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Chapter 6

Applications in Bionanotechnology ofSelf-Assembled Peptide Nanostructures

Yihua Loo, Elizabeth C. Wu, Anupama Lakshmanan, ArchanaMishra, and Charlotte A. E. HauserInstitute of Bioengineering and Nanotechnology, 31 Biopolis Way,The Nanos #04-01, 138669 Singaporechauser@ibn.a-star.edu.sg

6.1 Introduction

As early as 1959, Richard Feynman first presented the idea of

nanotechnology; envisioning “the possibility of maneuvering things

atom by atom” to provide a plethora of new opportunities for design

[1]. Since then, the advent of science and technology in providing

high-precision techniques and high-resolution tools for imaging,

production, and characterization has heralded the modern era of

nanotechnology. The older approach of “top-down” design, limited

by the starting material and its bulk properties, has given way to

the “bottom-up” approach that confers complete control of building

blocks for the desired end properties.

Bionanotechnology exploits lessons learnt by observing bio-

logical processes for designing molecular machinery to atomic

Self-Assembled Peptide Nanostructures: Advances and Applications in NanobiotechnologyEdited by Jaime Castillo-Leon, Luigi Sasso, and Winnie E. SvendsenCopyright c© 2012 Pan Stanford Publishing Pte. Ltd.ISBN 978-981-4316-94-1 (Hardcover), 978-981-4364-47-8 (eBook)www.panstanford.com

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148 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

specifications [2]. Self-assembly involves the spontaneous asso-

ciation and organization of individual components into coherent

and well-defined structures [3]. It occurs ubiquitously in nature at

macroscopic and microscopic scales and plays an important role in

many life processes. Cells are natural nanomachines that achieve

diverse functions ranging from replication, trafficking, recognition

and signaling through the self-assembly and organization of simple

biomolecules. The efficiency, high fidelity, and reproducibility of

cellular self-assembly processes make them ideal models for

designing nanostructures applied in bioengineering and medicine

[2].

Peptide and proteins are versatile building blocks for fabri-

cating supramolecular architectures such as synthetic membranes,

multilamellar structures, amphiphilic micelles, tubules and fibrillar

networks [4]. Precisely defined, hierarchical three-dimensional

(3D) structures can be obtained from the self-assembly of pep-

tides/proteins [5]. Peptide/protein self-assembly is highly specific

— the intermolecular interactions such as hydrogen bonding, ionic,

electrostatic, hydrophobic, and van der Waals interactions are

mediated by molecular recognition. The information needed for

proper folding is encoded in the amino acid sequence, bypassing

the need for chemical crosslinkers such as glutaraldehyde (which

is cytotoxic). The peptide sequence also determines the three-

dimensional structure (formation of α-helices, β-sheets, micellar,

lamellar, and fibrillar structures) and material properties (mechani-

cal strength, stimuli-sensitivity, assembly/disassembly reversibility,

and kinetics). As solid-phase peptide synthesis or genetic engi-

neered cells are used to produce the monomers, exact control of the

polypeptide/protein sequence, length, and hence 3D structure can

be achieved.

6.2 Peptide/Protein Hydrogels for Regenerative Medicine

6.2.1 Engineering Synthetic Extracellular Matrix

The extracellular matrix (ECM) plays an integral role in cell

patterning, migration, proliferation, and differentiation [6]. It not

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Peptide/Protein Hydrogels for Regenerative Medicine 149

only provides structural support as a passive scaffold substrate;

but also dynamically interacts with the cells through receptor-

mediated signalling, sequestration of growth factors (GFs), spatial

cues, and transduction of mechanical forces [7]. In this regard, a

major challenge is to produce a biomaterial that closely mimics

the structure and properties of the ECM while overcoming the

immunogenicity, non-specific interactions, chemical instability, and

large batch–batch variation of intact ECM proteins [8]. In summary,

ideal scaffolds should have good biocompatibility and proper

biodegradation, sufficient porosity, ability to promote cell prolif-

eration and cellular ECM production, while maintaining cellular

phenotype and function [9]. In addition, synthesis of the scaffold

biomaterial must be facile, scalable, reproducible, and cost-effective

for successful commercialization.

Peptide amphiphiles self-assemble to form complex, supramole-

cular three-dimensional architectures that provide specialized

microenvironments and niches for cell–cell adhesion, growth, pro-

liferation, migration, and morphogenesis necessary for functional

tissues [10]. These amphiphilic molecules typically refer to (i)

amphiphilic peptides consisting only of amino acids with a hy-

drophilic, polar headgroup, and a hydrophobic tail; (ii) hydrophilic

peptides conjugated to hydrophobic alkyl chains or fatty acids and;

(iii) peptide-based co-polymers [8]. Diverse nanostructures such as

vesicles, micelles, monolayers, bilayers, fibers, ribbons, and tapes

can be formed by the self-assembly of peptide amphiphiles [8]. By

altering the structural segments, the morphology, surface chemistry,

structural, and functional characteristics of the peptide can be

modulated [8], giving rise to “smart bio-materials” that respond

to physicochemical triggers such as concentration, pH, light, ionic

strength, solvent, and temperature [3].

Thus, peptide-amphiphiles serve as versatile tools for soft bioma-

terials. Their applications in regenerative medicine are realized by

their ability to provide a complex, 3D supramolecular architecture

resembling natural ECM. In addition, they are also used as invitro model systems for the characterization and understanding of

fundamental biological processes such as structure and function of

protein domains, cell adhesion, gene expression, receptor–ligand

interaction, and protein production [8].

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150 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

6.2.2 Peptide Hydrogels for Tissue EngineeringApplications

Combining the understanding of molecular and structural biology

with materials engineering and design will enable new strategies

to develop biological tissue constructs for clinical applications. Self-

assembled peptides are attractive candidates for tissue-engineered

scaffolds, particularly hydrogels that contain more than 99%

water, as they are biomimetic, and provide spatial and temporal

regulation [11]. Furthermore, self-assembling peptides capable of

in situ gelation serve as attractive candidates for minimally invasive

injectable therapies. The peptide hydrogels can also be tuned to

optimize their mechanical and physiochemical properties to match

the tissue of interest. A variety of studies using peptide amphiphiles

as scaffolds have demonstrated the induction of biomineralization

[12], reducing glial-scar tissue formation [13] and controlling

neuronal progenitor cell differentiation [14].

The first self-assembling peptide was discovered in Baker’s

yeast by Zhang and co-workers [15] while studying the protein

Zuotin, which had a repetitive peptide motif of 16 amino acids

(n-AEAEAKAKAEAEAKAK-c) that adopted a β-sheet configuration

by nature of self-complementary ionic interactions [16]. Similar

peptides were subsequently designed to form three-dimensional

(3D) nanofiber scaffolds for tissue culture applications. In particular,

RAD16, which is now commercially available as PuraMatrixTM

,

has been used in a wide range of biomaterial applications,

including cartilage tissue repair [17], osteoblast proliferation and

differentiation [18], hepatocyte differentiation [19], brain repair and

axon regeneration [20], and bone regeneration [21]. Interestingly,

the RAD16-I peptide scaffold lacks any peptide signalling motif.

This suggests that the 3D architecture of the peptide nanofibers

has intrinsic properties that promote cell growth, proliferation, and

migration [22]. RAD16 peptide hydrogel has also been evaluated

as a matrix for osteogenic differentiation of co-cultured bone

marrow stem cells in vitro and in vivo [23]. KLD12, another self-

assembling peptide, formed hydrogel scaffolds that were found

suitable for cartilage repair by fostering ECM production by bovine

chondrocytes [17]. The biocompatibility of these hydrogels were

also evaluated with rabbit mesenchymal stem cells [24] and nucleus

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Peptide/Protein Hydrogels for Regenerative Medicine 151

pulposus cells [9], allowing their potential application as tissue

engineered intervertebral discs. In addition, RAD16-I and (D-form)

d-EAK16 nanofiber barriers were also found to facilitate hemostasis

at surgical wound sites [25, 26].

β−sheet peptide hydrogels can also be formed from Lysβ-21

via triple-stranded β-sheets in the β-domain of the protein [27], as

demonstrated by Aggeli and colleagues. They also studied β-peptide

P11-4 (QQRFEWEFEQQ), which self-assembled into anti-parallel β-

tapes that stack together to form fibrils when the pH was changed

[28]. The resulting hydrogel was used as injectable scaffolds for

treating bone defects, dental hypersensitivity, and dental decay [29].

It was proposed that the peptides formed a fibril network within the

pores of the caries-like lesion, where the anionic groups of the side

chains could attract calcium, leading to the de novo precipitation of

the respective phosphate salts [30].

Pochan, Scneider, and colleagues have also discovered a class of

self-assembling β-hairpin peptides that are promising candidates

for injectable hydrogel scaffolds [31]. These peptides comprise

two β-strands with alternating hydrophobic valine and hydrophilic

lysine residues covalently bonded to a central tetra-peptide turn

sequence. The resulting solid hydrogels exhibited reversible shear-

thinning and consequent flow under an applied stress. This team

also designed a peptide that exhibited complete thermoreversible

self-assembly into a hydrogel network [32]. Such hydrogels can be

used to make smart scaffolds that respond to environmental cues.

6.2.3 α−Helical Peptide Hydrogels

Woolfson et al. rationally designed peptides that formed self-

assembling fibers (SAFs) based on α-helical coiled-coils [33–35]

enabling elucidation of design principles that underlie self-assembly

of such systems. Their design was based on two-component peptide

systems that have a coiled-coil heptad sequence repeat, abcdefg, in

which the a and d positions were occupied by isoleucine and leucine,

respectively. They recently designed a new class of hydrogelating

self-assembling fibers (hSAFs) that form α-helical hydrogels [36].

This was achieved by replacing specific amino acid residues of SAFs

involved in charged interactions with those that have a weaker and

more general interactions at all b, c, and f sites. The hydrogels that

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152 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

were composed of smaller, more flexible, and thinner fibers were

found to support bone growth and differentiation of rat adrenal

pheochromocytoma cells for sustained culture periods [36].

6.2.4 Aligned Monodomain Gels for 3D Cell Culture

Peptide amphiphiles, such as the hydrophilic peptide VVVAAA

EEE(COOH) linked to a C16 alkyl tail at the N-terminus, were

reported to alter their supramolecular architecture in response

to temperature changes [37]. These molecules self-assembled into

gels containing nanofibers in the presence of ions that screened

the charged amino acid residues. Heating solutions of randomly

entangled, isotropic, unscreened molecules aligned the system into

long filaments of bundled nanofibers. This unique property was

exploited to direct the orientation of cells in 3D environments,

forming monodomain fibrous gels with macroscopically aligned

arrays of cells. Stupp and colleagues are applying this technology

to develop aligned scaffolds that direct cell migration, growth

and spatial cell interconnections for brain, heart, and spinal cord

tissue engineering [37]. They have also developed a class of self-

assembling peptide amphiphiles that formed 3D nanofiber networks

[38]. These molecules consisted of a 6–12 amino acid peptide

segment coupled to a (10–22 carbon) fatty acid chain. The self-

assembly process has been exploited to entrap cells in the matrix,

without compromising the cell viability, proliferation, and motility

[38]. In addition, Hartgerink et al. designed bio-active elastic peptide

amphiphilic nanofibers that promoted cell-mediated degradation of

the gel through a matrix metalloproteinase specific cleavage site

[39].

6.2.5 Functionalized and Hybrid Peptide Hydrogels

Scaffolds have also been developed by using peptide motifs derived

from ECM proteins. For example, the α1(IV)1263-1277 collagen

sequence (called IV-H1), which promotes melanoma cell spreading

and adhesion, has been functionalized with C12, C14, and C16 mono-

and dialkyl chains to make collagen-mimetic peptide amphiphiles

with stable triple helical conformations [40]. The effect of changing

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Peptide/Protein Hydrogels for Regenerative Medicine 153

the length, number of alkyl chains, and temperature on the self-

assembly of model collagen-mimetic amphiphiles has been explored

[41]. Cell adhesion and spreading can be controlled by using

PEG-lipidated molecules of varying length to selectively mask the

bioactive amphiphilic peptide and alter ligand accessibility [42].

Self-assembling peptide sequences have also been modified at the N

and C terminus to incorporate functional groups for conjugation to

growth factors and cell recognition motifs, such as RGD. Palkans et al.

[43] investigated the effect of orientation and conformation of RGD

peptide amphiphiles on cell responses using two conformations of

the RGD peptide (linear and cyclic), as well as N- and C-grafted RGD

peptides. RGD incorporated into self-assembling peptide amphiphile

matrices have been applied as scaffolds for dental stem cells

[44]. Laminin epitope IKVAV conjugated to peptide amphiphile

nanofibers have also been used to encapsulate neural progenitor

cells. This nanofiber gel matrix induced rapid neuronal differenti-

ation while simultaneously inhibiting astrocyte development [14].

Lastly, electrospun scaffolds of self-assembling peptides with poly-

ethylene oxide have been developed for bone tissue engineering

in a recent approach that amalgamates different design strategies

[45].

6.2.6 Ultra Small Peptide Hydrogels

Recently, Hauser and colleagues reported a new class of very

small peptides that self-assembled to long fibers under aqueous or

physiological conditions [46, 47]. Interestingly, during self-assembly,

this peptide class adopted different structural configurations in a

transition process toward supramolecular fibrous structures. This

new class of systematically designed tri- to heptamer peptides

demonstrated one of the smallest non-aromatic structures that

self-assembled to hydrogels. The fibrous scaffolds assembled into

three-dimensional meshes that entrapped up to 99.9% water

and resembled collagen fibers in the extracellular matrix. The

biocompatible hydrogels were found to be heat-resistant up to

90◦C with high and tunable mechanical strength [47]. To date, a

variety of mammalian primary cells have been successfully cultured

on these peptide hydrogels. In light of their biocompatibility and

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154 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

high mechanical strengths of up to 90 kPa, potential applications

include injectable therapies for degenerative disc disease. There is

a strong clinical need for a non-invasive disc repair that can hinder

or impair early-stage disc damage. Considering the facile and cost-

effective synthesis of the peptide hydrogels, as well as their tunable

properties, this class of ultrasmall peptide hydrogels can serve

as attractive biomaterials for applications ranging from injectable

biomedical therapies to tissue-engineered scaffolds.

6.3 Delivery of Bioactive Therapeutics

6.3.1 Peptide and Protein-Based Hydrogel DrugDelivery Devices

The molecular architecture of biomaterials used in drug delivery de-

vices determines their physiochemical characteristics and biological

fate. Self-assembled peptides/proteins are of great interest as their

sequences, molecular weights, composition, and stereochemistry

can be precisely defined by solid phase synthesis or biosynthesis

using genetic engineered cells. This leads to well-characterized

assembly/dissociation kinetics, incorporation of biorecognition

motifs, controlled biodegradation, and stimuli sensitivity; properties

integral to controlled drug delivery.

Injectable drug delivery systems that form biodegradable net-

works in situ following injection, have been the focus of many

studies [48]. These systems typically consist of polymers that form

viscoelastic gels under physiological conditions, or in response to

stimuli such as UV exposure, changes in temperature, pH, and ionic

environment. A few of these systems are based on self-assembling

peptides and proteins.

Silk-elastin-like polymers (SELPs) are one such class of self-

assembling polypeptides that irreversibly self-assemble into hydro-

gels via hydrogen bond mediated crosslinks. These protein polymers

are composed of tandemly repeated silk-like (GAGAGS) and elastin-

like (GVGVP) amino acid blocks. The former forms β-sheets,

imparting thermal and chemical stability, while the latter increases

the flexibility and water solubility. Extensive characterization of

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Delivery of Bioactive Therapeutics 155

SELP hydrogels have been done to ascertain gel-formation, stimuli-

sensitivity, biodegradation, biocompatibility, and biorecognition

[49]. SELP hydrogels have been applied to the delivery of anti-tumor

drugs, as well as hydrophilic and ionic compounds. Capello et al. [50]

characterized the drug release kinetics from SELP-47K hydrogels.

The gel was used to deliver recombinant mitotoxin Pantarin,

demonstrating that encapsulation by SELP-47K did not affect the

bioactivity and slow, sustained drug release was feasible. In separate

studies, SELP-47K hydrogels were used to deliver theophylline,

vitamin B, and cytochrome C, hydrophilic compounds with varying

molecular weights [51, 52]. Controlled release of plasmid DNA and

adenoviral vectors for cancer gene therapy have also been explored

by Megeed et al. [53]. To increase their versatility, “smart” SELPs

have also been designed to render the hydrogel stimuli-responsive.

Nagarsekar et al. [54] reported that the substitution of glutamic

acid for valine in some of the elastin blocks conferred temperature

sensitivity. The resulting polypeptides demonstrated completely

reversible temperature-dependent gelation.

Elastin-based polypeptides (ELPs) with characteristic pentapep-

tide motif (VPGVG)m(VPGXG)n, are also capable of self-assembling

into micelles [55] and nanoparticles [56]. ELP diblock copolymers

consisting of hydrophilic and hydrophobic blocks exhibit thermally

triggered nanoscale self-assembly. Their phase transition tempera-

ture can be precisely controlled by amino acid sequence. Chilkoti

and co-workers have developed ELP micelles and nanoparticles for

anti-cancer drug delivery [57]. Hydrophobic drugs conjugated to

hydrophilic ELPs self-assemble into micelles [55], which can then

be systemically administered to the patient. Enhanced permeability

and retention at the tumors site result in micelle and hence drug

accumulation. Thermal targeting can also be achieved by exploiting

the temperature sensitivity of ELPs. Phase transition-triggered

aggregation of ELPs occurs when the tumors locale is heated.

Conjugation of tumors-targeting ligand further increases uptake by

cancer cells. Local delivery of anti-cancer drugs can be facilitated by

ELPs that coacervates following intratumorsal injection [58]. ELPs

are attractive macromolecular carriers for cancer therapeutics as

their biocompatibility, genetically encoded synthesis, and stimuli

responsiveness provide highly tunable properties that can be

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156 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

rationally optimized for a given drug [59]. ELPs can also be applied

to recombinant protein purification [60]. In this approach, phase

transition of ELP fusion proteins is triggered by the addition of

heat or salt. The ELP aggregates are then separated from the cell

lysate by centrifugation. Resolubilization of the aggregates in cold

buffer then allows the removal of insoluble protein contaminants.

Repetitions of the inverse transition cycling process further increase

the purity of the ELP fusion protein. This technology presents many

advantages over current chromatographic recombinant protein

purification techniques, including cost, technological simplicity, ease

of multiplexing, and high yields.

Hydrogels from self-assembled ABA polypeptide triblock copoly-

mers, where block A is a coiled-coil forming peptide and block B a

random coil, also demonstrate temperature and pH-responsiveness.

Minor modifications to the amino acids of the coiled-coil domains

have a significant impact on the folding and unfolding of the helical

structure. Consequently, reversible gelation and dissolution can be

modulated by temperature and pH changes [61].

Hybrid hydrogels are synergistic combinations of self-

assembling peptide/protein domains and synthetic polymers. The

peptide domain confers a level of control over the structure

formation at the nanoscale level, giving rise to novel biomaterials

with unique properties and structural organization [62]. Kopecek

and colleagues have developed several hybrid hydrogels based

on coiled-coil protein motifs grafted onto N -(2-hydroxypropyl)

methacrylamide (HPMA) copolymers [5]. Self-assembly of the

coiled-coil kinesin domains from their unfolded state to elongated

helices resulted in hydrogel formation [63]. Hybrid systems that

incorporated two or more different coiled-coil domains form gels

capable of stepwise transitions, with each step triggered by a differ-

ent temperature. This feature is extremely attractive for designing

multi-phasic or pulsatile drug delivery systems. The phase transition

is also influenced by the melting temperature of the protein

domains, which can be tuned by the amino acid sequence. Other

self-assembling hybrid systems utilized two oppositely charged

antiparallel heterodimeric coiled-coils grafted to HPMA [64] and

polyethylene glycol [65, 66].

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Delivery of Bioactive Therapeutics 157

6.3.2 Viral Peptide Self-Assembly in Gene Delivery andVaccine Development

The structural proteins of viral capsids self-assemble into robust

nanostructures ranging from 22 to 150 nm. Simple morphologies

include helical (such as the tobacco mosaic virus) and icosahedral

(such as adenovirus). In helical viruses, a single protein subunit

(capsomer) stacks around a central axis, resulting in rod-shaped

helices that interact electrostatically with the single-stranded viral

RNA and DNA. Icosahedral morphology is the most optimal method

of forming a closed shell from identical subunits and contains

multiple axes of symmetry. Enveloped viruses (such as influenza

and lentivirus) are able to use their viral capsid proteins to interact

with lipids, enabling them to further assemble an encapsulating lipid

envelope from the host membrane.

Regardless of their shape, viruses are genetically tailored to

encapsulate, protect, and deliver the viral genome. As such, modified

viruses are some of the most effective gene delivery vectors known

to date. Nucleic acids are regarded as a novel class of therapeutics

to treat a variety of inherited and acquired diseases. Plasmid DNA

can induce the regulated production of therapeutic proteins [67–

70], eliminating the need for intravenously administered substitutes

derived from donors and animals; while small interfering RNA

and anti-sense oligonucleotides can inhibit the expression of mis-

coded or harmful proteins. By virtue of evolution, viral vectors can

effectively enter their host cells, circumvent the various cellular

barriers and deliver the gene of interest to the nucleus for

expression. Viruses such as lentivirus are able to integrate the gene

of interest into the host genome, resulting in high-level, sustained

protein expression. To date, viral vectors are highly successful in the

laboratory. However, there is significant concern regarding their use

as gene delivery vectors due to their immunogenicity and ability to

cause insertional mutagenesis [71–73].

The immunogenicity of viral capsid proteins have also led to

their efficacy as adjuvants and antigens in vaccination. Currently,

attenuated and inactivated viruses are routinely used in vaccines

against infectious diseases such as influenza and smallpox. There

is increasing interest in using viruses as delivery vectors for DNA

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158 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

vaccination, in which the virus serves to deliver the gene sequence

encoding the antigenic peptide and as an adjuvant.

Virus-like particles (VLPs) are repeated peptide arrays assem-

bled from the structural proteins of viruses, but do not contain any

genetic material. Hence, VLPs are non-infectious. For instance, the

expression of the small envelope protein of the hepatitis B (HBV)

virus in yeast or mammalian cells resulted in the production of

22 nm VLPs that formed the basis of first generation HBV vaccines

[74]. More recently, VLPs assembled from recombinant L1 protein of

human papillomavirus (HPV) were used as a basis for HPV vaccines.

In the clinical trials, the HPV VLP vaccine was well tolerated and

highly immunogenic even in the absence of additional adjuvants,

with a majority of the recipients having serum antibody titers

that were approximately 40 times greater than observed in natural

infection [75]. The self-adjuvanting effects of VLPs are attributed to

their size, which facilitates uptake by dendritic cells (responsible for

MHC class II response).

VLPs are also used to display foreign antigenic epitopes or

targeting molecules. Synthetic peptides are of great interest as

antigens due to their precise chemical definition, allowing great

control over the exact epitopes against which an immune response

is to be raised. However, most synthetic peptides are poorly

immunogenic in the absence of a co-administered adjuvant [76].

VLPs provide the spatial structure for the display of conformational

epitopes and in doing so are more likely to mimic the native

virus structure, thereby enhancing the production of neutralizing

antibodies [74]. By modifying the VLP gene sequence, fusion

proteins of VLP and antigenic peptides are assembled into VLPs

during de novo synthesis. Some VLPs are also capable of presenting

multiple vaccine antigens. BioHepB, a recombinant HBV-derived VLP

from mammalian cells, is a good example [77]. The incorporation

of both the preS1 and preS2 viral envelope antigens with the

HBV self-assembling structural protein (HBsAg), resulted in VLPs

that better mimicked the virion envelope. The resulting vaccine

elicited antibody responses to preS1 and preS2 antigens, and

more importantly, enhanced T-cell mediated immunity, leading to

an earlier antibody response and improved seroprotection rate.

Foreign antigenic epitopes can also be chemically conjugated to

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Peptide Self-Assembly in Nanofabrication 159

pre-formed VLPs, enabling the presentation of non-protein antigens

such as nicotine [78].

6.4 Peptide Self-Assembly in Nanofabrication

6.4.1 Biomineralization

Biomineralization is the process by which hierarchically structured

materials are biologically produced for various purposes such

as mechanical strength and defense [79, 80]. A critical step in

biomineralization is the crystallization of inorganic compounds in

the presence of organic molecules [81]. This process has been

widely studied in order to develop strategies for the fabrication of

novel biomaterials based on synthetic organic–inorganic composites

[80]. Self-assembling amphiphilic peptides have been used in the

development of these synthetic methods to mimic the biological

system of mineral crystal nucleation, growth, and morphology

[82–84].

One mineral of interest is calcium carbonate (CaCO3), the most

abundant biomineral and an important structural biomaterial. Much

of synthetically produced CaCO3, however, does not strongly resem-

ble naturally occurring CaCO3 [79]. To address this issue, Volkmer

and colleagues used amphiphilic peptides comprising alternating

hydrophilic (aspartic acid) and hydrophobic (phenylalanine) amino

acid residues to imitate the epitopes of acidic proteins found in

calcified tissues [81]. They found that the amphiphilic peptides H-

(FD)2-OH and H-(FD)4-OH selectively interacted with two distinct

faces of calcite. In addition, scanning electron micrograph (SEM)

images revealed that the calcite crystals grown in the presence

of these amphiphilic peptides had similar morphological features

to the calcite crystals grown in solutions containing natural acidic

proteins.

Self-assembling peptide amphiphiles have been used to in-

vestigate the process of CaCO3 mineralization. Kros, Sommerdijk

and coworkers used an amphiphilic lipopeptide to investigate the

influence of structural adaption of the organic template on calcite

crystal growth [83]. They modified the water-soluble octapeptide

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160 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

(LE)4 with a phospholipid moiety, which self-assembled into a

flexible 2D β-sheet. Using this peptide as a flexible template for the

mineralization of CaCO3, nucleation of different crystal faces was

achieved. In addition, they discovered a new type of oriented calcite

crystal nucleated underneath the flexible amphiphilic lipopeptide

monolayer. This formation was observed to be in much less abun-

dance when a less flexible template was used, demonstrating the

importance of organic template flexibility in CaCO3 mineralization

[85].

The mineralization of hydroxyapatite for bone tissue engineering

can also be directed by self-assembling peptides [12, 83, 84].

The multiple levels of hierarchical organization present within

bone structure (such as the specific organization of organic and

inorganic nanophases [12, 86]) present challenges to synthesizing

materials that resemble bone. Stupp and colleagues attempted to

address these challenges by designing a lipopeptide to fabricate a

material that maintains the nanoscale orientation between collagen

and hydroxyapapite found in bone [12]. The peptide amphiphile

consisted of five key structural features: (i) a long alkyl tail to

impart a hydrophobic region to the molecule, (ii) four consecutive

cysteine residues to form disulfide bonds for polymerization of

the self-assembled structure, (iii) three glycine residues to provide

flexibility, (iv) a single phosphorylated serine residue to interact

with calcium ions and direct the mineralization of hydroxyapatite,

and (v) the cell adhesion ligand RGD. This specially designed

peptide amphiphile was found to self-assemble into fibers at pH

4, and subsequent oxidation of the cysteine thiol groups allowed

for stable fibers in alkaline solutions. Additionally, mineralization

of hydroxyapatite resulted in crystals that were co-aligned with

the long axes of the fibers. The resulting structure formed by

the nanofibers thus resembled the organization of hydroxyapatite

crystals in mineralized ECM, an important step in bone formation.

Many self-assembling materials are macroscopically disordered,

and the ability to control the placement and orientation of am-

phiphile peptide nanofibers may be of added benefit for biominer-

alization by adding another level of order. Hung and colleagues were

able to demonstrate the simultaneous self-assembly, alignment, and

patterning of peptide amphiphiles containing either RGD or IKVAV

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Peptide Self-Assembly in Nanofabrication 161

sequences by soft lithography [87]. The nanofibers oriented parallel

to the microchannels that were produced by an elastomeric stamp

as they assembled out of solution. This patterned surface containing

epitopes for promoting cell adhesion can be advantageous in

controlling cell behavior, and can be modified for use in templated

biomineralization.

6.4.2 Synthesis of Inorganic Nanoparticles

The synthesis and assembly of inorganic nanoparticles, such as Au

and CdS, are of great interest for biomedical imaging, therapeutics,

and sensing due to their unique optical, magnetic, and electronic

properties [88, 89]. Nanocomposites of these inorganic nanocrystals

with organic matrices can lead to enhanced properties not observed

in the individual components [90]. Additionally, 1D composites

of inorganic nanoparticles with organic nanofibers can be used

as components of electrical or optical biodevices [90]. The use

of peptide amphiphile nanofibers as organic matrices for the

fabrication of these inorganic nanoparticles has recently been in-

vestigated. Kelly and coworkers, for example, designed amphiphilic

peptidomimetics that self-assembled into 2D β-sheet monolayers at

the air–water interface [91]. They found that this easily synthesized

peptidomimetic amphiphile allowed for the nucleation of CdS

crystals. The Stupp group has also investigated the use of peptide-

based nanofibers for the nucleation and growth of CdS nanocrystals

[92]. Without the nanofiber template, they observed a different

polymorph of CdS in which the particles were no longer quantum

confined, indicating that peptide nanofiber templated CdS nucleated

on the nanofibers themselves. Peptide amphiphiles consisting of

a 14-carbon acid-based tail and a GG-COO− head group have also

been examined for synthesizing CdS nanoparticles [90]. Zhou and

colleagues found that these amphiphiles self-assembled into hollow

cylindrical structures in aqueous solutions and that the COO− head

group allowed for coordination with Cd2+ ions. The result was

the formation of uniformly dispersed CdS nanoparticles in the

membrane wall of the peptide amphiphile nanotube.

Stupp and coworkers also reported the one-dimensional as-

sembly of Au nanoparticles in apolar solvents using peptide-based

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

162 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

nanofibers [93]. The peptide nanofiber was formed by the co-

assembly of two different peptides: a tripeptide amphiphile and

a peptide amphiphile containing a thymine moiety as a binding

site. When a solution of Au nanoparticles functionalized with

diaminopyridine was added to the nanofiber suspension, linear

arrays of Au nanoparticles formed as a result of the specific

binding between the nanoparticles and the nanofibers. These results

indicate that peptide-based nanofibers can be used as a platform for

constructing 1D assemblies of lipophilic inorganic nanoparticles.

The synthesis of silica-based nanoparticles and nanotubes have

also garnered much interest in the field of bionanotechnology due to

their potential in drug delivery and biosensing [94]. In this regard,

an 11-residue peptide was designed by Meegan and colleagues to

assemble into 1D antiparallel β-sheets, which further assembled to

fibrils [95]. Tetraethylorthosilicate (TEOS) was added to the fibril

suspensions and it was observed that the fibrils became coated

with silica, resulting in silica nanotubes. More recently, Yuwono

and colleagues explored the use of peptide amphiphiles containing

four alanine residues followed by four lysine or histidine residues

as templates for TEOS polymerization in the formation of silica

nanotubes [96]. They found that the peptide amphiphile fibers acted

as a nucleating agent and catalyst for silica formation. In addition,

the thickness of the silica coating could be tuned by modifying

the fiber length. In another work, Xu and coworkers investigated

the use of ultrashort peptides that can self-assemble into stable

nanostructures for the fabrication of silica nanotubes [97]. They

found that the peptide I3K self-assembled into nanotubes and were

stable against heating or exposure to organic solvents. Furthermore,

the Lys groups on the inner and outer surfaces of the nanotube

were able to catalyze silicification, leading to the formation of silica

nanotubes.

6.5 Molecular Imaging

Molecular imaging has become a vital tool both in the research

setting for studying the molecular basis of diseases, and in the

clinical setting such as for locating a tumor in the body [98].

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Molecular Imaging 163

Although enormous strides have been made in imaging techniques,

there is still a need for the development of new biologically

compatible imaging probes and contrast agents that are specific for

certain cell markers or tissues [98]. Peptide amphiphiles can play

a role in the development of nanocarriers to target imaging probes

to the diseased site. For example, Tanisaka and coworkers devel-

oped nonionic amphiphilic copolypeptides comprising hydrophilic

poly(sarcosine) and hydrophobic poly(γ -methyl L-glutamate) blocks

[99]. These polypeptides were able to form vesicular assemblies, or

peptosomes, and could be labeled with a near-infrared fluorescence

(NIRF) probe. In vivo evaluation indicated that the peptosomes

had a long circulation time in the mouse. In addition, due to the

enhanced permeability and retention (EPR) effect, successful NIRF

imaging of a small cancer on the mouse using the peptosome

was achieved. In another example, Berti and colleagues developed

a thiol-reactive iodoacetyl polyhistidine linker for conjugation to

DNA [100]. The peptidyl linker allowed the DNA to self-assemble

with luminescent quantum dots. The quantum dot bioconjugates

were found to withstand strongly in reducing environments such as

the intracellular cytoplasm, indicating their potential as fluorescent

probes for cellular imaging.

In addition to the development of fluorescent probes, self-

assembling peptide amphiphiles have also been investigated as

carriers of contrast agents in magnetic resonance imaging (MRI).

The paramagnetic metal ion Gd(III) is widely used as a MR agent due

to its long electronic relaxation time. However, a high concentration

of Gd(III) is needed due to a lack of in vivo sensitivity [101, 102].

To increase sensitivity for obtaining contrast over an extended

period of time, the relaxivity of the MR agent needs to be increased.

This can be achieved by increasing the molecular weight of the

MR agent by conjugation to larger compounds [101]. Accardo and

colleagues approached this challenge by designing a mixed micelle

comprising a C18 hydrophobic moiety bound to an octapeptide

amide (CCK8) and a second amphiphilic compound containing a

chelating moiety or its Gd complex [102]. Evaluation of the micelle

showed that the bioactive peptide CCK8 was pointed toward the

external surface. This bioactive peptide can be used to target

receptors that are overexpressed in various cancers such as small

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164 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

cell lung and colon cancer. Additionally, relaxivity values of the

mixed micelles were also largely enhanced in comparison to the Gd

complex alone. In another work, Stupp and coworkers modified two

peptide amphiphiles with 1,4,7,10-tetraazacyclododecane-1,4,7,10

tetraactic acid (DOTA) for chelation of Gd(III) [101]. Self-assembly

of these peptide amphiphile contrast agents resulted in nanofibers

or spherical micelles. Integration of additional bioactive functions

and an increase in the T1 relaxation time indicates the potential

of peptide amphiphiles in the fabrication of target-specific contrast

agents.

6.6 Peptide Organogels

Organogels are applied in a wide variety of fields including

chemistry, pharmaceuticals, cosmetics, biotechnology, and food

technology. Organogels are viscoelastic systems comprising a

continuous non-aqueous liquid phase immobilized in a three-

dimensional network of self-assembled, crosslinked, or entangled

gelator fibers. The organogelators are either low molecular weight

(LMW) or polymers. The continuous phase is typically an organic

solvent, mineral, or vegetable oil. The formation of the gelling matrix

depends on the components’ physicochemical properties and their

resulting interactions [103, 104]. The organogelator entraps the

liquid phase by forming a network of crosslinked and entangled

chains in chemical and physical gels, respectively. Physical gels can

be further stabilized by weak inter-chain interactions such as hydro-

gen bonding, van der Waals forces, and π–π interactions. Depending

on the interactions between the organogelators and liquid phase,

either solid or fluid matrix organogels are obtained [103].

Solid matrix gels are formulated by dissolving the gelator in the

heated solvent, wherein the concentration can be as low as 0.1%

as reported in case of sugar-derived supergelators [105]. Aggregate

formation occurs when the affinity between the organogelator and

solvent decreases at lower temperatures. The solvent-aggregate

affinity further limits phase separation. Solid matrix gels are

also more robust and stronger compared to the fluid matrices,

as their fiber networks have relatively large (pseudo)crystalline

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Peptide Organogels 165

microdomains [103] as junction points. Other factors such as steric

effects, polarity, supramolecular chirality, thermoreversibility also

influence organogel formation.

Fluid matrix gels are formed when polar solvents are incor-

porated into organic solutions of organogelator. They consist of

fluid fibers that entrap the organic solvent. As the aggregate size

increases, these structures eventually entangle, immobilizing the

solvent via surface tension. These gels are weaker than solid

matrices due to the aggregate fluidity and the transience of junction

points. These structures are also referred to as “worm-like” or

“polymer-like” networks [103].

When dispersed in organic solvents, some peptides self-assemble

into macromolecular structures that entrap the solvent, forming

peptide organogels. In these systems, two types of interactions

stabilize their self-assembled structures — solvophobic interactions

with the solvent molecules and interactions between solvophobic

groups in the interior of the aggregate. Lyon et al. demonstrated

that the oxidized disulfide form of tripeptide glutathione (γ -

ECG) are low molecular weight organogelators. The tripeptide

adopted an intramoleular, antiparallel β-sheet-type conformation

that was further stabilized by the restricted rotation about the

disulphide bond. In bulk aqueous solutions of dimethyl sulfoxide,

dimethylformamide, and methanol, transparent thermoreversible

gels were formed [106]. Studies by Banerjee and co-workers

revealed the importance of π–π interactions between adjacent

phenylalanine residues in Boc-LVFFA-OMe pentapeptide for self-

assembly and gelation in organic solvents (benzene, toluene, m-

xylene, and 1,2-dichlorobnzene) [107]. Understanding the different

interactions that lead to peptide self-assembly in organic solvents

will facilitate the design and synthesis of new gelators.

Self-assembled micellar structures can also be obtained when

peptides are dispersed in organic solvents. Micelles of Boc-

protected isoleucine pentapeptide (Boc-IIIII-OMe) were stabilized

by intermolecular hydrogen bonding in presence of solvents like

chloroform and N, N -dimethylformamide. At higher peptide con-

centrations, the micelles formed fibrils with β-sheet conformation

()[108]. Other macrostructures obtained include β-turn fibers from

diphenylalanine dimer peptides [109].

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

166 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

Peptide organogels have also been formulated from peptide den-

drimers and synthetic peptide–polymer conjugates. Peptide-based

dendritic gels are a bridge between polymeric and low molecular

weight organogelators [110]. Smith and colleagues performed a

systematic study of lysine-based dendritic gelators. By altering the

spacer chain, generation of dendrons and dendrimers, solvents,

stereochemistry, stoichiometric ratio of the two components and

peripheral groups, the characteristics of the resulting organogels

can be modulated [111–114]. Photoreversible dendritic organogels

have been obtained from second generation poly(Gly-Asp) dendrons

containing an azobenzene focal point [115].

Like their hydrogel counterparts, organogels are of great interest

as drug delivery devices. l-alanine derivatives in safflower oil gel

in situ following subcutaneous injection, forming a depot for the

sustained delivery of leuprolide in prostate cancer treatment [116].

Acelofenac delivery using N -Lauroyl- l alanine derivatives (LA)

was described by Anne-Claude and co-workers. The fluid matrix

organogels were formulated using soya bean oil as the continuous

phase and ethanol as the cosolvent [117]. Palui et al. demonstrated

the use of dendrimers of l-aspartic acid and succinic acid in gelling

edible oils [110]. The ability to use peptides to structure edible oils

is of interest in the field of food technology.

In other fields, Yan et al. explored the use of peptide-based

organogels as stabilizers for storage of inorganic materials. Channon

and co-workers also presented interesting work in utilizing self-

assembling peptide fibers to formulate light-harvesting nanomate-

rials [118].

6.7 Conclusions

Taking inspiration from naturally occurring self-assembling pro-

teins such as collagen, elastin, silk, and viral capsids, scientists

experimented with various self-assembling peptide motifs in an

effort to understand the molecular interactions that drive self-

assembly. In the process, they discovered novel self-assembling

peptide sequences and developed new soft biomaterials [119,

120] for a variety of biomedical and technological applications

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

References 167

[4, 15, 121]. In this review, we focused on several major as-

pects of bionanotechnology in which self-assembling peptides

have lead to significant improvements in regenerative medicine,

delivery of bioactive therapeutics, nanofabrication, and molecular

imaging.

References

1. Feynman R P (1960). There’s Plenty of Room at the Bottom. AQ:

Please

provide

the

complete

details of

refer-

ence

1.

2. Goodsell D S (2004). Bionanotechnology: Lessons from Nature (New

Jersey: Wiley-Liss Inc).

3. Kyle S, Aggeli A, Ingham E, and McPherson M J (2009). Production

of Self-Assembling Biomaterials for Tissue Engineering, Trends inBiotechnology, 27, 423–433.

4. Zhang S G (2003). Fabrication of Novel Biomaterials through Molecular

Self-Assembly, Nature Biotechnology, 21, 1171–1178.

5. Kopecek J and Yang J (2009). Peptide-Directed Self-Assembly of

Hydrogels, Acta Biomaterialia, 5, 805–816.

6. Kelleher C M and Vacanti J P (2010). Engineering Extracellular Matrix

through Nanotechnology, Journal of the Royal Society, Interface / theRoyal Society, 7 (Suppl 6), S717–S729.

7. Prestwich G D (2007). Simplifying the Extracellular Matrix for 3-D

Cell Culture and Tissue Engineering: A Pragmatic Approach, Journal ofCellular Biochemistry, 101, 1370–1383.

8. Kokkoli E, Mardilovich A, Wedekind A, Rexeisen E L, Garg A, and Craig J

A (2006). Self-Assembly and Applications of Biomimetic and Bioactive

Peptide-Amphiphiles, Soft Matter, 2, 1015–1024.

9. Sun J, Zheng Q, Wu Y, Liu Y, Guo X, and Wu W (2010). Culture of

Nucleus Pulposus Cells from Intervertebral Disc on Self-Assembling

Kld-12 Peptide Hydrogel Scaffold, Materials Science and Engineering:C, 30, 975–980.

10. Mari-Buye N, Muinos M T F, and Semino C E (2010). Methods inBioengineering, ed (Berthiaume F. and M J. Massachussets: Artech

House).

11. Shastri V P (2009). In vivo Engineering of Tissues: Biological Con-

siderations, Challenges, Strategies, and Future Directions, AdvancedMaterials, 21, 3246–3254.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

168 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

12. Hartgerink J D, Beniash E, and Stupp S I (2001). Self-Assembly and

Mineralization of Peptide-Amphiphile Nanofibers, Science, 294, 1684–

1688.

13. Tysseling-Mattiace V M, Sahni V, Niece K L, Birch D, Czeisler C, Fehlings

M G, Stupp S I, and Kessler J A (2008). Self-Assembling Nanofibers

Inhibit Glial Scar Formation and Promote Axon Elongation after Spinal

Cord Injury, The Journal of Neuroscience, 28, 3814–3823.

14. Silva G A, Czeisler C, Niece K L, Beniash E, Harrington D A, Kessler J

A, and Stupp S I (2004). Selective Differentiation of Neural Progenitor

Cells by High-Epitope Density Nanofibers, Science, 303, 1352–1355.

15. Hauser C A E and Zhang S G (2010). Designer Self-Assembling Peptide

Nanofiber Biological Materials, Chemical Society Reviews, 39, 2780–

2790.

16. Zhang S, Holmes T, Lockshin C, and Rich A (1993). Spontaneous

Assembly of a Self-Complementary Oligopeptide to Form a Stable

Macroscopic Membrane, Proceedings of the National Academy ofSciences of the United States of America, 90, 3334–3338.

17. Kisiday J, Jin M, Kurz B, Hung H, Semino C, Zhang S, and Grodzinsky

A J (2002). Self-Assembling Peptide Hydrogel Fosters Chondrocyte

Extracellular Matrix Production and Cell Division: Implications for

Cartilage Tissue Repair, Proceedings of the National Academy ofSciences of the United States of America, 99, 9996–10001.

18. Horii A, Wang X, Gelain F, and Zhang S (2007). Biological Designer

Self-Assembling Peptide Nanofiber Scaffolds Significantly Enhance

Osteoblast Proliferation, Differentiation and 3-D Migration, PloS One,

2, e190.

19. Semino C E, Merok J R, Crane G G, Panagiotakos G, and Zhang S (2003).

Functional Differentiation of Hepatocyte-Like Spheroid Structures

from Putative Liver Progenitor Cells in Three-Dimensional Peptide

Scaffolds, Differentiation: Research in Biological Diversity, 71, 262–270.

20. Ellis-Behnke R G, Liang Y X, You S W, Tay D K, Zhang S, So K F, and

Schneider G E (2006). Nano Neuro Knitting: Peptide Nanofiber Scaffold

for Brain Repair and Axon Regeneration with Functional Return of

Vision, Proceedings of the National Academy of Sciences of the UnitedStates of America, 103, 5054–5059.

21. Misawa H, Kobayashi N, Soto-Gutierrez A, Chen Y, Yoshida A, Rivas-

Carrillo J D, Navarro-Alvarez N, Tanaka K, Miki A, Takei J, Ueda T, Tanaka

M, Endo H, Tanaka N, and Ozaki T (2006). Puramatrix Facilitates Bone

Regeneration in Bone Defects of Calvaria in Mice, Cell Transplantation,

15, 903–910.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

References 169

22. Semino C E (2008). Self-Assembling Peptides: From Bio-Inspired

Materials to Bone Regeneration, Journal of Dental Research, 87, 606–

616.

23. Ozeki M, Kuroda S, Kon K, and Kasugai S (2011). Differentiation of

Bone Marrow Stromal Cells into Osteoblasts in a Self-Assembling

Peptide Hydrogel: In vitro and in vivo Studies, Journal of BiomaterialsApplications, 25, 663–684.

24. Sun J, Zheng Q, Wu Y, Liu Y, Guo X, and Wu W (2010). Biocompatibility

of KLD-12 Peptide Hydrogel as a Scaffold in Tissue Engineering of

Intervertebral Discs in Rabbits, Journal of Huazhong University ofScience and Technology. Medical sciences, 30, 173–177.

25. Ellis-Behnke R G, Liang Y X, Tay D K, Kau P W, Schneider G E, Zhang

S, Wu W, and So K F (2006). Nano Hemostat Solution: Immediate

Hemostasis at the Nanoscale, Nanomedicine : Nanotechnology, Biology,and Medicine, 2, 207–215.

26. Luo Z L, Wang S K, and Zhang S G (2011). Fabrication of Self-

Assembling D-Form Peptide Nanofiber Scaffold D-Eak16 for Rapid

Hemostasis, Biomaterials, 32, 2013–2020.

27. Aggeli A, Bell M, Boden N, Keen J N, Knowles P F, McLeish T C B,

Pitkeathly M, and Radford S E (1997). Responsive Gels Formed by

the Spontaneous Self-Assembly of Peptides into Polymeric Beta-Sheet

Tapes, Nature, 386, 259–262.

28. Aggeli A, Bell M, Carrick L M, Fishwick C W, Harding R, Mawer P J,

Radford S E, Strong A E, and Boden N (2003). Ph as a Trigger of Peptide

Beta-Sheet Self-Assembly and Reversible Switching between Nematic

and Isotropic Phases, Journal of the American Chemical Society, 125,

9619–9628.

29. Ashley Firth A A, Julie L Burke, Xuebin Yang, and Jennifer Kirkham

(2006). Biomimetic Self-Assembling Peptides as Injectable Scaffolds

for Hard Tissue Engineering, Nanomedicine, 1, 189–199.

30. Kirkham J, Firth A, Vernals D, Boden N, Robinson C, Shore R C, Brookes

S J, and Aggeli A (2007). Self-Assembling Peptide Scaffolds Promote

Enamel Remineralization, Journal of Dental Research, 86, 426–430.

31. Yan C, Altunbas A, Yucel T, Nagarkar R P, Schneider J P, and Pochan D

J (2010). Injectable Solid Hydrogel: Mechanism of Shear-Thinning and

Immediate Recovery of Injectable B-Hairpin Peptide Hydrogels, SoftMatter, 6, 5143–5156.

32. Pochan D J, Schneider J P, Kretsinger J, Ozbas B, Rajagopal K, and Haines

L (2003). Thermally Reversible Hydrogels Via Intramolecular Folding

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

170 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

and Consequent Self-Assembly of a De Novo Designed Peptide, Journalof the American Chemical Society, 125, 11802–11803.

33. Bromley E H, Sessions R B, Thomson A R, and Woolfson D N (2009).

Designed Alpha-Helical Tectons for Constructing Multicomponent

Synthetic Biological Systems, Journal of the American Chemical Society,

131, 928–930.

34. Moutevelis E and Woolfson D N (2009). A Periodic Table of Coiled-Coil

Protein Structures, Journal of Molecular Biology, 385, 726–732.

35. Papapostolou D, Smith A M, Atkins E D, Oliver S J, Ryadnov M G,

Serpell L C, and Woolfson D N (2007). Engineering Nanoscale Order

into a Designed Protein Fiber, Proceedings of the National Academy ofSciences of the United States of America, 104, 10853–10858.

36. Banwell E F, Abelardo E S, Adams D J, Birchall M A, Corrigan A, Donald

A M, Kirkland M, Serpell L C, Butler M F, and Woolfson D N (2009).

Rational Design and Application of Responsive Alpha-Helical Peptide

Hydrogels, Nature Materials, 8, 596–600.

37. Zhang S, Greenfield M A, Mata A, Palmer L C, Bitton R, Mantei J R,

Aparicio C, de la Cruz M O, and Stupp S I (2010). A Self-Assembly

Pathway to Aligned Monodomain Gels, Nature Materials, 9, 594–601.

38. Beniash E, Hartgerink J D, Storrie H, Stendahl J C, and Stupp S I

(2005). Self-Assembling Peptide Amphiphile Nanofiber Matrices for

Cell Entrapment, Acta Biomaterialia, 1, 387–397.

39. Jun H W, Yuwono V, Paramonov S E, and Hartgerink J D (2005).

Enzyme-Mediated Degradation of Peptide-Amphiphile Nanofiber Net-

works, Advanced Materials, 17, 2612–2617.

40. Yu Y C, Berndt P, Tirrell M, and Fields G B (1996). Self-Assembling

Amphiphiles for Construction of Protein Molecular Architecture,

Journal of the American Chemical Society, 118, 12515–12520.

41. Gore T, Dori Y, Talmon Y, Tirrell M, and Bianco-Peled H (2001).

Self-Assembly of Model Collagen Peptide Amphiphiles, Langmuir, 17,

5352–5360.

42. Dori Y, Bianco-Peled H, Satija S K, Fields G B, McCarthy J B, and Tirrell

M (2000). Ligand Accessibility as Means to Control Cell Response

to Bioactive Bilayer Membranes, Journal of Biomedical MaterialsResearch, 50, 75–81.

43. Pakalns T, Haverstick K L, Fields G B, McCarthy J B, Mooradian D

L, and Tirrell M (1999). Cellular Recognition of Synthetic Peptide

Amphiphiles in Self-Assembled Monolayer Films, Biomaterials, 20,

2265–2279.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

References 171

44. Galler K M, Cavender A, Yuwono V, Dong H, Shi S T, Schmalz G,

Hartgerink J D, and D’Souza R N (2008). Self-Assembling Peptide

Amphiphile Nanofibers as a Scaffold for Dental Stem Cells, TissueEngineering Part A, 14, 2051–2058.

45. Brun P, Ghezzo F, Roso M, Danesin R, Palu G, Bagno A, Modesti

M, Castagliuolo I, and Dettin M (2011). Electrospun Scaffolds of

Self-Assembling Peptides with Poly(Ethylene Oxide) for Bone Tissue

Engineering, Acta Biomaterialia, 7(6), 2526–2532.

46. Hauser C A, Deng R, Mishra A, Loo Y, Khoe U, Zhuang F, Cheong D W,

Accardo A, Sullivan M B, Riekel C, Ying J Y, and Hauser U A (2011).

Natural Tri- to Hexapeptides Self-Assemble in Water to Amyloid

Beta-Type Fiber Aggregates by Unexpected Alpha-Helical Intermediate

Structures, Proceedings of the National Academy of Sciences of theUnited States of America, 108, 1361–1366.

47. Mishra A, Loo Y, Deng R, Chuah Y J, Hee H T, Ying J Y, and Hauser

C A E (2011). Ultrasmall Natural Peptides Self-Assemble to Strong

Temperature-Resistant Helical Fibers in Scaffolds Suitable for Tissue

Engineering, Nano Today, In Press, Corrected Proof AU:

Please

update

refer-

ence

47.

48. Haider M, Megeed Z, and Ghandehari H (2004). Genetically Engineered

Polymers: Status and Prospects for Controlled Release, Journal ofControlled Release, 95, 1–26.

49. Megeed Z, Cappello J, and Ghandehari H (2002). Genetically Engi-

neered Silk-Elastinlike Protein Polymers for Controlled Drug Delivery,

Advanced Drug Delivery Reviews, 54, 1075–1091.

50. Cappello J, Crissman J W, Crissman M, Ferrari F A, Textor G, Wallis

O, Whitledge J R, Zhou X, Burman D, Aukerman L, and Stedronsky

E R (1998). In-Situ Self-Assembling Protein Polymer Gel Systems for

Administration, Delivery, and Release of Drugs, Journal of ControlledRelease, 53, 105–117.

51. Dinerman A A, Cappello J, Ghandehari H, and Hoag S W (2002).

Swelling Behavior of a Genetically Engineered Silk-Elastinlike Protein

Polymer Hydrogel, Biomaterials, 23, 4203–4210.

52. Dinerman A A, Cappello J, Ghandehari H, and Hoag S W (2002). Solute

Diffusion in Genetically Engineered Silk-Elastinlike Protein Polymer

Hydrogels, Journal of Controlled Release, 82, 277–287.

53. Megeed Z, Haider M, Li D, O’Malley B W, Jr., Cappello J, and Ghandehari

H (2004). In vitro and in vivo Evaluation of Recombinant Silk-

Elastinlike Hydrogels for Cancer Gene Therapy, Journal of ControlledRelease, 94, 433–445.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

172 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

54. Nagarsekar A, Crissman J, Crissman M, Ferrari F, Cappello J, and Ghan-

dehari H (2003). Genetic Engineering of Stimuli-Sensitive Silkelastin-

Like Protein Block Copolymers, Biomacromolecules, 4, 602–607.

55. MacKay J A, Chen M, McDaniel J R, Liu W, Simnick A J, and Chilkoti A

(2009). Self-Assembling Chimeric Polypeptide-Doxorubicin Conjugate

Nanoparticles That Abolish Tumours after a Single Injection, NatureMaterials, 8, 993–999.

56. Wu Y, MacKay J A, McDaniel J R, Chilkoti A, and Clark R L

(2009). Fabrication of Elastin-Like Polypeptide Nanoparticles for Drug

Delivery by Electrospraying, Biomacromolecules, 10, 19–24.

57. McDaniel J R, Callahan D J, and Chilkoti A (2010). Drug Delivery to Solid

Tumors by Elastin-Like Polypeptides, Advanced Drug Delivery Reviews,

62, 1456–1467.

58. Liu W, MacKay J A, Dreher M R, Chen M, McDaniel J R, Simnick A J, Calla-

han D J, Zalutsky M R, and Chilkoti A (2010). Injectable Intratumoral

Depot of Thermally Responsive Polypeptide-Radionuclide Conjugates

Delays Tumor Progression in a Mouse Model, Journal of ControlledRelease, 144, 2–9.

59. MacEwan S R and Chilkoti A (2010). Elastin-Like Polypeptides:

Biomedical Applications of Tunable Biopolymers, Biopolymers, 94, 60–

77.

60. Hassouneh W, Christensen T, and Chilkoti A (2010). Elastin-Like

Polypeptides as a Purification Tag for Recombinant Proteins, CurrentProtocols in Protein Science, Chapter: Unit 6.11.

61. Petka W A, Harden J L, McGrath K P, Wirtz D, and Tirrell D A (1998).

Reversible Hydrogels from Self-Assembling Artificial Proteins, Science,

281, 389–392.

62. Vandermeulen G W and Klok H A (2004). Peptide/Protein Hybrid

Materials: Enhanced Control of Structure and Improved Performance

through Conjugation of Biological and Synthetic Polymers, Macromole-cular Bioscience, 4, 383–398.

63. Wang C, Kopecek J, and Stewart R J (2001). Hybrid Hydrogels

Cross-Linked by Genetically Engineered Coiled-Coil Block Proteins,

Biomacromolecules, 2, 912–920.

64. Yang J, Xu C, Wang C, and Kopecek J (2006). Refolding Hydrogels Self-

Assembled from N -(2-Hydroxypropyl)Methacrylamide Graft Copoly-

mers by Antiparallel Coiled-Coil Formation, Biomacromolecules, 7,

1187–1195.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

References 173

65. Yamaguchi N and Kiick K L (2005). Polysaccharide-Poly(Ethylene

Glycol) Star Copolymer as a Scaffold for the Production of Bioactive

Hydrogels, Biomacromolecules, 6, 1921–1930.

66. Vandermeulen G W, Hinderberger D, Xu H, Sheiko S S, Jeschke G,

and Klok H A (2004). Structure and Dynamics of Self-Assembled

Poly(Ethylene Glycol) Based Coiled-Coil Nano-Objects, Chemphyschem,

5, 488–494.

67. Williams D A and Baum C (2003). Medicine. Gene Therapy–New

Challenges Ahead, Science, 302, 400–401.

68. Cavazzana-Calvo M, Thrasher A, and Mavilio F (2004). The Future of

Gene Therapy, Nature, 427, 779–781.

69. Nishikawa M and Huang L (2001). Nonviral Vectors in the New

Millennium: Delivery Barriers in Gene Transfer, Human Gene Therapy,

12, 861–870.

70. Thomas M and Klibanov A M (2003). Non-Viral Gene Therapy:

Polycation-Mediated DNA Delivery, Applied Microbiology and Biotech-nology, 62, 27–34.

71. Kay M A, Glorioso J C, and Naldini L (2001). Viral Vectors for

Gene Therapy: The Art of Turning Infectious Agents into Vehicles of

Therapeutics, Nature Medicine, 7, 33–40.

72. Walther W and Stein U (2000). Viral Vectors for Gene Transfer: A

Review of Their Use in the Treatment of Human Diseases, Drugs, 60,

249–271.

73. Romano G, Marino I R, Pentimalli F, Adamo V, and Giordano A (2009).

Insertional Mutagenesis and Development of Malignancies Induced by

Integrating Gene Delivery Systems: Implications for the Design of Safer

Gene-Based Interventions in Patients, Drug News & Perspectives, 22,

185–196.

74. Grgacic E V and Anderson D A (2006). Virus-Like Particles: Passport to

Immune Recognition, Methods, 40, 60–65.

75. Harro C D, Pang Y Y, Roden R B, Hildesheim A, Wang Z, Reynolds M J,

Mast T C, Robinson R, Murphy B R, Karron R A, Dillner J, Schiller J T, and

Lowy D R (2001). Safety and Immunogenicity Trial in Adult Volunteers

of a Human Papillomavirus 16 L1 Virus-Like Particle Vaccine, Journalof the National Cancer Institute, 93, 284–292.

76. Rudra J S, Tian Y F, Jung J P, and Collier J H (2010). A Self-Assembling

Peptide Acting as an Immune Adjuvant, Proceedings of the NationalAcademy of Sciences of the United States of America, 107, 622–627.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

174 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

77. Madalinski K, Sylvan S P, Hellstrom U, Mikolajewicz J, Zembrzuska-

Sadkowska E, and Piontek E (2001). Antibody Responses to Pres

Components after Immunization of Children with Low Doses of

Biohepb, Vaccine, 20, 92–97.

78. Maurer P, Jennings G T, Willers J, Rohner F, Lindman Y, Roubicek K,

Renner W A, Muller P, and Bachmann M F (2005). A Therapeutic

Vaccine for Nicotine Dependence: Preclinical Efficacy, and Phase I

Safety and Immunogenicity, European Journal of Immunology, 35,

2031–2040.

79. Sommerdijk N A J M and With G d (2008). Biomimetic CaCO3

Mineralization Using Designer Molecules and Interfaces, ChemicalReviews, 108, 4499–4550.

80. Estroff L A (2008). Introduction: Biomineralization, Chemical Reviews,

108, 4329–4331.

81. Volkmer D, Fricke M, Huber T, and Sewald N (2004). Acidic Peptides

Acting as Growth Modifiers of Calcite Crystals, Chemical Communica-tions, 1872–1873.

82. Dickerson M B, Sandhage K H, and Naik R R (2008). Protein- and

Peptide-Directed Syntheses of Inorganic Materials, Chemical Reviews,

108, 4935–4978.

83. Cavalli S, Albericio F, and Kros A (2010). Amphiphilic Peptides and

Their Cross-Disciplinary Role as Building Blocks for Nanoscience,

Chemical Society Reviews, 39, 241–263.

84. Zhao X B, Pan F, Xu H, Yaseen M, Shan H H, Hauser C A E, Zhang S G, and

Lu J R (2010). Molecular Self-Assembly and Applications of Designer

Peptide Amphiphiles, Chemical Society Reviews, 39, 3480–3498.

85. Cavalli S, Popescu D C, Tellers E E, Vos M R J, Pichon B P, Overhand M,

Rapaport H, Sommerdijk N, and Kros A (2006). Self-Organizing Beta-

Sheet Lipopeptide Monolayers as Template for the Mineralization of

CaCO3, Angewandte Chemie — International Edition, 45, 739–744.

86. Weiner S and Wagner H D (1998). The Material Bone: Structure

Mechanical Function Relations, Annual Review of Materials Science, 28,

271–298.

87. Hung A M and Stupp S I (2007). Simultaneous Self-Assembly,

Orientation, and Patterning of Peptide-Amphiphile Nanofibers by Soft

Lithography, Nano Letters, 7, 1165–1171.

88. Daniel M-C and Astruc D (2003). Gold Nanoparticles: Assembly,

Supramolecular Chemistry, Quantum-Size-Related Properties, and

Applications toward Biology, Catalysis, and Nanotechnology, ChemicalReviews, 104, 293–346.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

References 175

89. Michalet X, Pinaud F F, Bentolila L A, Tsay J M, Doose S, Li J J, Sundaresan

G, Wu A M, Gambhir S S, and Weiss S (2005). Quantum Dots for Live

Cells, in vivo Imaging, and Diagnostics, Science, 307, 538–544.

90. Zhou Y, Kogiso M, He C, Shimizu Y, Koshizaki N, and Shimizu T

(2007). Fluorescent Nanotubes Consisting of CdS-Embedded Bilayer

Membranes of a Peptide Lipid, Advanced Materials, 19, 1055–1058.

91. Bekele H, Fendler J H, and Kelly J W (1999). Self-Assembling

Peptidomimetic Monolayer Nucleates Oriented CdS Nanocrystals,

Journal of the American Chemical Society, 121, 7266–7267.

92. Sone E D and Stupp S I (2004). Semiconductor-Encapsulated Peptide-

Amphiphile Nanofibers, Journal of the American Chemical Society, 126,

12756–12757.

93. Li L S and Stupp S I (2005). One-Dimensional Assembly of Lipophilic

Inorganic Nanoparticles Templated by Peptide-Based Nanofibers with

Binding Functionalities, Angewandte Chemie — International Edition,

44, 1833–1836.

94. Slowing I I, Trewyn B G, Giri S, and Lin V S Y (2007). Mesoporous

Silica Nanoparticles for Drug Delivery and Biosensing Applications,

Advanced Functional Materials, 17, 1225–1236.

95. Meegan J E, Aggeli A, Boden N, Brydson R, Brown A P, Carrick L,

Brough A R, Hussain A, and Ansell R J (2004). Designed Self-Assembled

Beta-Sheet Peptide Fibrils as Templates for Silica Nanotubes, AdvancedFunctional Materials, 14, 31–37.

96. Yuwono V M and Hartgerink J D (2007). Peptide Amphiphile

Nanofibers Template and Catalyze Silica Nanotube Formation, Lang-muir, 23, 5033–5038.

97. Xu H, Wang Y M, Ge X, Han S Y, Wang S J, Zhou P, Shan H H, Zhao X

B, and Lu J A R (2010). Twisted Nanotubes Formed from Ultrashort

Amphiphilic Peptide I3k and Their Templating for the Fabrication of

Silica Nanotubes, Chemistry of Materials, 22, 5165–5173.

98. Weissleder R and Pittet M J (2008). Imaging in the Era of Molecular

Oncology, Nature, 452, 580–589.

99. Tanisaka H, Kizaka-Kondoh S, Makino A, Tanaka S, Hiraoka M, and

Kimura S (2008). Near-Infrared Fluorescent Labeled Peptosome for

Application to Cancer Imaging, Bioconjugate Chemistry, 19, 109–117.

100. Berti L, D’Agostino P S, Boeneman K, and Medintz I L (2009). Improved

Peptidyl Linkers for Self-Assembly of Semiconductor Quantum Dot

Bioconjugates, Nano Research, 2, 121–129.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

176 Applications in Bionanotechnology of Self-Assembled Peptide Nanostructures

101. Bull S R, Guler M O, Bras R E, Meade T J, and Stupp S I (2005).

Self-Assembled Peptide Amphiphile Nanofibers Conjugated to MRI

Contrast Agents, Nano Letters, 5, 1–4.

102. Accardo A, Tesauro D, Roscigno P, Gianolio E, Paduano L, D’Errico

G, Pedone C, and Morelli G (2004). Physicochemical Properties of

Mixed Micellar Aggregates Containing CCK Peptides and Gd Complexes

Designed as Tumor Specific Contrast Agents in MRI, Journal of theAmerican Chemical Society, 126, 3097–3107.

103. Hentschel J and Borner H G (2006). Peptide-Directed Microstructure

Formation of Polymers in Organic Media, Journal of the AmericanChemical Society, 128, 14142–14149.

104. Vintiloiu A and Leroux J C (2008). Organogels and Their Use in Drug

Delivery–a Review, Journal of Controlled Release, 125, 179–192.

105. Gronwald O and Shinkai S (2001). Sugar-Integrated Gelators of Organic

Solvents, Chemistry, 7, 4328–4334.

106. Lyon R P and Atkins W M (2001). Self-Assembly and Gelation of

Oxidized Glutathione in Organic Solvents, Journal of the AmericanChemical Society, 123, 4408–4413.

107. Banerjee A, Palui G, and Banerjee A (2008). Pentapeptide Based

Organogels: The Role of Adjacently Located Phenylalanine Residues in

Gel Formation, Soft Matter, 4, 1430–1437.

108. Jayakumar R, Murugesan M, Asokan C, and Aulice Scibioh M (2000).

Self-Assembly of a Peptide Boc-(Ile)5-Ome in Chloroform and N, N -

Dimethylformamide, Langmuir, 16, 1489–1496.

109. Yan X, Cui Y, He Q, Wang K, and Li J (2008). Organogels Based

on Self-Assembly of Diphenylalanine Peptide and Their Application

to Immobilize Quantum Dots, Chemistry of Materials, 20, 1522–

1526.

110. Palui G, Garai A, Nanda J, Nandi A K, and Banerjee A (2009).

Organogels from Different Self-Assembling New Dendritic Peptides:

Morphology, Rheology, and Structural Investigations, The Journal ofPhysical Chemistry B, 114, 1249–1256.

111. Hirst A R, Smith D K, Feiters M C, and Geurts H P M (2004).

Two-Component Dendritic Gel: Effect of Spacer Chain Length on the

Supramolecular Chiral Assembly, Langmuir, 20, 7070–7077.

112. Hirst A R, Smith D K, Feiters M C, Geurts H P M, and Wright A C (2003).

Two-Component Dendritic Gels: Easily Tunable Materials, Journal ofthe American Chemical Society, 125, 9010–9011.

April 19, 2012 18:17 PSP Book - 9in x 6in 06-Edith-Svendsen-c06

References 177

113. Love C S, Hirst A R, Chechik V, Smith D K, Ashworth I, and Brennan C

(2004). One-Component Gels Based on Peptidic Dendrimers: Dendritic

Effects on Materials Properties, Langmuir, 20, 6580–6585.

114. Partridge K S, Smith D K, Dykes G M, and McGrail P T (2001).

Supramolecular Dendritic Two-Component Gel, Chemical Communica-tions, 319–320.

115. Ji Y, Kuang G-C, Jia X-R, Chen E-Q, Wang B-B, Li W-S, Wei Y,

and Lei J (2007). Photoreversible Dendritic Organogel, ChemicalCommunications, 4233–4235.

116. Plourde F, Motulsky A, Couffin-Hoarau A C, Hoarau D, Ong H, and

Leroux J C (2005). First Report on the Efficacy of l-Alanine-Based insitu-Forming Implants for the Long-Term Parenteral Delivery of Drugs,

Journal of Controlled Release, 108, 433–441.

117. Couffin-Hoarau A C, Motulsky A, Delmas P, and Leroux J C (2004). In

Situ-Forming Pharmaceutical Organogels Based on the Self-Assembly

of l-Alanine Derivatives, Pharmaceutical Research, 21, 454–457.

118. Channon K J, Devlin G L, and MacPhee C E (2009). Efficient Energy

Transfer within Self-Assembling Peptide Fibers: A Route to Light-

Harvesting Nanomaterials, Journal of the American Chemical Society,

131, 12520–12521.

119. Hamley I W and Castelletto V (2007). Biological Soft Materials,

Angewandte Chemie — International Edition, 46, 4442–4455.

120. Langer R and Tirrell D A (2004). Designing Materials for Biology and

Medicine, Nature, 428, 487–492.

121. Lehn J M (2002). Toward Self-Organization and Complex Matter,

Science, 295, 2400–2403.

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