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Solid-binding peptides: smart tools for nanobiotechnology Andrew Care1*, Peter L. Bergquist1,2, and Anwar Sunna1 1 2

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia Department of Molecular Medicine & Pathology, Medical School, University of Auckland, Auckland, New Zealand

Over the past decade, solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to the surfaces of a diverse range of solid materials including metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers, and minerals. They can direct the assembly and functionalisation of materials, and have the ability to mediate the synthesis and construction of nanoparticles and complex nanostructures. As the availability of newly synthesised nanomaterials expands rapidly, so too do the potential applications for SBPs. Peptides that bind to nanomaterials With advances in nanotechnology, a multitude of novel nanomaterials (see Glossary) have been designed and synthesised for diagnostic and therapeutic applications (e.g., upconversion nanocrystals [1]). Many of these nanomaterials suffer from low solubility and poor biocompatibility, which present potential safety concerns for in vivo applications. Furthermore, the conventional bioconjugation techniques originally developed for labelling proteins {e.g., EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and reactive NHS (N-hydroxysuccinimide) esters and maleimides} that are used to functionalise nanomaterials are often laborious and inefficient, and may also interfere with the recognition efficiency of the immobilised protein towards its specific receptor/molecular partner. Several new approaches have been developed to achieve a much greater control over the bioconjugation and, eventually, the optimal and functional display of biomolecules on the surface of nanomaterials [2,3]. SBPs are short amino acid sequences that display binding affinity for the surfaces of solid materials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilisation and orientation of nanoscale entities onto solid supports without impeding their functionality [4]. These biological molecules have been referred to variously as genetically engineered peptides for inorganics Corresponding author: Sunna, A. ([email protected]). Keywords: solid-binding peptides; functionalisation; bioconjugation; nanomaterials; biomaterials. * Current address: ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, Australia. 0167-7799/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.02.005

(GEPIs), material-binding peptides and inorganic-binding peptides (IBPs). We will use the term SBPs to refer to all of these types of peptides. SBPs have been employed in numerous nanobiotechnological applications (Table 1). These include the controlled synthesis of nanomaterials and nanostructures [5], formation of hybrid biomaterials [6], immobilisation of functional proteins [7], and improved nanomaterial biocompatibility [8]. There have been several general reviews on SBPs from recognised contributors in the field [9–11]. However, they did not emphasise the wide range of applications that take advantage of these peptides and new trends and developments in this emerging field. There are no reviews of the current status of this type of molecular tool, and we will provide a summary of recent and prospective practical applications of SBPs in nanobiotechnology and, in particular, their use as molecular building blocks for the self-assembly of functional nanoparticles and other nanomaterials. Solid-binding peptides as ‘molecular linkers’ A multitude of novel nanomaterials with unique properties (e.g., physical, chemical, optical, magnetic, and mechanical) Glossary Binding: adsorption of biomolecules (protein, peptides) to a surface. Binding affinity: the adsorption strength of a biomolecule (e.g., proteins and peptides) for a specific material. Binding selectivity: the ability of a biomolecule to discriminate between two or more material surfaces. Also referred to as surface recognition. Bioactive peptide motifs: short amino acid sequences that have a diverse range of unique biological functions, for example antimicrobial, cell penetrating, and tumour-homing. Bioconjugation: the linkage of biomolecules to one another or to a material by chemical or biological means. Biomolecules: molecules from living organisms, such as nucleic acids, proteins, enzymes, and antibodies. Biopanning: an affinity-driven selection procedure in which peptides that bind with affinity to a target substrate (e.g., solid materials and biomolecules) are isolated from a combinatorial peptide display library (e.g., phage display, cell surface display) during consecutive rounds of incubation, washing, elution, amplification, and re-selection. Functionalisation: the addition of new functions, capabilities, or properties to a material by altering its surface. Nanomaterials: materials with a size range of approximately 1–100 nm. Nanoscale: comprising dimensions less than 100 nm. Self-assembly: the spontaneous process by which molecules undergo specific organisation without the intervention of external forces. Self-assembling monolayers (SAMs): organic molecules that spontaneously organise themselves on a surfaces via adsorption, and then facilitate the covalent attachment of biomolecules to these surfaces. Solid-binding peptides (SBPs): short peptide sequences of 7–12 amino acids (which may be repeated) that recognise and bind to solid materials with high selectivity; also referred to as genetically engineered peptides for inorganics (GEPIs), material-binding peptides, or inorganic-binding peptides (IBPs).

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Table 1. SBPs mentioned in this review and their applications Charge b

Applications mentioned in this review

SBP source Ref c

Refs in this review d

8.52

+1

[71]

[19–47]

WALRRSIRRQSY

12.00

+4

[72]

[21,47,73]

WAGAKRLVLRRE LKAHLPPSRLPS VSGSSPDS

11.71 11.00 3.80

+3 +2 0

[72] [74] [75]

[47,72] [26] [35,57]

Palladium

TSNAVHPTLRHL

9.47

+1

[76]

[51,52]

Platinum

PTSTGQA TLTTLTN SSFPQPN CSQSVTSTKSC AYSSGAPPMPPF NPSSLFRYLPSD RPRENRGRERGL RKLPDA

5.96 5.19 5.24 8.06 5.57 5.84 11.82 8.75

0 0 0 +1 0 0 +3 +1

Biomolecule immobilisation; biocompatibility; multi-material fabrication Biomolecule immobilisation; biocompatibility Biocompatibility Material-specific antibody generation Biomaterial production; multi-material fabrication Nanostructure synthesis; controlled catalysis SBP binding studies Nanostructure synthesis Nanostructure synthesis Biocompatibility Biomaterial production Biomaterial production Biocompatibility Multi-material fabrication; nanostructure synthesis

[9] [78] [78] [9] [50] [50] [47] [79]

[77] [5] [5] [47] [6] [46,61] [47] [54]

RRTVKHHVN ACTARSPWICG

12.01 8.11

+3 +1

Biomolecule immobilisation Biomolecule immobilisation; biocompatibility

[23] [8]

[7,23] [8]

9.59

+1

[22]

[7,22]

11.22 7.10 8.75

+6 0 +1

[80] [81] [79]

[35] [35,36] [45,55,56]

5.95

0

[82]

[6,47,73,74]

10.90 8.86 12.01

+3 +1 +3

Biomolecule immobilisation; nanostructure synthesis Multi-material fabrication Multi-material fabrication Multi-material fabrication; nanostructure synthesis Biomolecule immobilisation; biocompatibility; biomaterial production; multi-material fabrication Biomolecule immobilisation Material-specific antibody generation Biomolecule immobilisation

[83] [84] [85]

[1,24–32] [27] [33]

KDVVVGVPGGQD

4.21

1

[86]

[64]

NPYHPTIPQSVH

6.92

0

Biomolecule immobilisation; biomaterial production Biomaterial production

[87]

[6]

EPLQLKM

6.10

0

[37]

[6,37]

HSSYWYAFNNKT

8.50

[88]

[6,36,38,89]

DYFSSPYYEQLF DSPHTELP

3.67 4.35

2 2

Biomolecule immobilisation; biomaterial production Biomolecule immobilisation, biomaterial production; multi-material fabrication Biocompatibility Biomaterial production

[90] [58]

[48] [58–60]

Semiconductors Cadmium sulphide

CTYSRKHKC

9.39

+3

[91]

[53]

Gallium arsenide Zinc sulphide

AQNPSDNNTHTH CGPAGDSSGVDSRSVGPC

5.97 4.21

0 1

[92] [62]

[93] [6,62]

CNNPMHQNC LRRSSEAHNSIV

6.72 9.61

0 +1

Multi-material fabrication; nanostructure synthesis SBP binding studies Biomolecule immobilisation; biomaterial production Biomaterial production Multi-material fabrication; nanostructure synthesis

[94] [94]

[65] [53]

Target solid material

Sequence

Metals Gold

MHGKTQATSGTIQS

Silver Titanium

Metal oxides Iron oxide Lanthanide oxide and upconversion nanocrystals Silica

MSPHPHPRHHHT SSKKSGSYSGSKGSKRRIL HPPMNASHPHMH RKLPDA

Quartz

PPPWLPYMPPWS

Zeolites Zinc oxide

VKTQATSREEPPRLPSKHRPG EAHVMHKVAPRP RPHRK

Minerals Calcium phosphate Hydroxyapatite Carbon Materials Graphene Single-walled carbon nanotubes

pI a

a

Isoelectric points (pI) were calculated using the ‘Compute pI/Mw tool’ (http://web.expasy.org/compute_pi/).

b

Calculated by subtracting the number of positively charged residues (R and K) from the number of negatively charged residues (D and E).

c

Reference in which the SBP sequence was originally identified.

d

References (in this review) in which the SBP sequence was used in an application.

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have been designed and synthesised as a result of recent advances in nanotechnology. The exceptional molecular recognition properties of biomolecules are fundamental to a variety of potential diagnostic and therapeutic applications such as targeting, sensing, imaging, and delivery. Hence, the conjugation of biomolecules onto nanomaterials to impart biological functions is of growing significance in many fields of biology and biomedicine. The ideal bioconjugation strategy would be non-toxic, chemically and biologically stable, material-specific, and should immobilise biomolecules onto surfaces in a manner that best preserves their biological activity. Currently, most biomolecules are immobilised onto nanomaterials by weak interactions (e.g., hydrophobic, electrostatic, and hydrogen bonding), direct coupling reactions (e.g., EDC, sulfo-NHS, and glutaraldehyde) or coupling reactions on self-assembled monolayers (SAMs), for example using thiol- or silane-based SAMs to

functionalise metal or oxide surfaces. However, with these procedures biomolecules may attach to surfaces with altered conformations and in random orientations, which can cause a reduction or loss of biological activity. Some bioconjugation processes (those on SAMs in particular) are highly complex and require harsh chemical reagents such as organic solvents and high temperatures, which may reduce the stability of biomolecules and the biocompatibility of nanomaterial surfaces. By contrast, SBPs act as ‘molecular linkers’ (Figure 1) conferring directionality and orientation to the immobilisation of biomolecules without impeding their activities. They recognise materials under ambient conditions via specific non-covalent interactions with the aqueous material interface and without the need for extensive chemical modifications or physical treatments, and are stable in biological surroundings (Box 1). Accordingly, SBPs represent a class of molecular linkers that offer a facile and

Box 1. How do SBPs bind to nanomaterials? SBPs bind to their corresponding materials through multiple noncovalent interactions (e.g., electrostatic, polar, hydrophobic, hydrogen bonds). They can exhibit high affinity and selectivity with equilibrium binding constants in the nM to sub-mM range, and large negative binding energies similar to those observed with analogous SAMs [95]. Many of the mechanisms underlying SBP recognition, selectivity, and binding affinity remain poorly understood partly due to the complexity of the peptide–material interface [50] in the surrounding solution (e.g., water or solvent). This complexity is the result of the inherent variability of each component [3,43]. The peptide has a variety of conformations, structures, chemistries, polarities, electrostatic charges, or binding affinities. Likewise, the material surface can have a variety of facets, oxidation states, surface charges, crystallographic orientations, or defects. They all have been shown to contribute and affect the binding interactions (Table I). Further complexity arises from the dynamic nature of the assembled peptide–material surface interface in which peptides are in constant motion, tending to diffuse, reorient, and adapt themselves to the lowest-energy structures. The interactions between SBPs and solid materials are based on the high affinity that chemical groups within amino acid residues have for solid surfaces. SBPs that bind to the same class of materials have highly similar amino acid compositions. For example, peptides that bind to noble metals predominantly contain hydrophobic and hydroxyl-containing polar residues [95] whereas peptides that bind

to carbon-based materials are enriched in aromatic residues [89]. Despite these compositional trends, the presence of particular amino acids does not always guarantee the binding avidity of an entire peptide to a specific material, and the exact amino acid sequence is considered to be more important than composition in defining SBP function [93]. The structural properties of SBPs as encoded by their amino acid sequences play a crucial role in determining their recognition of and binding to solid surfaces. For example, SBPs that retain their secondary structures in both linear and cyclic forms also retain similar binding affinities to their target substrate. By contrast, their binding affinities vary greatly [72,77] when the secondary structures of SBPs differ between their linear and cyclic forms. SBPs must adopt molecular conformations that promote maximum contact between key residues and solid surfaces for effective binding interactions [95,96]. Accordingly, conformational preferences of the peptide play an important role in binding to material surfaces. SBPs that have comparable compositions but different sequences tend to bind to different materials as a result of differences in structure and conformational flexibility (i.e., rigid or flexible), which can either promote or hinder the simultaneous binding of key residues on a surface [97]. A comprehensive understanding of how SBPs selectively recognise and bind to materials will contribute towards the tailored design and engineering of novel SBPs for applications in nanobiotechnology.

Table I. Some of the tools used to investigate the binding interactions between peptides and solid materials Tool

Peptide–material properties investigated

Affinity Selectivity Kinetics and Surface Composition Structure and thermodynamics coverage and conformation sequence * * Fluorescence microscopy * * * Site-directed mutagenesis * * Competitive binding assays * * * * Surface plasmon resonance * * * * Quartz crystal microbalance * * * * Isothermal titration calorimetry * * * * Atomic force microscopy * * Circular dichroism spectroscopy * Nuclear magnetic resonance * Fourier-transform infrared spectroscopy * * * * * * Computer models and simulations * Electron microscopy Dynamic light scattering Raman spectrocopy X-ray photoelectron spectroscopy

Surface Refs properties

*

* * * * *

[8] [8,93] [98] [15] [95] [33] [15] [51] [99] [100] [97] [5] [86] [101] [92]

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Anbody-binding proteins

+

Fluorescent proteins

Anbody fragments

Solid material

Angens

Enzymes

RG D

Bifunconal solid-binding pepdes

+

Bioacve pepde mofs

SA Bion/Streptavidin (SA) Solid-binding pepde (SBP) = TRENDS in Biotechnology

Figure 1. Illustration depicting the use of solid-binding peptides (SBPs) as ‘molecular linkers’. SBPs can mediate the immobilisation of different types of biomolecules (e.g., enzymes, antibodies, and bioactive peptides) onto the surfaces of solid materials (e.g., nanoparticles) without compromising their function.

biologically-friendly alternative to conventional chemical conjugation procedures. Nanofabrication technologies (e.g., lab-on-a-chip, microfluidics, and microarrays) rely on the highly-selective and spatially controlled functionalisation of multiple materials patterned on surfaces at the nanoscale. However, conventional bioconjugation processes are unable to discriminate between different materials that are in close proximity. Many of the active headgroups that direct the formation of SAMs on substrates exhibit low materialselectivity. An example is thiols that couple to gold and silver by similar means [12], which reduces the capacity of SAMs to differentiate between materials that share a surface and restricts their use to a limited range of nanomaterials [13]. Conversely, SBPs recognise and bind to nanomaterials with high selectivity, allowing discrimination between different materials at the micro- and nanoscale [14,15]. This ability has been well-demonstrated, using peptides with opposite binding affinities towards gold and silica to direct the highly selective co-assembly of different fluorescent materials such as quantum dots and fluorescein simultaneously on a shared nanopatterned gold/silica surface [15]. Enzymes The incorporation of enzymes into nanomaterials can improve enzyme loading, activity, and stability [16]. Accordingly, nanomaterials have attracted interest as solid supports for enzyme-based diagnostics involving biosensors and bioassays, and in industrial processes such as bioremediation and biofuel production [17]. A variety of methods including adsorption, entrapment, and covalent attachment have been developed for their immobilisation [18]. However, these methods may involve conformational 4

changes or the use of functional groups that distort the active sites of enzymes and compromise their catalytic function. SBPs are able to direct the immobilisation of enzymes onto nanomaterials without compromising their catalytic function [7,19–21]. For example, a gold-binding peptide (GBP1) was used to direct the self-assembly of organophosphorus hydrolase (OPH) onto a gold nanoparticle-coated graphene chemosensor [20]. In contrast to unmodified OPH, GBP1–OPH retained its native conformation upon immobilisation and displayed better orientation and higher activity. The GBP1–OPH-functionalised sensor displayed faster and more sensitive detection of the organophosphorus pesticide paraoxon than the unmodified enzyme. The attachment of enzymes to magnetic nanomaterials allows their straightforward recovery and reuse in multiple reactions, maximising their economic value [17]. Peptides that bind to either silica [22] or iron oxide [23] have been used to facilitate the immobilisation of a bioremediation-related enzyme onto silica-coated or uncoated iron oxide magnetic nanoparticles [7]. In these cases, the enzyme remained active but its reuse in more than a single reaction was not reported. Many SBPs may be unstable under industrial conditions (e.g., extremes of pH and temperature) and incompatible with such processes. Nevertheless, some SBPs bind to their respective materials over a wide range of pH values and at high temperatures. For example, one silica-binding peptide has been shown to mediate the immobilisation of industrially-relevant thermostable enzymes on silicacontaining materials at pH values from 5 to 9 at 808C [24]. The application of SBPs for the immobilisation of enzymes in industrial processes warrants further research with respect to their stability under industrial conditions as well as their capacity to be co-immobilised into pathways and to allow the reuse of the enzymes. Antibodies The functionalisation of nanomaterials with antibodies is fundamental to many therapeutic and diagnostic technologies such as sensing, targeting, and imaging. Unfortunately, only a handful of antibodies that are able to recognise materials have been generated owing to their low immunogenicity [25]. A novel approach to the production of material-binding antibodies using SBPs has been established [26]. SBPs were grafted onto antibody fragments through genetic engineering to form a fusion protein, followed by phage display and selection. The antibodies then were subjected to directed evolution to improve their binding avidity and specificity. Recently, these antibodies have been used to mediate the self-assembly of hybrid nanomaterials and the functionalisation of immunosensors [26,27]. Even though antibodies offer a structural scaffold for SBPs that can improve their stability and binding avidity, this approach is complex and suffers from a lack of versatility because it requires the specific modification of antibodies. The conventional techniques for antibody immobilisation (e.g., adsorption and covalent binding) often lead to random antibody orientations that decrease the density

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Review and availability of antigen-binding sites. Alternatively, antibody-binding proteins (ABPs, such as proteins A and G of bacterial origin) rapidly bind to the Fc region of antibodies, ensuring their correct orientation [28]. Therefore, SBPs have been fused genetically to ABPs [29,30] to avoid the specific modification of antibodies and to increase versatility. The SBP directs the immobilisation onto specific nanomaterials, while the ABP couples to antibodies and retains their biological activity. This approach has been shown to improve significantly the density and orientation of antibodies immobilised on material surfaces when compared to covalent methods [29]. We have implemented this strategy in the form of a fusion protein, linker–protein G (LPG), that comprises two functionally-distinct regions: (i) a peptide linker sequence, which binds to a broad range of silica-containing nanomaterials; and (ii) Streptococcus protein G’, which binds to antibodies. We have utilised LPG as an anchoring point for the oriented immobilisation and functionalisation of nanomaterials with antibodies in a range of applications (i.e., capture, detection, and imaging [31,32]), including functionalisation of lanthanide-doped upconversion nanocrystals (UCNCs). UCNCs have a tendency to aggregate after functionalisation, severely limiting their biological applications. This limitation can be overcome by LPG, which mediates the oriented immobilisation of antibodies on silica-coated UCNCs within minutes and without the need for complex chemical modification of the surface [1]. Antigens Nanoparticles have been used to deliver tumour-specific antigens into dendritic cells (DCs) for the induction of antitumour immunity and for tumour immunotherapy. However, common conjugation chemistries can alter the specificity and stability of tumour-specific antigens and reduce the functionality of DCs. A ZnO-binding peptide (ZBP) can facilitate the assembly of carcinoembryonic antigen (CEA) on multimodal Fe3O4–ZnO core–shell nanoparticles, which are suitable for both magnetic resonance (MRI) and fluorescence imaging [33]. The ZBP–CEA-functionalised particles were recognised and rapidly internalised by dendritic cells. Mice immunised with the modified DCs displayed enhanced CEA-specific T cell responses, suppression of tumour growth, and prolonged survival in comparison to control mice. The ZBP was shown to remain strongly associated to the nanoparticles in culture medium for up to 4 h, despite binding in a non-covalent manner, before slowly disassociating over the next 2 days. The authors suggested that the slow and continuous release of antigens inside DCs mediated by the disassociation of the ZBP may enhance their capacity to initiate antigenspecific immune responses. Bioactive peptide motifs Bioactive peptide motifs are short amino acid sequences that have diverse biological functions and serve as alternatives to antibodies as biorecognition elements [34]. Unlike antibodies, they are stable under a wide range of conditions, have low complexities, and can be engineered easily. However, bioactive peptide motifs contain multiple reactive groups that promote non-specific coupling and

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uncontrolled orientation during bioconjugation procedures. SBPs have been used to control the oriented immobilisation of bioactive peptide motifs in nanomaterialbased sensors via the formation of bifunctional peptides. These peptides are chemically synthesised typically with glycine-rich spacers between both functional regions to provide flexibility and to prevent spatial constraints [35– 38]. Combination of a bioactive peptide motif with an SBP does not guarantee the retention of both expected functions in the resulting bifunctional peptide, and thus sequence alteration may be required to improve flexibility, solubility, or selectivity [36]. Graphene holds great promise in chemical and biological sensing applications owing to its remarkable electric, optical, mechanical, and thermal properties [39]. Unfortunately, graphene lacks functional groups as well as established self-assembly monolayer chemistries for the attachment of biomolecules. Covalent functionalisation of graphene often results in the degradation of its delicate structure and loss of electronic properties as a result of localised breakage of lattice symmetry [40]. There is considerable interest in identifying robust non-covalent bioconjugation strategies for graphene surfaces. Graphenebinding peptides have mediated the non-covalent assembly of unique bioactive peptide motifs in several graphenebased sensing devices. For example, an antimicrobial peptide motif that can recognise and bind to pathogenic bacteria was isolated from the skin of the frog species Odorrana grahami and then conjugated to a graphenebinding peptide [38]. This bifunctional peptide was shown to functionalise the surface of a graphene-based wireless biosensor without degrading its electronic sensing properties. The graphene was printed on water-soluble silk such that the sensor could be transferred easily to other biological surfaces (e.g., skin and bone). Similar strategies have been employed to construct graphene-based chemical sensors. For example, a trinitrotoluene (TNT)-binding peptide motif isolated from the binding domain of the honeybee odorant-binding protein ASP1 was fused to a peptide that binds graphene in phage display [37]. The resulting peptide retained its dual functionality when used to modify surfaces in a graphene fieldeffect transistor device that allowed detection of parts per billion levels of TNT in the vapour phase. Solid-binding peptides as ‘material synthesisers and constructors’ Whereas the term ‘molecular linkers’ refers to the ability of SBPs to facilitate the functionalisation of nanomaterials (Figure 1), the concept of ‘material synthesisers and constructors’ refers to their capacity to mediate the synthesis and construction of ordered nanostructures (Figure 2). Improvement of nanomaterial biocompatibility and toxicity Several nanomaterials, such as quantum dots [41], carbonbased nanomaterials [42], and lanthanide oxide plus upconversion nanocrystals [8], have been shown to cause cellular stress and toxicity in vitro and in vivo. Their surface properties including charge, solubility, structure, 5

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(A)

Nanofabricaon

(B)

Nanomaterial synthesis Nucleaon

Top-down + +

+ +

+ +

Bulk

+ +

+

Growth

+

Powder Shape Size Nanostructure

Molecules

(C)

Biocompable coangs

Atoms Boom-up

Inorganic–inorganic nanostructures

(D) Core–shell

(E)

Mul–layered

Nanocomposites

Inorganic–organic hybrid biomaterials Inorganic coangs on amyloid nanofibres

Phage–assembled nanowires

Inorganic core–protein shell nanoparcles

TRENDS in Biotechnology

Figure 2. Illustration depicting the use of solid-binding peptides (SBPs) as ‘material synthesisers and constructors’. (A) SBPs represent a ‘bottom-up’ approach towards the fabrication of functional nanostructures. (B) SBPs can regulate the properties of the nanomaterials they synthesize, and (C) improve their biocompatibility. (D) SBPs can also facilitate the construction of inorganic–inorganic nanostructures and (E) inorganic–organic hybrid biomaterials.

and stability are key determinants of their ability to interact safely with biological systems [43]. As a result, nanomaterials are commonly coated with proteins and peptides to improve their biocompatibility and minimise toxicity (Figure 2C). They have been used to produce biocompatible coatings on the surfaces of nanomaterials in a simple and highly-specific manner because SBPs function under biological conditions and are usually unable to cause immunogenic or cytotoxic responses. Recently, a short peptide (RE-1) with high binding affinity for lanthanide oxide and upconversion nanocrystals was identified from phage 6

display experiments [8]. It formed a stable coating on the surface of the nanocrystals, thus eliminating their potent cytotoxicity by preventing sedimentation and reducing non-specific interactions with cells, both in vitro (HeLa cells) and in vivo (mice). Another measure taken to enhance the biocompatibility of nanomaterials includes the encapsulation of nanoparticles inside self-assembled protein cages such as ferritin [44]. The incorporation of SBPs into protein cages enables the directed encapsulation of nanoparticles and their directed delivery onto solid surfaces [45], as well as the

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Review peptide-mediated synthesis of nanoparticles within the confines of the protein cage [46]. In other reports, the conjugation of polyethylene glycol (PEG) to an SBP allowed the directed formation of PEG coatings on nanomaterials [47]. This strategy has been used to reduce the aggregation of nanomaterials in vivo [48] and to produce bio-inert surfaces for potential medical applications [47]. Solid-binding peptides in the synthesis of designer nanomaterials and nanostructures Nanostructured materials can be manufactured using ‘top-down’ techniques (e.g., soft- and photolithography) in which special tools are used to fabricate nanoscale structures from macroscopic objects, or ‘bottom-up’ approaches, which rely on molecular recognition and self-assembling biomolecules to form larger hierarchical structures (Figure 2A). ‘Bottom-up’ approaches provide higher spatial resolution (below 20 nm), high turnover, less surface defects and degradation (e.g., roughness), and lower costs than conventional ‘top-down’ approaches [49]. SBPs bind to and lower the surface energies of their target solids, providing a thermodynamic driving force for the nucleation and growth of solid materials [50] (Figure 2B). They have attracted interest as versatile material synthesisers for the ‘bottom-up’ fabrication of well-ordered bioinorganic hybrid materials and functional nanostructures with nanoscale precision. There is considerable interest from biomaterial scientists in developing efficient methods to control the size and shape of inorganic nanostructures because their functionality is highly dependent on these properties. SBPs can act as capping agents that allow the highly controlled synthesis of designer nanostructures, as exemplified by two peptides isolated by phage display that selectively bind to and lower the surface energies of different faces (i.e., Pt-100 and Pt111) of platinum nanocrystals. They were shown to mediate the synthesis of platinum nanocrystals with distinctly different shapes to form nanocubes and nanotetrahedrons [5]. Post-synthesis transformation between each shape was achieved by simply switching the peptides under ambient conditions. Although combinatorial display technologies permit the isolation of peptides that bind to selected materials, they do not allow the isolation of peptides that can best direct material synthesis. Nevertheless, the rational modification of SBPs modulates their surface-binding characteristics to control the final properties and functionality of synthesised materials. For example, an isolated palladium (Pd)-binding peptide mediated the synthesis of peptide-capped palladium nanoparticles, which were shown to function as nanocatalysts for the formation of C C bonds using the Stille coupling reaction [51,52]. Simple changes to the Pdbinding peptide sequence modulated its surface-binding characteristics and allowed selective changes in the size and catalytic activity of the synthesised nanoparticles. SBPs have been used to synthesise slightly more complicated core–shell nanostructures. For example, peptides that promoted the growth of either CdSe or ZnS nanocrystals were fused to one another and were shown to facilitate the synthesis of luminescent CdSe/ZnS core–shell quantum dots [53].

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Inorganic–inorganic nanocomposites and complex hierarchical nanostructures Various ‘bottom-up’ approaches for fabricating conductive nanostructures (e.g., nanowires and nanofibres) have gained attention because of their potential applications in nanoelectronics (e.g., computer processors, power cells, and optical fibres). SBPs can mediate the synthesis and/or construction of inorganic-inorganic nanocomposites (Figure 2D). In a recent work, a self-assembling cageshaped protein (CDT1) was modified genetically to codisplay 12 single-walled carbon-nanotube (SWNT)-binding and TiO2-mineralising peptides [54]. This bifunctional protein supramolecule mediated the rapid and precise formation of thin TiO2 layers on SWNTs. All proteins were eliminated readily by sintering at high temperatures, yielding a highly-conductive nanocomposite. It was applied in dye-sensitised solar cell (DSSC) photoelectrodes, which displayed enhanced electric current densities and photoelectric conversion efficiencies when compared to TiO2only DSSCs. A SBP-mediated layer-by-layer fabrication process was developed for the construction of much more complex hierarchical nanostructures [55]. Nanoparticle-loaded ferritin cage proteins were modified to display an SBP that had the capacity to bind to and mineralise specific inorganic materials. Multilayered nanostructures composed of layers of protein-encapsulated nanoparticles and intercalating minerals were fabricated in a highly controlled manner by alternating between the binding and mineralisation activities. Cage proteins were removed by UV oxidation. This process was used recently to self-assemble multiple nanoparticle layers of ferrihydrite bionanodots (Fe-BND) separated by layers of mineralised silica to produce a charge storage node in a metal oxide-semiconductor flash memory device. This device displayed memory operation, and an increase in the number of nanoparticle layers resulted in an increase in its charge storage capacity [56]. Heterogeneous nanoparticle multilayers may be constructed using this process because ferritin can incorporate different nanoparticles into its cage-like structure, enabling applications in various nanoelectronic devices. Inorganic–organic hybrid biomaterials There is growing interest in controlling the self-assembly of inorganic–organic hybrid biomaterials (Figure 2E). Biological scaffolds that can be reprogrammed genetically (e.g., virus-like particles and extracellular matrix proteins) to contain SBPs show promise as templates for the production of biomaterials. There are extensive reports on the use of M13 phage particles that have been genetically engineered for the multivalent display of SBPs as robust biological scaffolds that can serve as templates for the synthesis and organisation of inorganics. This approach has been used to produce conductive and fluorescent biomaterials for applications in lithium-ion batteries [57], photovoltaic cells [58], and imaging agents [59,60]. Another example is silk proteins. They are useful biological scaffolds that have high mechanical strength and can be processed into films, fibres, and 3D frameworks. A peptide that binds and mineralises silver nanoparticles was incorporated into recombinant silk proteins from the spider, 7

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Review Nephila clavipes. This resulted in chimeric proteins that template, both in solution and on films, the formation of various silver nanostructures that demonstrated antimicrobial activity [61]. Inorganic core–protein shell nanoparticles have been fabricated using SBPs and show great potential in biomedical applications [62]. For example, calcium phosphate (CaP) nanoparticles are known adjuvants [63] that can be synthesised using SBPs. A fusion protein between a CaP-binding peptide (situated within the protein scaffold TrxA) and an antigen was shown to fabricate CaP nanoparticle adjuvants in a single step with antigen-bearing surfaces [64]. Mice vaccinated with these particles exhibited stronger antigen-specific and long-lasting CD8+ T cell responses in comparison to the unbound fusion protein. Thus, a novel self-assembling vaccine formulation technology has been developed by exploiting the synthesising and binding activities of an SBP. Amyloid protein nanofibres have been exploited as biological scaffolds for the production of novel biomaterials. E. coli cells have been bioengineered for programmable production and extracellular self-assembly of amyloid protein nanofibres [65]. The integration of functional SBPs into these nanofibres permitted the organisation and growth of inorganic materials. For example, amyloid nanofibres that displayed a ZnS-mineralising peptide were shown to form a template for the mineralisation of fluorescent ZnS nanocrystals. In a similar approach, E. coli was programmed to produce amyloid-based biofilms that displayed functional SBPs [6]. In one example, a silver-mineralising peptide promoted the binding and growth of silver nanoparticles on amyloid fibres within an extracellular biofilm matrix. These experiments represent the first steps towards the programmable and self-replicating fabrication of multifunctional biomaterials at the nanoscale with properties that cannot be achieved with existing nanomaterials. Hence, SBPs may play a significant role in the future development and application of novel biomaterials. Concluding remarks and future perspectives As an ever-increasing number of new nanomaterials with diverse surface chemistries and physical properties emerge, the development of bioconjugation strategies best suited for their functionalisation continues to lag behind them. SBPs can be selected readily for virtually any nanomaterial via combinatorial display technologies, and represent a biological route towards nanomaterial-specific bioconjugation. They can provide improved solubility, colloidal stability, and biocompatibility to nanomaterials, overcoming many of the technical issues that prevent their translation from the laboratory bench to the market. However, in some cases, the stability and functionality of SBPs are compromised within living systems, resulting in dissociation from their target substrates via exchange with free host proteins or complete degradation by host proteases [24]. A more application-orientated approach would include the isolation of SBPs using combinatorial display libraries that bind to nanomaterials during the biopanning procedure while in the presence of the appropriate biological fluid (e.g., whole blood, serum, urine) that they will come into contact with during their application 8

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in vivo or ex vivo. This action will go a long way to ensure that both the stability and binding function of the isolated SBPs are retained within complex biological environments, consequently improving their feasibility in biological applications. Another important point to consider is that combinatorial display technologies are not without their disadvantages. Peptide libraries display some compositional, positional, and expression bias, which can lead to sequences with desirable properties being under-represented [66]. General biopanning protocols performed against solid materials demonstrate intrinsic bias for positively charged sequences that bind via electrostatic interactions, and discriminate against sequences that bind via non-electrostatic interactions, thus many strong-binding SBPs are not isolated and remain unknown [67]. Online databases are currently available for specific classes of bioactive peptides (e.g., anticancer peptides [68] and antimicrobial peptides [69]) and offer details on their characteristics and properties. Although some SBPs have been deposited into databases that contain a broad range of peptides (e.g., the MimoDB 2.0 database [70]), there is no specialised depository for SBPs. We propose the construction of a comprehensive database that provides researchers with easy access to a full spectrum of SBPs and complete information on their physical, chemical, and biological properties and functions that are relevant to their application (e.g., sequence, structure, binding affinity, and selectivity). Furthermore, information from this database combined with molecular modelling and computational simulations will further the understanding of the mechanisms that govern SBP binding, enabling the rational design of new SBPs with properties and functions tailored to their specific application in nanobiotechnology, reducing the reliance on combinatorial display technologies. References 1 Lu, Y. et al. (2014) Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photon. 8, 32–36 2 Avvakumova, S. et al. (2014) Biotechnological approaches toward nanoparticle biofunctionalization. Trends Biotechnol. 32, 11–20 3 Sapsford, K.E. et al. (2013) Functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology. Chem. Rev. 113, 1904–2074 4 Sengupta, A. et al. (2008) A genetic approach for controlling the binding and orientation of proteins on nanoparticles. Langmuir 24, 2000–2008 5 Chiu, C-Y. et al. (2011) Platinum nanocrystals selectively shaped using facet-specific peptide sequences. Nat. Chem. 3, 393–399 6 Nguyen, P.Q. et al. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 4945 7 Johnson, A. et al. (2008) Novel method for immobilization of enzymes to magnetic nanoparticles. J. Nanopart. Res. 10, 1009–1025 8 Zhang, Y. et al. (2012) Tuning the autophagy-inducing activity of lanthanide-based nanocrystals through specific surface-coating peptides. Nat. Mater. 11, 817–826 9 Sarikaya, M. et al. (2003) Molecular biomimetics: nanotechnology through biology. Nat. Mater. 2, 577–585 10 Tamerler, C. et al. (2010) Molecular biomimetics: GEPI-based biological routes to technology. Pept. Sci. 94, 78–94 11 Baneyx, F. et al. (2007) Selection and analysis of solid-binding peptides. Curr. Opin. Biotechnol. 18, 312–317 12 Niemeyer, C.M. (2001) Nanoparticles, proteins, and nucleic acids: biotechnology meets materials science. Angew. Chem. Int. Ed. 40, 4128–4158

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