Self-assembly and applications of biomimetic and

REVIEW

www.rsc.org/softmatter | Soft Matter

Self-assembly and applications of biomimetic and bioactive peptideamphiphiles Efrosini Kokkoli,*a Anastasia Mardilovich,a Alison Wedekind,a Emilie L. Rexeisen,a Ashish Garga and Jennifer A. Craigb Received 29th June 2006, Accepted 16th August 2006 First published as an Advance Article on the web 18th October 2006 DOI: 10.1039/b608929a Peptide-amphiphiles are amphiphilic structures with a hydrophilic peptide headgroup that incorporates a bioactive sequence and has the potential to form distinct structures, and a hydrophobic tail that serves to align the headgroup, drive self-assembly, and induce secondary and tertiary conformations. In this paper we review the different self-assembled structures of peptide-amphiphiles that range from micelles and nanofibers, to patterned membranes. We also describe several examples where peptide-amphiphiles have found applications as soft bioactive materials for model studies of bioadhesion and characterization of different cellular phenomena, as well as scaffolds for tissue engineering, regenerative medicine, and targeted drug delivery.

1. Introduction Recently, much attention has been given to developing biomimetic systems that promote desirable interactions with cells and interact selectively with a specific cell type through biomolecular recognition events. In particular, extracellular matrix (ECM) proteins, which are known to have a capacity to regulate cell behavior, such as adhesion, spreading, growth and migration, have been used widely to enhance cell–material interactions.1–6 However, the effects observed for a given protein vary significantly depending on the properties and a

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA. E-mail: [email protected] b Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA

Efrosini Kokkoli received a Diploma in Chemical Engineering from the Aristotle University of Thessaloniki, Greece in 1992. In 1994 she received her MS from the University of Illinois at Urbana-Champaign under the supervision of Frank van Swol, and in 1998 she earned her PhD in Chemical Engineering from the University of Illinois at Urbana-Champaign under the direction of Charles F. Zukoski. She completed Efrosini Kokkoli her postdoctoral work with Matthew Tirrell in the Department of Chemical Engineering and Materials Science at the University of Minnesota, and the Department of Chemical Engineering at the University of California, Santa Barbara. She is a McKnight Land-Grant Assistant Professor in the Department of Chemical Engineering and Materials Science, and a Graduate Faculty of the This journal is ß The Royal Society of Chemistry 2006

nature of the underlying substrate and the method of protein immobilization.7–9 To avoid complications associated with intact proteins, such as immunologic response, undesired activity, and thermal and chemical instability, short amino acid sequences that mimic the adhesion domains of ECM proteins can be employed for surface modification.10–14 Using these minimal peptide sequences, specific cellular interactions can be targeted while eliminating the possibility of delivering undesired activity that intact proteins may promote. Significant progress has been made in incorporating adhesion peptides into practical biomaterials that might be used as implants, and different approaches including incorporation into biodegradable copolymers15–17 and polyethylene glycol hydrogels,18,19 grafting onto solid surfaces,20,21 cross-linking to natural polymers,22 and covalently attaching to self-assembled monolayers,23–26 have been used to fix peptides on surfaces.

Department of Biomedical Engineering at the University of Minnesota. Current research interests include biomimetic peptide-amphiphiles and biopolymers for bionanotechnology, targeted drug delivery, and regenerative medicine. Anastasia Mardilovich (born in Minsk, Belarus in 1978) is a PhD candidate in the Department of Chemical Engineering and Materials Science at the University of Anastasia Mardilovich Minnesota. She completed her BS degree at the Belarus State Technological University in 2000, and started her graduate studies under the supervision of Prof. Efrosini Kokkoli in 2001. Her research interests include selfassembling colloidal systems, nanostructured biomimetic materials and their interfacial properties, and characterization of biomolecular interactions using atomic force microscopy. Soft Matter, 2006, 2, 1015–1024 | 1015

Fig. 1

Building blocks of peptide-amphiphiles. Adapted from Kunitake.37

Furthermore, it has been demonstrated that linking peptides to synthetic hydrophobic tails (peptide-amphiphiles) enables them to self-assemble into biomimetic films with highly organized interfaces, and enhances their ability to attain welldefined secondary and tertiary conformations, that effectively promote cell adhesion, spreading, migration, growth and differentiation.27–35 The term ‘‘peptide-amphiphile’’ can be used to describe amphiphilic peptides consisting only of amino acids, hydrophilic peptides linked to hydrophobic alkyl chains or lipids, and peptide based copolymers.36 In this review we will discuss the various self-assembled structures, and applications of peptide-amphiphiles in biomaterials consisting of a hydrophilic peptide headgroup and an alkyl tail.

2. Self-assembled structures of peptide-amphiphiles One of the first designs of synthetic peptide-amphiphiles was proposed by Kunitake37 and consisted of a hydrophobic tail, a linker, a spacer, and a hydrophilic head (Fig. 1). By changing any of the structural segments of an amphiphilic molecule, one can control the morphology, characteristics, surface chemistry, and function of the molecule.38 Amphiphiles can self-assemble into a variety of different structures such as micelles, vesicles, monolayers, bilayers, nanofibers, nanotapes, ribbons, and twisted ribbons, to minimize unfavorable interactions with their surroundings.39 The self-assembly of the hydrophobic tails of the peptideamphiphiles has been shown to induce and/or stabilize the

Alison Wedekind received her BS in Chemical Engineering from the University of Delaware in 2005. She is currently a graduate student in the Department of Chemical Engineering and Materials Science at the University of Minnesota. Her current research, under the guidance of Prof. Efrosini Kokkoli, focuses on the targeted drug delivery of stealth liposomes for the treatment of cancer. Alison Wedekind

Ashish Garg

Emilie L. Rexeisen received her Bachelors in Chemical

Ashish Garg received his Bachelors and Masters degrees in Materials Science from the Indian Institute of Technology Bombay in 2003. He has been a graduate student in Prof. Kokkoli’s group in the Department of Chemical Engineering and Materials Science at the University of Minnesota since 2003. His research interests include functionalizing liposomes with peptides and polymers for targeted drug delivery with applications to cancer tissues.

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Emilie L. Rexeisen

Engineering from Purdue University in 2004. As an undergraduate student, Emilie worked with Profs Gil U. Lee and Daniel Suter characterizing the topology of a neuronal growth cone with an atomic force microscope. She is currently a graduate student in Prof. Kokkoli’s group in the Chemical Engineering and Materials Science Department at the University of Minnesota. Her current research interests are biomimetic peptide-amphiphiles and their applications in regenerative medicine.

Jennifer Craig is working to complete a MS in Biomedical Engineering at the University of Minnesota, following the BS in Chemical Engineering that she received from the University of Notre Dame. Her current research in the group of Prof. Kokkoli is in testing human umbilical vein endothelial cells’ responses to unique fibronectin-mimetic peptide sequences. Jennifer A. Craig

This journal is ß The Royal Society of Chemistry 2006

three-dimensional structure of the peptide headgroup into triple helices, and a-helices, giving rise to protein-like molecular architectures.28–30,34,35,40–42 For example, the a1(IV)1263–1277 collagen sequence (called IV-H1), which promotes melanoma cell adhesion and spreading, has been functionalized with C12, C14, and C16 mono- and dialkyl tails to create collagen-like peptide-amphiphiles. Results showed that while the [IV-H1] peptide did not exhibit any positive ellipticity similar to a polyPro II helix, the peptideamphiphiles investigated were all in triple-helical conformations.28 Moreover, the triple-helical peptide-amphiphiles were very stable. The difference, for example, in the denaturation temperatures between the (Gly-Pro-Hyp)4-[IV-H1]-(Gly-ProHyp)4 peptide and the corresponding C12 dialkyl peptideamphiphile was about 36 uC.35 More recently, a 16-residue peptide was studied to determine if the addition of monoalkyl tails will promote an a-helical conformation. The 16-residue peptide alone did not form a distinct structure in solution, whereas when connected to a C6 or C16 monoalkyl hydrocarbon chain it adopted predominantly an a-helical structure in solution.42 The thermal stability of the a-helix was greater for the C16 peptide-amphiphile compared with the C6 chain, and the authors attributed this to the extent of aggregation induced by the respective hydrocarbon chains.42 Similarly, the a-helical propensity of a peptide incorporating the angiogenesis-inducing 119–122 sequence from SPARC (secreted protein acidic and rich in cysteine), and peptideamphiphiles with C6–C18 monoalkyl hydrocarbon chains, has been examined.41 The 21 residue peptide alone did not form any distinct structure in solution, whereas the peptideamphiphile versions of it adopted a predominantly a-helical structure with a thermal stability that increased with increasing hydrocarbon chain length.41 Gore et al., have investigated the effect of tail length, number of tails, and temperature on the self-assembly of model collagen mimetic peptide-amphiphiles.43 They connected a (Gly-Pro-Hyp)4-[IV-H1] peptide headgroup to either monoalkyl (single) or dialkyl (double) tails and tested several tail lengths including C12, C14, C16, C18, and C20. Their cryo-TEM images and small angle neutron scattering (SANS) data indicated that the resulting geometry is sensitive to the tail length, to the number of tails (single versus double tail peptide-amphiphiles), and for amphiphiles with certain tail length to the temperature, which in turn was shown to disrupt the triple-helical structure of the peptide headgroup.43 For example, peptide amphiphiles with single tails of various lengths, and C12, C14 double tails formed close-packed spheroidal micelles organized in a lattice (Fig. 2a). Increasing the length of the double tail to C16, and C18 gave rise to disklike micelles that stacked up to form extended strandlike structures at room temperature (Fig. 2b), that upon heating and cooling formed spheroidal aggregates.43 Overall, the results of this study indicate that even though such model collagen peptide-amphiphiles follow some of the trends observed for other more common surfactants, they also show dependence of aggregate geometry on thermal history and the formation of extended strands, which might be a result of interactions between peptide headgroups.43 This journal is ß The Royal Society of Chemistry 2006

Fig. 2 Cryo-TEM images of double-tail (Gly-Pro-Hyp)4-[IV-H1] peptide-amphiphiles: (a) C16 double tails form close-packed spheroidal micelles organized in a lattice, (b) C18 double tails form disklike micelles that stack up to form extended strandlike structures. Reprinted with permission from ref. 43. Copyright 2001, American Chemical Society.

A decapeptide that self-assembles into 0.5 mm diameter hollow tubules and helices ranging in length from tens to hundreds of microns depending on formation conditions was synthesized by Lee et al.44 The structure formed is called a high-axial-ratio microstructure or HARM. The linker used was glutamic acid (Glu), with a bis(tetradecylamide) tail. The decapeptide head group was modified to contain a trypsinlabile cleavage site.44 Recently, DNA-binding peptide-amphiphiles with a GCN4 region, a short coiled-coil nucleation sequence, and a tail containing a polymerizable methacrylic group were used to investigate the effect of the peptide secondary structure on aggregation behavior.45 Results revealed a variety of morphologies. In solution the peptide-amphiphiles formed helical ribbons and tubules (hollow cylinders) and upon DNA binding they formed lamella.45 This study demonstrated that the specific interaction of the headgroup is important in determining the structure of the self-assembled material. Amphiphiles with a conical shape that have a bulkier headgroup than the tail section tend to form cylindrical micelles, which are termed nanofibers. Fig. 3A shows a schematic of a nanofiber formed from monoalkyl tail peptide-amphiphiles where the peptide headgroup includes

Fig. 3 (a) Schematic representation of the self-assembly of peptideamphiphiles into cylindrical nanofibers. Reprinted with permission from ref. 47. Copyright 2001, American Association for the Advancement of Science. (b) TEM image of a C10 single-tail CCCCGGGS(PO4)RGD peptide-amphiphile, negatively stained with phosphotungstic acid. Reprinted with permission from ref. 46. Copyright 2002, Proceedings of the National Academy of Sciences USA.

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sheet-forming segments, charged residues for solubility, and biological epitopes that mimic extracellular matrix proteins and promote cell adhesion or differentiation through cell signaling.46–49 Using different triggers such as pH changes, or ion concentration, these molecules self-assemble in aqueous solution into nanofibers.46,47 It has also been shown that two different peptide-amphiphiles with different epitopes and complementary charge, or peptide sequences with reverse polarity can be coassembled into nanofibers.50,51 Furthermore, dip-pen nanolithography was used to control the surface morphology of peptide amphiphile nanofibers by producing aligned arrays and double-layer patterns of isolated and closepacked nanofiber patterns on silicon substrates.52 The internal structure of peptide-amphiphile nanofibers was studied by incorporating into the peptide headgroup a tryptophan or pyrene chromophore that allowed for fluorescent quenching experiments to determine how accessible different positions in the nanofiber are to small aqueous molecules.53 Different peptides were used with the chromophore positioned at either the N- or C-terminus of the peptide, and they also designed one molecule to use pyrene as the hydrophobic section of the molecule. This study showed that the nanofiber interior remains well-solvated, suggesting that the peptide-amphiphile aggregates maintain high degrees of free volume. In addition, they showed that fluorophores within a nanofiber can interact with the external environment, and that all accessible chromophores may interact with small molecule quenchers to different degrees despite their depth within the nanostructure.53 This offers the possibility that other small molecules will have similar access thus making peptide-amphiphile nanofibers suitable for delivery vehicles. The effect of amino acid sequence and alkyl tail modification on the formation of nanofibers with varying morphology, surface chemistry, and potential bioactivity was investigated by Hartgerink et al.46 Results showed that short peptide amphiphiles with a tail length of C6 or with no tail did not form gels at any pH. Longer molecules with tail lengths C10, C16, and C22 formed self-supporting gels under slow acidification. At pH 7, the peptide-amphiphiles that were examined had a net negative surface charge and acidification eliminated electrostatic repulsion thus allowing the assembly of nanofibers. Another finding from this study was that disulfide bonds should be fully reduced in order for nanofibers to form, as partial oxidation of the cysteine residues will create intermolecular bonds and will prevent the formation of nanofibers. The cysteine residues were found to be unessential to nanofiber formation, but gels without cysteine residues exhibited some phase separation after a couple of days. The formation and structure of the nanofibers was independent of the starting concentration, but in higher concentrations, the nanofibers tended to align in bundles (Fig. 3B), which is necessary for gel formation. All of the peptide-amphiphiles with different cell binding domains formed gels under acidification, but TEM images showed that the length of the nanofiber or stiffness of the gel could vary with various peptide chemistries. Since acidification is not a convenient method for formation of biocompatible gels in vivo the authors also demonstrated that gels could be formed by 1018 | Soft Matter, 2006, 2, 1015–1024

adding a divalent cation, such as Ca2+.46 More recently Paramonov et al. studied the role of hydrogen bonding and amphiphilic packing in the self-assembly process of peptideamphiphiles into nanofibers.54 Their work demonstrates that the four amino acids closest to the nanofiber core form b-sheet hydrogen bonds that are necessary for the formation of nanofibers. Disruption of these hydrogen bonds favors the formation of spherical micelles instead of elongated cylindrical structures.54 Tsonchev et al., studied the formation of peptide-amphiphile nanostructures, via an all-atom empirical force field calculations and Monte-Carlo simulations.55–58 The authors assumed that in absence of other interactions the peptide-amphiphiles will self-assemble into their minimum energy configuration, which is usually the highest density structure, and for peptideamphiphiles with truncated cone structures they showed that they would fit more densely in spherical shapes, in contrast to a cylindrical structure predicted by the critical packing parameter.39 Their model showed that self-assembly was directed by hydrophobic and electrostatic interactions. When electrostatic interactions were the dominant driving force cylindrical micelles were formed, as determined experimentally, whereas when these forces were removed the peptide-amphiphiles formed spherical micelles. Langmuir–Blodgett (LB) films of peptide-amphiphiles permit control over the properties of the interface at the molecular scale, and as such can be used as model surfaces for studying a variety of fundamental phenomena in cell adhesion. The LB transfer of peptide-amphiphiles to hydrophobic surfaces and the subsequent effect of the surface ‘‘template’’ on peptide conformation and orientation have been studied extensively. LB pressure-area isotherms for peptide-amphiphiles (both pure and mixed with other lipids) at high pressures show an ordered solid phase at an area per molecule that is consistent either with the area of the alkyl lipid tail59,60 or with the area of the secondary headgroup structure, such as a triplehelical architecture.31,32,35 Peptide-amphiphile monolayers have high collapse pressures indicative of well-ordered single monolayers.31,59–61 Parameters that can affect the shape of the LB isotherms of pure peptide-amphiphiles or their mixtures with other lipidated molecules are temperature, the tail length, the bulkiness, polar character, and sequence of the peptide headgroup that can either completely prevent the formation of stable monolayers or affect the occurrence of various phases during the monolayer compression.31,62–67 In addition, LB isotherms of peptide-amphiphiles can be affected by different conformations of the peptide headgroup induced by the hydrophobic tail,31 by ions present in the aqueous subphase,68,69 or by metals that complexate with the polar headgroup.70 Atomic force microscopy (AFM) of peptide-amphiphile/ lipid LB films, shows the appropriate height ‘‘differential’’ for peptide-amphiphiles and lipids.32,34,59,71 Fourier-transform infrared spectroscopic data for RGD-containing peptideamphiphiles33 and neutron reflectivity data for triplehelical peptide-amphiphiles32,72 demonstrated that peptide headgroups are oriented perpendicular to the air–water interface and retain the same conformation as in solution. Similar This journal is ß The Royal Society of Chemistry 2006

conclusions have been reported on the basis of Johnson– Kendall–Roberts (JKR) contact-mechanics-based adhesion measurements between the a5b1 integrin receptor and LB membranes of RGD-containing peptide-amphiphiles.60 Varying ligand organization at the nanoscale can regulate cell motility. Therefore, methods by which surface density, presentation, organization, and accessibility of a peptide ligand can be controlled may provide valuable insights into cell adhesion. The methodologies associated with peptidefunctionalized LB films allow the aforementioned variables to be modified. When designing materials for biosensing devices, LB films of peptide-amphiphiles mixed with different lipids provided ideal configurations for precise delivery of the bioactive molecule at the sensing interface.73 Model LB membranes of mixtures of peptide-amphiphiles with lipidated polyethylene glycol were used to investigate the effect of ligand accessibility to a receptor, either by mixing peptide-amphiphiles with lipidated molecules, or by varying the deposition pressure.32,61,71 Dori demonstrated that twocomponent membranes could be transformed from homogeneous to heterogeneous patterned surfaces simply by changing the number of methylene groups in the tails of the respective peptide-amphiphiles.74 Thus, utilizing the synthetic nature of peptide-amphiphiles may allow a biointerface to be tailored to higher degrees than a chemically modified substrate. In addition, it was recently shown that twocomponent mixtures of peptide-amphiphiles with other lipidated molecules can give rise to well-mixed or patterned interfaces simply by changing the concentration of the mixture or the deposition pH.59 In this study, the mixing behavior of peptide-amphiphiles and lipidated short polyethylene glycol 120 (PEG120) at different concentrations and pH was investigated using thermodynamic analysis of the surface pressure–area isotherms and AFM imaging. Results demonstrated, for example, that for RGD-PHSRN containing peptide-amphiphiles at pH 6.5–7 patterned formation was observed at concentrations of less than 40%. The patterns ranged from round-shape domains of varying size to flowerlike formations of crystalline PEG in a peptide-amphiphile liquidlike continuous matrix (Fig. 4).59 Peptide concentration and

Fig. 4 3-D view of an AFM phase image of a LB membrane containing 75% PEG–25% RGD-PHSRN containing peptide-amphiphile.59 The flowerlike isolated patterns consist of PEG whereas the continuous phase consists of peptide-amphiphile and smaller PEG domains.


subphase pH are parameters that can easily be controlled, and as such can be employed in creating cell-responsive surfaces with desired patterns of different functionalities.

3. Biomimetic peptide-amphiphiles for functional biomaterials One important aspect of biomaterials research is the bioactivity of materials and devices. Especially in the fields of drug delivery, tissue engineering, and regenerative medicine, cell binding capability is of particular interest. Peptideamphiphiles represent a promising way of functionalizing interfaces and incorporating cell binding ability and recognition into biomaterials. These molecules have gained attention in recent years because they represent an attractive alternative to the incorporation of whole proteins into soft materials. The long alkyl chain(s) characteristic of peptide-amphiphiles provide a convenient mechanism for self-assembly and association with other hydrophobic substrates. Thus, the composition and orientation of molecules on the surface is well controlled, enabling optimal presentation of active binding sites. Further, these long chains can also improve the stability and activity of the peptide relative to the parent protein by imparting secondary structure.75 Current and past research into the utility of peptide-amphiphiles has focused on several aspects, including, but not limited to, binding specificity; the effect of surface composition, and ligand availability on binding; factors affecting cell spreading and migration; and the effect of ligand structure on binding capability. One of the most desirable qualities in any bioactive material is the ability to interact specifically with cells of interest. Thus, the focus of recent research is in constructing biomimetic synthetic membranes, which could be used to tether molecules to a surface, and thus could lead to the development of a new class of molecular sensors, diagnostic devices, or model systems for the characterization of fundamental biological phenomena such as receptor–ligand interactions and cell–cell binding. An inherent flexibility of self-assembly processes in producing chemically heterogeneous structures is the major aspect that drives an increasing interest in model membranemimetic systems composed of synthetic amphiphilic molecules with groups that exhibit biological activity.76,77 Traditionally, interactions between membrane-bound receptors and their ligands are measured using signals from a secondary molecule/event, such as for example, a fluorescent probe, radioactive labeling, or as in the case of channel proteins—electrical conductance changes.78,79 Using biomimetic membrane-like structures, on the other hand, allows measuring of the interactions between ligands and their receptors directly, without the need of any additional labels. For example, Heyse and coworkers used integrated optics to measure receptor–ligand interactions in situ. They have used phospholipid bilayers attached covalently to waveguides as a hydrophobic template for the deposition of a complete bilayer of functionalized lipids or lipid-anchored peptides (peptideamphiphiles) by either vesicle fusion or lipid self-assembly from mixed lipid/detergent micelles.80 The monolayer formation was monitored by an integrated optics technique which allowed the determination of the optical thickness and Soft Matter, 2006, 2, 1015–1024 | 1019

refractive indices of the adsorbed organic layers, and thus the evaluation of the quality of the forming bilayer.80 As a result, they have been able to study recognition reactions between ligands and their transmembrane receptors without the need of labels, and have determined that the synthetic lipid bilayer not only served as a matrix for the incorporation of membraneanchored receptors, but also helped to reduce the nonspecific binding of proteins. Their results indicate that the detection limit of such label-free integrated optic measurements is much higher than that of the traditional fluorescence detection technique. Studies with model LB membranes of glycine peptideamphiphiles have provided fundamental measurements on the effect of pH, electrostatic interactions, and hydrogen bonding on bioadhesion.81–83 These measurements have important implications in studies of protein stability and the design of functional self-assembled biomaterials. Furthermore, LB membranes of peptide-amphiphiles were used to show that ligand accessibility plays a critical role in controlling ligandreceptor interactions and cell response to functionalized surfaces.32,61,71 When human skin fibroblast cells (NB1RGB) were cultured on LB membranes and cast membranes of lipidated peptides and ECM proteins, the cell density was found to be approximately the same on LB and cast membranes of various lipidated peptide membranes, and the concentration and production of interferon-beta (IFN-b) were higher on the LB membranes than on the cast membranes of the ECM proteins.84,85 Adhesion and spreading of melanoma cells on supported LB membranes of triple-helical collagenmimetic (Gly-Pro-Hyp)4-[IV-H1] peptide-amphiphiles demonstrated that different cellular responses (adhesion and spreading) can be promoted by altering ligand accessibility via selective masking of the bioactive peptide-amphiphile headgroup with PEG lipidated molecules of varying length.32 For PEG molecules much longer than the peptide-amphiphile, human melanoma cells did not adhere to the surface; for slightly shorter PEG molecules the cells bound but did not spread; and for the PEG molecules much shorter than the peptide-amphiphile, cells were able to both bind to and spread on the surface due to the ability of the peptides to correctly spatially orient for binding.32 Different deposition pressures that give rise to a more ‘‘solid’’- or ‘‘fluid’’-like membrane were used as a means to control ligand accessibility and give more space for the bioactive headgroups to bend and expose the binding sequence at the interface.61,71 Collagen-mimetic self-assembling systems have also been used as model systems to study receptor–ligand interactions.14,42 For example, the role of a specific receptor binding in activation of a particular cellular response has been elucidated using peptide-amphiphiles.86,87 The effect of mixtures of peptide-amphiphiles in promoting endothelial cell behaviors comparable to extracellular matrix components was investigated using multicomponent films of the a-helical SPARC (angiogenesis-inducing sequence) peptide-amphiphiles, the a2b1 integrin binding site from type I collagen-derived triple-helical peptide-amphiphiles, and a pseudolipid.41,87 The pseudolipid was required for the spatial distribution of the peptide-amphiphiles that allowed for optimal cellular interactions.41 In addition, type IV 1020 | Soft Matter, 2006, 2, 1015–1024

collagen-derived triple-helical peptide-amphiphiles have been used to evaluate the role of the a2b1 integrin and CD44/CSPG receptor on human melanoma cell activation.86 These studies demonstrated that growing cells on such triple-helical collagen-mimetic peptide-amphiphiles can be used as a general in vitro model system to study and monitor gene expression and protein production changes induced by binding to a specific receptor. To investigate the effects of orientation and conformation of RGD peptide-amphiphiles on cellular responses, Pakalns et al. designed two conformations of the RGD peptide, one linear and one looped, and two orientations of the linear RGD, N- and C- grafted, which were independently presented to cells. The surface density of RGD was adjusted by dilution using a looped RGE amphiphile, or a methylated amphiphile (RGE and methylated amphiphiles did not support cell adhesion). Results from this study demonstrated that the membranes of looped RGD supported adhesion and spreading of M14#5 human melanoma cells in a dose-dependent and specific manner, and inhibition assays identified a3b1 as the primary receptor mediating cell adhesion.33 The effect of peptide concentrations was investigated using RGD-based linear and cyclic peptide-amphiphiles as a model system for the a5b1 integrin, and results demonstrated that RGD lipopeptides can support efficient cell binding and spreading at peptide ligand loadings as low as 0.1 mol%.88 More recently LB membranes of peptide-amphiphiles were used to examine the effect of RGD secondary structure and the synergy site PHSRN, on cell adhesion, cell spreading, integrin binding affinity, and specific integrin engagement via cell studies, JKR and AFM collective studies (multiple interactions), and AFM single-molecule force spectroscopy experiments.60,61,64,89,90 Dillow et al. employed biomimetic bilayer membranes as model surfaces to investigate specific adhesive interactions between fibronectin-derived peptide-amphiphiles and isolated a5b1 integrins using the JKR apparatus.60 Their study utilized both the GRGDSP peptide sequence (the primary binding site for a5b1) as well as the PHSRN peptide (the synergy binding site for a5b1). Through JKR bioadhesion studies, they clearly showed that the ability of the a5b1 integrin to bind to peptide-amphiphile surfaces composed of GRGDSP, PHSRN, and short PEG molecules attached to phospholipids, is dependent on membrane composition, density, and temperature that affects peptide mobility.60 Kokkoli et al., have used bioartificial membranes constructed from GRGDSP, and PHSRN peptide-amphiphiles to directly measure their interaction with isolated a5b1 integrins immobilized on the AFM tip at the collective and single-molecule level.89 These results demonstrated that the separation of the receptor-ligand pairs at the collective level proceeds through a combination of multiple unbinding and stretching events that can be described by the wormlike chain model of polymer elasticity. However, at a single-molecule level, no such stretching was observed, and the dissociation of single a5b1– GRGDSP pair under a range of loading rates revealed the presence of two activation energy barriers in the unbinding process.89 Mardilovich and Kokkoli have explored the effect of the synergy site PHSRN on the strength and dynamics of a5b1 This journal is ß The Royal Society of Chemistry 2006

integrin adhesion at the molecular level with a fibronectinmimetic peptide-amphiphile using AFM studies.61 They reported that surfaces functionalized with a single peptideamphiphile consisting of both RGD and PHSRN at a spatial configuration that mimics the adhesion domain of the fibronectin showed marked improvement in integrin binding over RGD peptide-amphiphiles alone.61 The results of this work demonstrate that accessibility and appropriate spatial orientation of the PHSRN sequence is crucial for designing fibronectin-mimetic surfaces and can be used as a means to control interaction with integrins. In a recent study Mardilovich et al. have designed a novel fibronectin-mimetic peptide-amphiphile that has been shown to outperform fibronectin.90 The peptide-amphiphile (called PR_b) had four building blocks: a spacer, RGDSP, PHSRN, and a linker. The linker was designed to mimic two important criteria: the distance and the hydrophobicity/hydrophilicity between RGD and PHSRN in fibronectin. This was the first study demonstrating that a peptide gives stronger human umbilical vein endothelial cell (HUVEC) adhesion than fibronectin for 1–24 h, and equivalent cell adhesion after 48 h at which point high amounts of secreted ECM fibronectin had covered surfaces.90 In addition, cells were spread the most on PR_b, and results from immunocytochemical studies showed that compared to fibronectin, the PR_b peptide-amphiphile can promote stronger cytoskeletal organization and focal adhesion formation (Fig. 5).90 Peptide-amphiphiles have been used to study the structure and function of isolated domains of integral transmembrane proteins, and the antibacterial activity of different peptide sequences.75,91,92 For example, in a recent report the bactericidal peptide SC4 (KLFKRHLKWKII) was conjugated to a fatty acid chain of a C12 or C18 length to make peptideamphiphiles. It was found that these amphiphile versions of SC4 had up to 30-fold increased activity against Gram-positive bacteria, even the Staphylococcus aureus strain which is resistant to conventional antibiotics.75 The authors used

Fig. 5 Confocal image of HUVECs on a LB membrane of PR_b peptide-amphiphile. HUVECs were seeded for 24 h in the absence of fetal bovine serum. Actin stress fibers are shown in red, vinculin in green, and the nucleus in blue. Reproduced with permission from ref. 90. Copyright 2006, American Chemical Society.


NMR studies and CD to probe the structure of the molecule, and concluded that when acylated, the SC4 peptide-amphiphiles assumed a more helical structure than the SC4 peptide alone. Since it had been shown previously that stabilization into helical structures is an important step in the activity of anti-microbial peptides, the authors determined that the use of peptide-amphiphile technology in this case helped a potentially bactericidal peptide assume the conformation necessary to lead to lysis of bacterial membranes. Peptide-amphiphiles were also used as bullets for targeted drug delivery, or as markers in the construction of a bioanalytical device. For example, (Gly-Pro-Hyp)4-[IV-H1] peptide-amphiphiles were used to functionalize liposomes decorated with PEG and were targeted to mouse fibroblast cells.93 Results showed that the IV-H1 peptide-amphiphile maintained its native triple helical structure when it was incorporated into the liposomal membrane, and its presence enhanced uptake of the IV-H1 functionalized liposomes over non-targeted liposomes.93 As a first step in the development of a fractalkine-targeting drug delivery system Kokkoli et al. investigated the binding of a peptide-amphiphile (NTFR), derived from the N-terminus of the fractalkine receptor, to fractalkine expressing inflamed endothelial cells.71 The goal of this study was to demonstrate that the NTFR peptideamphiphile binds specifically to fractalkine and not to any other receptors, such as integrins, found on inflamed or healthy cell surfaces. The results indicated that the NTFR peptide-amphiphile binds to its target fractalkine with strength and specificity similar to the binding of RGD peptideamphiphiles to the a5b1 integrin, and that NTFR does not bind significantly to integrins such as the a5b1.71 Cyclic RGDcontaining lipopeptides were incorporated into giant liposomes and showed strong selective adhesion to endothelial cells thus demonstrating that peptide-amphiphiles can be used to improve the efficiency of liposomes for the purpose of drug delivery.94,95 Vesicles decorated with a cyclic peptide-amphiphile that contains the RGD sequence were shown to bind to the aIIbb3 integrin reconstituted into phospholipid vesicles forming regularly spaced bridges between the two kinds of vesicles.96 Polymerizable RGD peptide-amphiphiles were assembled into vesicles and were stabilized via light-induced polymerization.97 The resulting functionalized vesicles exhibited interesting chromatic responses when binding to a5b1 integrins in solution and can be used for label-free detection of interactions with other molecules in solution. Similar polymerizable RGD peptide-amphiphiles were also used for the formation of stable surface coatings that showed increased stability to air and were used to manipulate cell adhesion and spreading.98 RGD functionalized peptide-amphiphile nanofibers were used to direct mineralization of hydroxyapatite to form a composite material that resembled the lowest levels of hierarchical organization of bone,47 were shown to influence attachment, proliferation, and osteogenic differentiation of mesenchymal stem cells,99 and to effectively encapsulate pyrene, a small hydrophobic molecule.100 In addition, peptide-amphiphiles covalently functionalized with pyrene in the hydrophobic portion of the tail exhibited excimer formation upon self-assembly into nanofibers, a property that Soft Matter, 2006, 2, 1015–1024 | 1021

was not observed in similar peptide-amphiphiles that formed spherical aggregates instead of cylindrical nanofibers.100 Peptide-amphiphile nanofibers functionalized with the laminin epitope IKVAV were used to encapsulate neural progenitor cells.48 Relative to laminin or soluble peptides the gel nanofiber matrix induced very rapid differentiation of cells into neurons, while discouraging the development of astrocytes.48 In another study MC3T3-E1 cells were encapsulated in a peptide-amphiphile nanofibrillar matrix. Cells survived in culture for at least three weeks, and cell spreading and motility depended on the cell adhesive molecules of the matrix.101 In addition, bioactive elastic peptide-amphiphile nanofibers were designed that could promote cell-mediated degradation of the gel by incorporating a matrix metalloproteinase (MMP2) specific cleavage.102 A branched peptide-amphiphile system was designed for enhanced recognition of biotin on peptide-amphiphile nanofibers.103 The new design could present more than one epitope per molecule. Guler et al., found that nanofibers formed by self-assembly of biotinylated branched peptide-amphiphiles enhanced recognition by the avidin receptor relative to similar nanostructures formed by linear peptide analogues.103 Branched anionic and cationic peptide-amphiphiles were used to encapsulate carbon nanotubes thus enhancing the solubility of the carbon nanotubes without degrading their structural, electronic, and optical properties caused by covalent functionalization, and without the need to incorporate peptide sequences that specifically bind to nanotube surfaces.104 Peptide-amphiphiles were used in a variety of other applications such as contrast agents for MRI.105 Also, peptide nucleic acid/peptide-amphiphile conjugates (PNA–PA) that self-assemble into fiber-shaped nanostructures were designed to bind oligonucleotides with high affinity and specificity.106 The PNA–PA DNA, and PNA–PA RNA systems were shown to bind more strongly than the corresponding DNA–DNA and RNA–RNA duplexes.

4. Summary In this review we focused on the self-assembly mechanisms and generated structures of peptide-amphiphiles, and on their applications in biomaterials research and biomedical surface engineering. The self-assembly of the hydrophobic tails of the peptide-amphiphiles is a driving force that stabilizes the threedimensional structure of the peptide headgroup into triple helices, and a-helices, giving rise to protein-like molecular architectures. Furthermore, depending on the overall geometry of the peptide-amphiphile structure, and the interactions of the building blocks of the molecule under different environmental conditions, peptide-amphiphiles can self-assemble into a variety of soft materials such as spheroidal micelles, disklike micelles that stack up to form extended strandlike structures, helical ribbons, tubules, lamella, nanofibers, and patterned membranes. Biomimetic peptide-amphiphiles are versatile tools for soft biomaterials and have found applications as model systems for the characterization and understanding of fundamental biological phenomena such as the structure and function of protein domains, receptor–ligand interactions, cell adhesion, gene expression, and protein production. Moreover, 1022 | Soft Matter, 2006, 2, 1015–1024

peptide-amphiphiles have also been used as ligands for targeted drug delivery, as markers in the construction of a bioanalytical device, or as scaffolds for the generation of tissue engineered materials. Thus, the self-assembly of peptideamphiphiles is a novel and promising method of engineering biomimetic peptides onto biomaterial structures with distinct 3-D structures. Their self-assembling properties and their ability to incorporate multifunctional peptide sequences makes them an attractive tool for the design of bioengineered soft materials.

Acknowledgements Acknowledgement is made to the Donors of the American Chemical Society Petroleum Research Fund for partial support under 39120-G4. JAC acknowledges partial support through a 3M Science and Technology Fellowship.

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