. These peptides can be used either on subsequent residues to hydrogen bond together, holding the their own (self complementary) or in pairs of opposite charge. peptide strand into a tight hairpin. The MAX1 (Table 3, They fold into (twisted) sheets that may further assemble into entry 1) peptide consists of alternating polar lysine and apolar super-helices. Responsiveness of these systems can be tuned by valine residues either side of the turn-inducing residues, rational incorporation of acidic and basic amino acids, while resulting in an amphiphilic hairpin structure able to self- the stability towards temperature can be controlled by tuning assemble and form a hydrogel. The lysine residues are hydrophobicity of the uncharged residues. Primary sequences protonated at physiological pH and the resulting charge– have been modified with bioactive peptide regions, to render charge repulsion inhibits b-hairpin and hydrogel formation. the system responsive to enzymes. Applications of responsive Gelation may be triggered either by increasing the pH to 9.0 to

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Fig. 4 Self-supporting hydrogels formed from aggregates of b-hairpin molecules.

Table 3 Responsive systems based on b-hairpin formation System Stimulus Response Ref. Applications

D 1 MAX1 VKVKVKVKV PPTKVKVKVKV-NH2 pH (9) Sol-to-gel 31 Biomedical and tissue engineering D 2 MAX1 VKVKVKVKV PPTKVKVKVKV-NH2 Ionic strength Sol-to-gel 32 Tissue engineering/regeneration D MAX2 VKVKVKVKV PPTKVKTKVKV-NH2 (150 mM NaCl) D MAX4 KVKVKVKVK PPSVKVKVKVK-NH2 D MAX5 VKVKVKVKV PPSKVKVKVKV-NH2 D 3 MAX1 VKVKVKVKV PPTKVKVKVKV-NH2 Heat (y25 uC) Sol-to-gel 33 Stimuli responsive materials D MAX2 VKVKVKVKV PPTKVKTKVKV-NH2 Heat (y40 uC) D MAX3 VKVKVKTKV PPTKVKTKVKV-NH2 Heat (y60 uC) D 4 MAX7 VKVKVKVKV PPTKVKXKVKV-NH2 Light Sol-to-gel 34 Tissue engineering regeneration, (X = Cys or Cys(a-carboxy-2-nitrobenzyl) drug delivery

neutralise the lysine residues,31 or at pH 7 by increasing the forming b-sheets.29 Studies using a peptide amphiphile ionic strength of the solution to 150 mM to screen their whose polar section includes several cysteine residues and a charges.32 Self-assembly of MAX1 and the closely related well known cell binding epitope (Table 4, entry 1) show that

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. MAX2 and MAX3 sequences may also be controlled by when an aqueous solution of the peptide amphiphile is changing the temperature, hairpin formation and assembly acidified, self-assembly occurs; a process which is reversed at being triggered by elevated temperatures (Table 3, entries 2 neutral or basic pH.36 Once the fibres are self-assembled, the and 3).33 A modified version of MAX1 resulted in photo- inclusion of cysteine residues allows them to be reversibly sensitive hydrogelation (Table 3, entry 4). Here a cysteine polymerised by oxidative cross-linking (top panel, Fig. 5) to residue was incorporated in place of a valine and decorated enhance their stability and diminish their pH sensitivity. with a charged, photo-sensitive molecule. With the charged Further studies have shown the self-assembly of supramole- a-carboxy-2-nitrobenzyl molecule in place, interaction between cular nanofibres can be initiated by electrolyte solutions or hydrophobic faces is disrupted and self-assembly inhibited. changes in pH, and these reactions determine the bulk Photo-cleavage of the polar side chain removed this inhibition, properties of the macroscopic gel that is formed. triggering self-assembly.34 Recently, Hartgerink et al. demonstrated a peptide amphiphile molecule (Table 4, entry 2) containing a cell binding 5. Systems based on amphiphiles epitope and a matrix metalloprotease cleavable sequence, which mimics the ability of the extracellular matrix to degrade Peptide amphiphiles consisting of a polar peptide region and by the action of cell-mediated enzymes. Gel formation was an apolar aliphatic tail constitute a versatile class of molecules triggered by the addition of calcium ions, then the gel was which undergo dynamic self-assembly to form a variety of dissolved by type IV collagenase.37 Using the PA-1 peptide peptide nanofibres. The wedge-shaped monomers align with (Table 4, entry 3), Stupp and co-workers demonstrated peptide the narrower hydrophobic tails inwards and the bulkier polar amphiphile self-assembly triggered by screening of charged region outwards to form fibres.35 The surface of the fibres aspartate sidechains by di- and tri-valent metal ions or displays the peptide sequence, making these materials good neutralisation by pH adjustment.38 A molecule in this series candidates for the construction of responsive biomaterials. bearing a laminin epitope (Table 4, entry 4) was gelled by These surface peptides may be further stabilised laterally by electrolyte action on mixing with murine neural progenitor

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Table 4 Responsive systems derived from amphiphilic monomers System Stimulus Response Ref Application

1 12 Peptides, e.g. PA-4: CH3(CH2)14CO- pH (Various acidic) Sol-to-nanofibres 36 Cell culture, regenerative medicine, CCCCGGGS(PO4)RGD Di- and Tri-valent metal biomineralisation ions (20 mM) 2+ 2 (CH3(CH2)14CO- GTAGLIGQRGDS Ionic stength (0.1 M Ca ) Sol-to-gel 37 Cell culture, regenerative medicine Type IV collagenase Gel-to-sol 3 PA-1: CH3(CH2)14CO-AAAAGGGS- pH (,9) Sol-to-gel 38 Regenerative medicine 2+/3+ (PO4)KGE Ionic strength (.30 mM M ) 4CH3(CH2)14CO- AAAAGGGIKVAV Cell culture media (DMEM) Sol-to-gel 39 Cell culture, neuroregenerative medicine 5 KK(DOTA-e-K-e-K) LLCCCK- pH (.7) Sol-to-gel 40 Magnetic resonance imaging, (CO(CH2)14CH3) metabolic studies KK(DOTA-e-KGRGDS) LLLAAA- (CO(CH2)14CH3) 6 GG-(CO(CH7)nCO)-GG pH (8) Tubes to ribbons 41 Drug delivery 7 VV-(CO(CH2)nCO)-VV Divalent metal ions (10 mM) Sol-to colloid 42 Nanomaterials research Sol-to-gel 8 Cyclohexane-(FG)3 pH (.5) Sol-to-gel 43 Drug delivery 9 Cyclohexane-(MH)3 pH (.6) Sol-to-gel 43 Drug delivery

cells in physiological fluids.39 Neural progenitor cells encap- interactions, the attractive interactions between p-electrons in sulated by the gelation process survived and were induced to aromatic rings, in addition to hydrogen bonds and ionic rapidly differentiate into neurons. Further molecules synthe- interactions. The use of p-stacking as a driving force for self- sised by the same group (Table 4, entry 5) incorporate groups assembly has been practised for decades in supramolecular 40 44 which strongly chelate gadolinium(III) ions. The contrast chemistry, generally in organic solvent systems. This enhancing spin properties of the gadolinium species allows the technique was recently rediscovered for use in aqueous decay products of implanted gels to be easily traced in three solutions. A first example of self-assembly of short peptides dimensions by magnetic resonance imaging. A variation of through p-stacking has been described by Reches and Gazit, this type of dynamic self-assembly is exhibited by bola- who observed that aromatic dipeptides (Table 5, entry 1) could amphiphiles; molecules in which two or more hydrophilic self-assemble into straight nanotubes,45a hollow spherical groups are connected by hydrophobic functionalities. One structures45b and amyloid-like fibres45c upon dilution from a such molecule (Table 4, entry 6) has been shown to adopt fluorinated organic solvent. To rule out electrostatic interac- different structures under differing pH conditions, forming tions between terminal carboxylic acids and amines as the crystalline tubules at low pH and helical ribbons at higher driving force for nano-tube formation in diphenylalanine a values (lower panel, Fig. 5).41 Once formed, these structures number of N– and/or C– terminal capped analogues may be directly interconverted. The different structures are were tested. The formation of tubular structures was still proposed to be determined by the strength of acid–acid and observed, demonstrating that the self-assembly process

Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39. acid–acetate pairs, depending on the pH, and the curvature must instead be explained in terms of p–p interactions. they allow the polymeric tape to adopt. Kogiso et al. describe Xu and co-workers reported that certain fluorenylmethoxy- the gelation of divaline (Table 4, entry 7) bola-amphiphiles by carbonyl (Fmoc) -protected amino acids and dipeptides a selection of divalent metal ions, to form colloidal suspensions spontaneously formed fibrous scaffolds upon application of and hydrogels depending on the pH.42 A natural extension of a pH switch. Ulijn et al.46 and Xu et al.47 collectively studied a this structure leads to three-armed amphiphiles such as small library of Fmoc–dipeptides made up of combinations of

those investigated by van Bommel et al. These C3 symmetric the amino acids serine, threonine, glycine, alanine, leucine, scaffolds are based on cyclohexane rings modified by three phenylalanine encompassing a range of hydrophobicities identical, pendant pairs of amino acids to create molecules (Table 5, entries 2 and 3). The pH value at which gelation that can form hydrogels in response to either acidic (Table 4, took place varied with the amino acid sequence and no gel entry 8) or basic (Table 4, entry 9) conditions.43 formation was observed by Fmoc-glycine-phenylalanine and The responsiveness of amphiphile based peptide materials Fmoc-glycine-threonine peptides under any of the conditions is mainly driven by the hydrophobic effect and therefore tested. Three peptide gels that were stable at neutral pH were allows great flexibility in the peptide structures that are used. found to support 3D cell culture of chondrocytes for periods of 46 Hence, these systems are ideally suited for displaying bioactive up to three weeks. In addition to being temperature and pH peptides for bio-recognition and enzyme responsiveness in gel responsive, gel-to-sol transition upon binding to a small scaffolds. molecule ligand, vancomycin, was demonstrated. The molecular recognition event dramatically increased the elasticity of 48 6. Systems based on aromatic interactions these gels. Whilst the L,L diastereoisomers of Fmoc– dialanine and pyrenyl–dialanine were found to be unrespon- A number of very short peptide motifs containing aromatic sive, the D,D diastereoisomers formed a gel.49 Fluorescence groups have been found to self-assemble in aqueous condi- spectroscopy has been used to provide evidence for p–p tions. These systems are believed to be stabilised, through p–p interactions within the gels. In Fmoc and pyrenyl systems, the

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Fig. 5 Responsive systems based on self-assembled peptide amphiphiles.

emissions from monomeric residues were visible and in a to be super-helical in nature. FT-IR spectroscopy analysis of number of cases red shifted emissions were assigned to dimeric dried samples of three different diphenylalanine urethane species (excimers). Circular dichroism spectra were used to derivatives—butoxycarbonyl (Boc), carboxybenzyl (CBz) and further unravel the molecular arrangements, generally thought Fmoc (Table 5, entry 1)—suggested significantly different

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Table 5 Responsive systems derived from amphiphilic monomers bearing aromatic groups System Stimulus Response Ref. Application

1 FF Proteinase K Disassembly of 45 Nanowire templating, nanotubes micro/nano electronics 2 Fmoc-GG,AA,FG,GF,FF,FF/Ka, FF/GGa pH 4-8 Sol-to-gel 46 3D cell culture No gel formed for Fmoc-GF 3 Fmoc-AA (L,L and D,D) , GG, GA, GS pH 3–5 Sol-to-gel 47 Sensing No gel formation for Fmoc-GT 4 Fmoc-AA, pyrenyl-AA (L,L and D,D); Presence of ancomycin Gel-to-sol 49 Sensing 5 Naphthalene-FFGEY i. Phosphatase i. Sol-to-gel 51 in vivo Gelation, ii. Kinase ii. Gel-to-sol regenerative medicine 6 Fmoc-XFF and Fmoc-LLL where Thermolysin (gelation by Suspension-to-gel 52 3D Cell culture X = F, A, V, L . No gel formation reverse hydrolysis) observed when X = G or P a 50:50 (mol/mol) ratios were used.

structures depending on the N-protecting group, ranging entry 1). Enzymatic sol-to-gel transitions were demonstrated from a-helical for Boc-diphenylalanine to b-sheets for CBz- by Xu and co-workers in (de)phosphorylation of Fmoc– diphenylalanine.45c A more complete picture of the interac- tyrosine47b,50 systems and more recently on napthyl–pentapep- tions in these short peptide gels is likely to emerge in the future tide (See Fig. 6).51 In this case, a pair of enzymes with as research in this area continues. complementary and opposite activities were used assembly and Enzyme-responsive assembly or disassembly was demon- disassembly. Tyrosine residues were de-phosphorylated by a strated by Reches and Gazit, who used proteinase K to trigger phosphatase to induce gelation and a kinase was used to the hydrolysis of diphenylalanine nanotubes45a (Table 5, reverse the process (Table 5, entry 5). Electrostatic repulsion of Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39.

Fig. 6 Enzyme responsive materials featuring aromatic interactions.

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negatively charged phosphate groups prevented gelation of the temperature phase transition, becoming less soluble and precursors, while a combination of p-stacking interactions forming aggregates as their temperature is elevated above a between phenyl and napthyl groups and hydrogen bonds transition point. This point is dependant on the guest residues (b-sheets) triggers hydrogel formation. Since the enzyme and the number of repeats in the primary sequence and may be reactions proceed under thermodynamic control, it is thought tailored to the desired application.53 that this method results in fewer defects in the resulting self- The transition temperature of ELPs has been exploited by assembled structures. Indeed, more uniform nanotubular Chilkoti and co-workers in the developments of drug carriers structures were obtained when comparing the enzymatically for hyperthermic cancer treatments. In these regimes, local obtained dephosphorylated peptide gel to that of the gel heating (y42 uC) is applied to a cancerous tissue. By designing triggered by pH switching.51 ELP systems with a transition temperature around 39 uC the A recent contribution from our laboratory demonstrated carrier is more soluble in general circulation (37 uC) and less so the use of a protease in reverse, to catalyse peptide synthesis at the site of the tumour. Enhanced uptake of a fluorescently- (condensation) instead of hydrolysis, to produce amphiphilic tagged thermally responsive polypeptide by tumour cells has Fmoc–peptide hydrogelators that spontaneously form been demonstrated both in vitro54a and in vivo.54b,c Further nano-fibrous gel structures (Table 5, entry 6).52 We have work resulted in doxorubicin-ELP conjugates with a range of demonstrated that the thermodynamic stabilisation of transition temperatures, which demonstrated equivalent cyto- Fmoc–peptides upon self-assembly, relative to non-assembling toxicity to that of free doxorubicin, but were found to localise Fmoc–amino acid and dipeptide pre-cursors, provides a differently in tumour cells.54d,e Thermoresponsive gel swel- sufficient driving force to trigger formation of supramolecular ling,55 the thermally triggered aggregation of protein–ELP hydrogels. conjugates for expressed protein purification,56 and the In summary, systems in which aromatic interactions play aggregation of ELP-modified gold nanoparticles57 have also key roles have been studied in the last few years and include been reported by the same group, as have ELP modified those that respond to pH, enzymes, and small molecules. In surfaces for the binding of ELP-tagged proteins58 and silica– these systems, much shorter peptide sequences have been used ELP hybrids resulting in temperature dependant permeable 59 compared to those in any of the other categories. membranes. Recently elastin-like polypeptides were com- bined with leucine-zipper type helices which were entwined 7. Elastin-like polymers (ELPs) with monomeric kinesin-1 units to yield fused biomotor- protein assemblies (Fig. 7). Using repeated helical segments, A final category of peptide-derived responsive materials is multiple, cooperative motive units were incorporated into the based on a template derived from the naturally occurring same molecule. When these multivalent motor arrays were protein elastin. These structures consist of pentad repeats used to coat a cover slip increased microtubule gliding where four of the constituent amino acids, the first, second, velocities relative to monovalent systems were observed.60 third and fifth are conserved, and the fourth is variable. The Elastin-like polypeptides are designed using a well under- conserved residues are valine, proline, glycine and glycine stood template and through their predictable temperature and the ‘‘guest’’ residue may be any amino acid except responsive behaviour have found utility in drug delivery, proline. Polypeptides based on this template display inverse protein sensing and purification and biomotor alignment. Published on 25 August 2006. Downloaded by University of California - Santa Barbara 03/02/2016 22:03:39.

Fig. 7 Elastin-like polypeptides and coiled-coils create a backbone to form multivalent kinesin macromolecules.

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