Recently, there has been a growing demand to have the ability of fabricating nanosized structures that are 3D in orientation, produced in large quantities and yield uniform shapes and sizes. Biomimetic assembly has been given attention in that it relies on the use of bio-inspired materials that are characteristically organized from the macroscale all the way to the nanoscale. Peptides are one of nature&’s phenomenon that have the ability to take an active role in self-assembly and that can further be integrated to consequently yield the self-organization of structures with interesting properties in high quantities. Our approach relies on the robust self-assembly nature of a collagen-like triple helical peptide together with a Au-streptavidin conjugated nanoparticle to generate 3D nanoparticle superstructures. In this study, first, micron-sized assembly of streptavidin-functionalized Au nanoparticles and biotinylated collagen peptides into cubic structures was demonstrated as assembled peptide frameworks incorporate nanoparticles in the exact position of a unit cell, and then other fluorescent molecules or nanoparticles with biotin moieties were co-assembled to generate complex 3D nanoparticle assemblies. Energy transfer and excitonic lifetime changes of these assemblies were investigated. This type of robust large-scale 3D material assembly technique with precise positioning could be beneficial for future bottom-up device assembly such as solar cells, batteries, and metamaterials.

In natural systems, a single protein can be used to perform multiple functions through interactions with other proteins at well-defined epitope binding sites to form a variety of functional co-assembled structures. Inspired by the biological use of epitope recognition to control the interactions between proteins and enable diverse yet specific functions, we present Template Engineering Through Epitope Recognition (TEThER), a strategy that takes advantage of molecular recognition and assembly between different components to achieve functional versatility from a single protein template. Engineered TEThER peptides are rationally designed to assemble with a protein scaffold and serve as molecular bridges between the protein and the surrounding environment in a localized, specific, and versatile manner. The peptides discussed in this work are bifunctional sequences that bind to specific recognition sites on the protein scaffold and serve as sites for localized bio-enabled nucleation and growth of inorganic materials through inorganic recognition sequences. We functionalized self-assembled clathrin protein cages at specific sites through co-assembly with designed TEThER peptides to achieve protein/peptide hybrid nanostructures with three tailored chemical functionalities: the bio-enabled synthesis of gold, cobalt oxide, and anatase titanium dioxide nanoparticles in aqueous solvents at room temperature and ambient pressure. Compared with previous demonstrations of bio-enabled inorganic synthesis, the TEThER strategy relies solely on defined, noncovalent interactions without requiring any genetic or chemical modifications to the protein template. Therefore, this design strategy represents a mix-and-match, biomimetic approach to achieve versatile and site-specific functionalization that can be broadly applied to other protein templates to generate structures with a range of functionalities.

The ability to control self-assembly of complex 3D architectures from functional building blocks could allow further development of complex device configurations.Genetically engineered peptides with a variety of functional building blocks such as metal NPs can be applied to design new materials with the specificity of assembled structure, the robustness of assembly, and the versatility of the superstructure. Here, we present large-scale (µm3 - mm3) biomimetic 3D assemblies using nanoscale collagen peptides as building blocks. In this strategy, biotylated collagen peptides and ligand-functionalized nanoparticle hubs are self-assembled into 3D microcrystals in controlled structures and NP density with the precise nanoscale interparticle distance. This robust fabrication protocol produces high yields of 3D materials in controlled shapes, promising ease and flexibility in manufacturing future functional devices. The reconfigurability of 3D directed assembly was also demonstrated by modifying peptides with genetic engineering. In this reconfiguration functionality, conformation change of peptide building blocks induced by pH could trigger disassembly of the hybrid NP-peptide cube and undergo the reassembly into different shapes. Here we would also like to introduce a new concept of energy device with harvesting energy from reconfiguration of self-assembly of peptides; peptide self-assembly is used as an energy source and metal-organic framework (MOF) as an energy storage, and this hybrid MOF can be powered by re-self-assembly of peptides on the MOF. Here we developed autonomous biochemical motors by integrating MOF and self-assembling peptides. MOF is applied as an energy-storing cell that assembles peptides inside nanoscale pores. The robust assembling nature of peptides enables reconfiguring their assemblies at the water-MOF interface, which is converted to fuel energy. The re-organization of hydrophobic peptides could create the large surface tension gradient around the MOF and it efficiently powers the translation motion of MOF. This system moves 30 times faster than existing hydrogel-based motors and this peptide engine could power macroscopic plastic boats in motion for longer than 20 minutes.

The development of materials derived from renewable resources that are capable of neutralizing pathogenic microorganisms is highly desirable. Although many self-decontaminating materials have been produced during the last decade, the preparation of these materials often requires extensive manufacturing facilities, sophisticated chemical synthesis techniques, and highly skilled personnel. Furthermore, many of the antimicrobial materials and surface treatments currently available are ineffective against or slow to kill bacterial spores, such as those of the Bacillus genus. In our work, we report on the development of a facile approach to create potent antimicrobial materials from polypeptide-based textiles, films, fibers, and powders. In this process, the nitrogen moieties present in proteins and peptides may be chlorinated to produce halamine compounds. Our results demonstrate that chemically modified fibroin-, keratin-, and peptide-based materials induce >5 Log reduction in the colony forming units of Escherichia coli, Staphylococcus aureus, and Bacillus thuringiensis cells, as well as B. thuringiensis spores within 10 min of organism-material contact. The effects of processing parameters on the antimicrobial efficacy and mechanical strength of the treated protein-based materials will be presented. It is anticipated that the results discussed in this presentation may be extended to produce protein-based antimicrobial materials from a variety of inexpensive polypeptide sources. Such materials may find use in self-decontaminating coatings, textiles, filters, and protective equipment.

A broad range of peptides that have been shown to form stable nanowires or nanotubes are based on amyloid proteins attributed to diseases such as Alzheimer&’s and Type II diabetes. Such peptide nanotubes have robust self-assembly and have been used as building blocks for applications including electronics and as templates for growth of inorganic material. In this study, we have examined the ability to sublime different peptides and the subsequent deposition of peptide nanotubes and nanowires using Plasma Enhanced Chemical Vapor Deposition (PECVD). Dipeptides were sublimed into a reactive plasma species using a home-built reactor and allowed to deposit onto downstream substrates. The aromatic dipeptides, diphenylalanine and dityrosine are peptide monomers which have been used in this study. The morphology of peptide nanostructures and characteristics such as their length, thickness, density have been controlled by varying parameters during the deposition process as well as the effects of using different substrates has been studied. Plasma used in the PECVD process allows control over the composition of the nanotube growth imparting unique surface properties without modifying the bulk material properties of the substrate. Parameters during the deposition process such as pressure in the plasma chamber, time of deposition, flow rate of the ionizing species and the RF plasma power source conditions have been varied and their effects on the nanotube formation have been studied. We have used a variety of techniques such as SEM, TEM, contact angle measurements, nanoindentation and AFM to quantify the effects of the deposition conditions on the peptide nanostructures. Mechanical testing techniques such as nanoindentation have been used to demonstrate the mechanical stability and hardness of the deposited nanotubes. AFM techniques have been used to study the details of the tubular outer shell with tapping and electrostatic force modes, while also probing the mechanical integrity. We have sampled single nanotubes as well as vertically aligned 3D arrays. I-V characterization was carried out via electrical contacts deposited on the nanotube ends by focused ion beam deposition. The electrical conductivity characteristics have been studied using the Scanning Kelvin Probe Microscopy and conductive AFM as well. Thermal responses of the nanotubes to localized tip-side heating using the scanning thermal probe techniques have been studied. Some of the synthesized nanotubes show quantum confinement and are fluorescent in the blue region (400 - 500 nm) as shown by photoluminescence measurements.

Artificially engineered protein hydrogels provide an attractive platform for biomedical materials due to their similarity to components of the native extracellular matrix. Engineering responsive transitions in these materials could potentially enable gels that have both shear-thinning injectable phases and tough phases to be produced as novel injectable biomaterials. To engineer a gel with such transitions, a triblock copolymer with thermoresponsive polymer endblocks and an artificially engineered protein gel midblock is designed. Poly(N-isopropylacrylamide) (PNIPAM) endblock association forms a large length scale network, while association of coiled-coil repeat domains within the protein midblock leads to self-assembly of a short length scale network. Temperature is used to trigger a transition from a single network protein hydrogel phase to a two network phase with both protein and block copolymer networks present. The presence of protein associating domains in the polymer midblocks has a large effect on the phase behavior of the block copolymer gels. Associating midblock domains decrease the thermal transition temperature of the PNIPAM endblocks, promoting the formation of a microphase separated gel phase. In addition, both larger molecular weight PNIPAM endblocks and higher densities of associating groups in the protein midblock promote syneresis above the thermal transition temperature. The formation of the second network is shown to produce a 5 to 10-fold increase in the elastic modulus, where the magnitude of modulus enhancement depends strongly on the molecular design. In all cases the increase is highly nonlinear, suggesting a dramatic increase in the fraction of elastically effective chains above the thermal transition temperature. The gels also show enhancements in creep compliance, increased toughness, and a dramatically reduced erosion rate, suggesting that thermoresponsive reinforcement can effectively produce injectable materials with improved durability in the physiological environment.

The mechanism of amyloidosis of amyloid β (1minus;42) (Aβ(1minus;42)) was investigated by the well-defined glycocluster interface. We prepared monovalent, divalent, and trivalent 6-sulfo-N-acetyl-D-glucosamine (6S-GlcNAc) immobilized substrates. The morphology and secondary structure of Aβ(1minus;42) aggregates on the substrates were investigated by dynamic-mode AFM and FTIR-RAS. Aβ(1minus;42) interactions with multivalent sugars were evaluated by surface plasmon resonance, and the cytotoxicity of Aβ(1minus;42) to HeLa cells was evaluated by MTT assay. Morphological images showed, interestingly, that Aβ(1minus;42) aggregates had a tendency to form globules rather than fibrils as the valency of 6S-GlcNAc on the substrate was increased. The SPR measurements indicated that this morphological change of Aβ(1minus;42) was related to the change of binding mode, and the binding mode was dependent on the multivalency of the sugar. Globular Aβ (1minus;42) was more toxic than fibrillar Aβ (1minus;42) to HeLa cells. These results suggested that the multivalency of sugars for the amyloidosis of Aβ (1minus;42) was significant in its morphology and aggregation effects at the surface of the cell membrane mimic.

Many animals change their skin colors, to communicate, to express mood, for camouflage, or to respond to environmental changes. Inspired by nature, sensors are being developed that change color in response to target chemicals by employing biomimetic structures and mechanisms. However, a significant drawback of previous structural color sensors is their limited intrinsic affinity for specific targets of interest (e.g., explosives and pathogens) and resulting poor selectivity against analytes with similar chemical structures. Current there is various kinds of methods to promote target specificity into structural color sensor, but incorporating the analyte-responsive elements into the sensing device is still challenging because it requires complex designs and multistep synthetic pathways. In this MRS presentation, we developed a novel bioinspired colorimetric sensor for the explosive and environmental material detection using hierarchical bacterial virus (M13 phage) nanostructures. Using self-templated materials assembly process, we could fabricate phage-based matrices with controlled bundled structure exhibit specific tunable colors. The resulting color matrices could be utilized as colorimetric sensors responding rapidly upon exposure to target materials. With the aid of a common handheld device, we could detect down to 100 ppb concentration of target explosives and environmental toxicants in a selective manner. Our novel colorimetric sensors can be useful for the detection of a wide variety of harmful toxicants and pathogens to protect human health and national security.

Self-assembled nanocapsules have been studied intensively because of their controlled assembly and disassembly, and storage capacity. In nature many examples of protein cages are known, including nanovaults, viruses and ferritin. Spherical viruses generally consist of several hundreds of subunits, which self-assemble to encapsulate their genetic material for storage and transport. However, many virus capsid proteins can be induced to assemble even without their natural cargo. Cowpea chlorotic mottle virus (CCMV) is an excellent example of a virus whose assembly can be exquisitely controlled in the absence of its viral RNA. At high pH (7.5) empty CCMV capsids dissociate into capsid protein (CP) dimers and at low pH (5.0) they reassemble. Virus-like particles (VLPs) of CCMV have been used as nanoreactors or as template for constrained synthesis of nanomaterials. However, these VLPs are only stable at lower pH, which limits their applications. Templated assembly has been described as an approach to generate VLPs at different conditions. However, nanoparticle-templated capsids are already filled and cannot accommodate additional material, limiting their applicability. We realized that if the assembly of the CPs could be controlled by an external trigger this would allow much greater control over assembly of CPs. Herein we report the construction and assembly properties of a block copolymer in the form of a fusion protein consisting of the CP of CCMV with an N-terminal short stimulus-responsive elastin-like polypeptide (ELP) block. In this design, the properties of both blocks are combined: the CP ability to form well-defined VLP morphologies and the stimulus-responsive character of the ELP fragment. Two types of highly homogeneous self-assembled structures can be formed using a single capsid protein: (1) pH-induced assembly into 28 nm VLPs and (2) ELP-induced assembly into 18 nm VLPs . The latter can only be accessed via the emergent properties of the ELP-CPs.

Vertically aligned nanostructures have recently attracted increasing interest due to their unique properties and they have exploited for variety of applications including field emission devices, filter media, superhydrophobic surfaces, synthetic membranes, intracellular gene delivery devices, biosensors, composites, logic and memory devices and numerous others. Fabrication of well-ordered and aligned nanostructures may provide number of advantages in any application where the surface area and the alignment of structures are considered key parameters. Besides synthetic materials, utilization of biological molecules for creating novel nanostructures is a promising approach due to their biocompatibility, extraordinary stability, functional flexibility and molecular recognition capability. In particular, dipeptides, which can spontaneously form variety of nanostructures within a certain solvent medium, serve as an excellent model for the possible applications in biological and non-biological fields. Herein, we demonstrated the fabrication and application of well-ordered and vertically aligned dipeptide nanostructures based on a simple gas phase deposition. Deposited nanostructures were exhibited superhydrophobic property with a very low sliding angle. Highly reproducible SERS data have also been obtained after combining deposited films with a thin layer of gold. Directional peptide films have been also fabricated based on oblique angle deposition (OAD) technique. We believe that such bio-inspired materials would have a great impact for several technological applications involving catalysis, tissue engineering, and biosensors.

In recent times much focus has been drawn to magnetic nanoparticles (MNPs) and there potential commercial uses, including many in the field of medicine, technology and industry. Magnetotactic bacteria have been found to produce MNPs in the form of chains of nanosized magnetite (Fe₃O₄) particles. This biosynthesis of magnetite is the earliest known example of biomineralisation, having first occured some two billion years ago. Despite this, much of the detailed atomistic mechanism by which the process occurs is unknown. Therefore, we have begun to develop an atomistic model for the system, in an attempt to understand the processes involved. The Magnetotactic bacteria strain of M. magneticum AMB-1was the focus of our work alongside the work of experimentalists. It was previously discovered that the Mms6 protein was found to be linked to the control of the morphology and size of the magnetite crystals within the M.magneticum AMB-1. The acidic C-terminal region of this sequence is of particular interest due to its potential link to iron binding. Atomistic model development for such a system is complicated, as the force field must accurately reproduce all related interactions involved in the system, including the inorganic magnetite crystal, the peptides to be attached to this crystal, their interactions with water, along with the interactions between each other. Additionally, as the complexity of the peptide increases so does the number of potential interactions with the surface and so it would be impossible to accurately describe the peptide/mineral interface using brute force dynamics alone. Constrained dynamics using an amended version of DL_POLY [1] were performed to illustrate the interactions of the small chain peptides with the mineral surface and how they compare with each other on different surfaces. In order to do this, force fields were developed to correctly reproduce the interactions between the short chain peptides and the surface of the magnetite. These were modelled on the ClayFF [2] and ff99SB [3] force fields respectively. Subsequently AMBERTOOLS was used to build solvated peptides based on sequences investigated experimentally. Future work will validate the interactions between the peptides and the magnetite {1 0 0} and {1 1 1} surface, enabling us to succesfully fit the required organic/ inorganic cross parameters to the system. The acidic C-terminal group shall be examined thoroughly on both surfaces. It shall also be investigated within a solution of iron to see if there are preferential areas of iron attachment along the sequence. References [1] S. Kerisit et al, J. Am. Chem. Soc., 2004, 126 (32), 10152 [2] S. Kerisit, Geo Cosmo Acta, 2011, 75(8), 2043 [3] V. Hornak, Proteins: Structure, Function, and Bioinformatics, 2006, 65(3), 712

There is a growing demand for surface materials and fabrication techniques that can control the adhesion, growth and interconnectivity of nerve cells. Here we use an Atomic Force Microscopy (AFM) based nanolithography technique (nanoshaving) to immobilize extracellular matrix proteins at well-defined locations on 2-dimensional surfaces. We show that these protein patterns confine neuronal cells and guide axonal growth and the formation of neuronal synapses. We also investigate the interaction between neuronal cells and the protein substrates via combined AFM-force spectroscopy and fluorescence microscopy experiments, and quantify the role that different types of substrate-assembled protein cues play in neuronal growth. Finally, we discuss the implications of these results for engineering nerve-material interfaces and nerve prosthetic devices. Funding: NSF-CBET 1067093

We demonstrate how to emulate skeletal muscle structure to achieve the rapid and large muscle-like contraction function. We have developed a method of a stepwise construction of a gel consisting of (i) astral-shaped actin filaments with their plus end connected on photo-responsive polymer beads and (ii) myosin filaments as linkers in order to mimic sarcomeric structure for the artificial muscle. In the method, firstly, 4 mu;m diam. beads were prepared from an acrylate polymer containing azobenzene moiety [1] by a good-solvent evaporation technique. Next, gelsolin, which servers and remains bound to the plus end of an actin filament, was adsorbed and then immobilized on the bead surface by exposure to light from blue LEDs, and then fluorescent actin filaments were mixed with the beads. Star-like, astral actin filaments on the beads were observed to be formed in fluorescent microscopy. The filament was fixed vertically on the bead surface, indicating that the gelsolin immobilized on the surface worked well to cap the plus end of the actin filament. Finally, when the beads with actin filaments were mixed with myosin mini filaments with ca. 1 mu;m in length, dozens of the beads were found to be assembled into a gel form. After adding ATP to the gel solution, the gel was slowly contract up to 60% comparing with its original volume, suggesting that linker myosin filaments moved on the actin filaments toward the plus end on the beads. The method will provide promising applications not only for artificial muscle, but micromechanical system and/or active optical element. This work was supported by JSPS KAKENHI 22510121. [1] T. Ikawa et al., Langmuir, 22, 2747 (2006); ibid. 26, 12673 (2010).

Molecular self-assembly has emerged as a powerful tool for the fabrication of molecular materials with a wide variety of properties. In recent years, considerable advances have been made in using simple oligopeptides as building blocks for the production of novel biomaterials. In particular being able to trigger the self assembly of these small molecules by an external stimulus such as light, pH or ionic strength is attractive as a route for the fabrication of soft solids i.e: hydrogels. Enzyme are another family of triggering stimuli that can be used. Recently, we have reported the enzyme catalysed synthesis and gelation of ionic complementary oligopeptides from non-gelling tetrapeptide precursor FEFK (F: phenylalanine, K: lysine, E: glutamic acid) via reverse hydrolysis [1]. We showed that for an initial tetrapeptide concentration higher than a critical concentration octapeptides are the dominant product of the reverse hydrolysis reaction. These longer sequences self-assembled into β-sheet rich fibres leading to the development of a dense fibrillar network and a hydrogel. Here we have focussed, based on this recent work, on the development of a simple protocol for the encapsulation and injection of cells for 3D cell culture applications. Rhelogy was used to characterise the mechanical properties of the hydrogels. Tetrapeptide/enzyme solution containing cells were injected into cell culture plate with subsequent gelation of the materials leading to encapsulation of the cells into a 3D network. This system was evaluated for the 3D cell culture of human dermal fibroblasts (HDF&’s). Microscopy showed that cells were uniformly distributed within the gel matrix. Cell counting and live/dead staining showed proliferation of HDF's with limited cell death over 10 days. REFERENCES: [1] J.B Guilbaud et al (2010) Langmuir 26:11297-11303. ACKNOWLEDGMENTS: Peptide Supply: Peptisyntha (a member of the Solvay Group), Belgium (http://www.peptisyntha.com) Funding: BBSRC Vacation Bursary Scheme and EU-FP7 NMP BIOSCENT Project

Self-assembling materials are gaining interest for their potential uses in tissue engineering and drug delivery devices. Recent work has shown that Fmoc or naphthalene N-protected di- and tri-peptides can self-assemble into hydrogel-forming fibrous structures. This process is putatively driven by π-π interactions and β-sheet-like interactions between adjacent aromatic protecting groups and dipeptides, respectively. For these gels to be used practically as biomaterials, they should be easily degradable by simple hydrolysis rather than by an enzyme-mediated process. To this end we have synthesized Fmoc-Alanine-Lac, a depsipeptide analog of the known gelator Fmoc-Alanine-Alanine (Fmoc-AA). Here, lactic acid, the α-hydroxy analog of alanine, is bonded via a hydrolytically degradable ester to alanine. Since ester bonds are unable to interact with other esters via hydrogen bonding, this system allows us to directly assess whether β-sheet-like hydrogen bonding is a requirement for the self-assembly of short conjugated peptides. Our results show that when triggered via pH change or solvent exchange, Fmoc-ALac solutions indeed form self-supporting gels at very low concentrations (< 1% w/v) and TEM images show that Fmoc-ALac gels are composed of nanofiber structures comparable in size to those of Fmoc-AA gels. The similarity in size may indicate similarity in the supramolecular structure of the component nanofibers for both systems, pointing toward a similar self-assembly mechanism that is not dependent on the formation of β-sheet-like interactions between molecules. FT-IR absorption data show that Fmoc-ALac gives no strong absorption signals in the amide I characteristic region whereas Fmoc-AA displays strong absorptions at 1644 cm-1 and 1686 cm-1, frequencies that correspond to unordered (polyproline II) and antiparallel β-sheet type interactions, respectively. Circular dichroism measurements are quite similar between the two systems; in both cases, dichroism increases over the course of gelation induced by the slowly hydrolyzing acid glucono-δ-lactone, and negative peaks at ~228 and ~278 nm and a positive peak at ~255 nm are seen in both Fmoc-AA and Fmoc-ALac gels. This may indicate that the two systems self-assemble in a similar manner and form final assemblies in which individual molecules take on similar preferred conformations. Finally, time-sweeps in rheometry illustrate quantitatively the difference in assembly time, with Fmoc-ALac gels taking nearly 4 times longer than Fmoc-AA to reach a steady state storage modulus value. This work validates the continued use of depsipeptides as the basis for peptide-like, nonspecifically degradable biomaterials, and suggests that β-sheet forming capability may not be necessary but likely plays a role in the rate of self-assembly. Future work will include investigations into the self-assembly of longer depsipeptide conjugates with side chain sequences analogous to known bioactive peptides.

The increasing utility of polymer-protein conjugates in medicine, biotechnology and nanotechnology has driven the need for generating homogeneous and well defined biohybrid materials. Of importance for such applications are stimuli responsive or “smart” materials which present the ability to reversibly alter their structure and physico-chemical properties when subjected to an external stimulus such as temperature or pH.1-4 In this presentation we will focus on designing temperature responsive polymer-peptide based hydrogels. As the bare scaffold we use ionic complementary oligopeptides as these short peptides are known to self-assemble into β-sheets and their subsequent fibrillation gives physically cross-linked networks and ultimately self-supporting hydrogels.5 To introduce an internal responsive transition into these hydrogels , the peptidic segments have been coupled to thermoresponsive poly-N-isopropylacrylamide (pNIPAAm) polymer, which dis-plays a reversible phase transition in aqueous solution from a swollen, hydrated state to a col-lapsed, dehydrated state depending on the temperature. pNIPAAm&’s with defined architecture have been synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization allowing control over the molecular weight and chain end functionality.6 This provides control over the physical properties of the polymeric segments i.e. the temperature at which the transition occurs. Through this convergent synthesis approach, we have been able to synthesize a new generation of smart hydrogels in an efficient and quantitative manner. Here we will show how these novel materials present unique physical properties which can be tuned simply by adjust-ing the peptide/polymer ratio or by adjusting the length of the polymeric segment. They also display singular changes in behavior when subjected to an environmental cue making them attractive candidates in a range of biomedical applications including drug delivery and tissue engineering. References 1. S. K. Ahn, R. M. Kasi, S. C. Kim, N. Sharma and Y. X. Zhou, Soft Matter 4 (6), 1151-1157 (2008). 2. A. K. Bajpai, J. Bajpai, R. Saini and R. Gupta, Polym. Rev. 51 (1), 53-97 (2011). 3. F. Stoica, C. Alexander, N. Tirelli, A. F. Miller and A. Saiani, Chem. Commun. (37), 4433-4435 (2008). 4. A. Maslovskis, N. Tirelli, A. Saiani and A. F. Miller, Soft Matter 7 (13), 6025-6033 (2011). 5. A. Mohammed, A. F. Miller and A. Saiani, Macromol. Symp. 251, 88-95 (2007). 6. B. Yu, J. W. Chan, C. E. Hoyle and A. B. Lowe, J. Polym. Sci. Pol. Chem. 47 (14), 3544-3557 (2009).

Inorganic semi-conductor nanoparticles possess a range of tunable optical fluorescence depending on their chemical composition and shapes (sphere or rods) whereas the surface ligands can be optimized to tailor interaction with the surrounding environment. Rod like semi-conductor particles or Quantum rods (QR) are ideal building block in order to create new 3d structures for optical materials (Dif A., Henry E. et al, Nano letters, 2012, 11(12), 5443) We describe here the surface chemistry and a method for self-assembling QR into 3D crystals at a macroscopic scale by using dynamical template upon controlled slow rate evaporation of water. QRs functionalization was achieved using a peptide-based anchor and carrying a tunable function such as a carboxylic acid or an amide (Dif A. et al, J.A.C.S., 2009, 131(41): 14738). Ligand exchange was characterized by electrophoreses, NMR and FTIR. Following this step, 3D smectic B like microcrystals of nanorods were obtained in capillaries and then upscale to macrocrystal using a microstructured PDMS pattern and a controlled rate of water evaporation, (Hamon C. et al, ACS Nano, 2012, 6(5): 4137), as demonstrated by TEM and SAXS experiments. These processes ensure the high quality of the crystal and offer opportunities to generate crystals with new symmetries upon QR size and ligands modulation.

Peptide hydrogels have gained special interest as an emerging class of biomaterials due to their ability to be degraded by naturally occurring proteolytic enzymes as well as possessing mechanical properties that can be fine-tuned to mimic a chosen tissue type. Coupling of these two properties has been achieved through exploration of chirality effects in hydrogels composed of self-assembling β-hairpin peptides. Oscillatory shear rheology shows that L-MAX forms a mechanically rigid hydrogel under physiological conditions. Surprisingly, when its enantiomer, D-MAX1, is doped into the gel a non-additive, synergistic enhancement of these mechanical properties is observed leading to a large increase in material rigidity. In the racemic system, a 1 wt % hydrogel results in a 4-fold increase in material rigidity with a storage modulus of 800 Pa, compared to hydrogels composed of only one enantiomer, either L-MAX1 or D-MAX1, in which both gels have storage moduli of 200 Pa. The basis for this increase in rigidity has been explored on both the network and molecular levels. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) studies of the racemic gel revealed fibrillar networks with fibril heights and widths corresponding to the expected dimensions of a fiber composed of a bilayer of assembled hairpins. To determine if L-MAX1 and D-MAX1 co-assemble within a given fibril or self-sort to form homogeneous fibrils within a racemic hydrogel, peptides were orthogonally labeled post-assembly with 10 nm (L-MAX1) and 5 nm gold nanoparticles (D-MAX1). Once labeled with gold nanoparticles, the location of the peptide enantiomers within the fibrillar network was observed by TEM. Molecular level interactions of the enantiomeric peptides were further investigated using isotope-edited infrared spectroscopy. Additionally, rates of hydrogel degradation by trypsin can be controlled by varying the molar stoichiometry of enantiomers in the hydrogel formulation as assessed by oscillatory rheology, HPLC, and mass spectroscopy. The use of chirality as a design tool to simultaneously control material degradation and rigidity presents a new way to prepare tunable hydrogels.

Self-assembling peptide hydrogels are rapidly becoming established candidates for new tissue engineering scaffolds. P11-4, designed at Leeds, is one such peptide (Ac-QQRFEWEFEQQ-Am): it is already finding application as a treatment for early stage dental caries. Like many of the other self-assembling peptide classes available, P11-4 does not incorporate sequences that specifically promote cell interaction. Nonetheless, these can be attached to a self-assembling peptide. Unmodified peptides can then be mixed with modified peptides to produce functionally decorated scaffolds for tissue engineering. A common modification is the incorporation of the RGD sequence found in ECM proteins, like fibronectin. RGD is recognised by cell integrins, facilitating cell adhesion. Cyclic RGD (cRGD) enhances interactions with integrins over the linear form [1]. However, almost all of the RGD-modified self-assembling peptide studies to date have used linear RGD modifications. Several cRGD variants of P11-4 have been synthesised, differing in charge and the nature of the C terminus (free versus amidated) and the size of the cRGD ligand. These peptides was characterised by microscopy (TEM and AFM), both individually and co-assembled with P11-4 in different ratios. Differences were apparent in the structures formed by the modified cRGD peptides alone when compared with those of unmodified P11-4. However, when mixing cRGD-P11-4 with unmodified P11-4, structures more like those of P11-4 alone were observed. A fluorinated phenylalanine was also included in the cyclic ligand so that peptide self-assembly could be studied by 19F NMR. Initial NMR studies provide further evidence that the cRGD-P11-4 is capable of co-assembling with unmodified P11-4 to form mixed scaffolds. Scaffolds were assessed in vitro with an ATPLite-M assay of primary human dermal fibroblasts seeded within peptide scaffolds. P11-4 and several mixtures of P11-4 with cRGD-P11-4 (1:200, 1:100 and 1:50 ratios) were compared with collagen. Even with no RGD modification, P11-4 itself performed as well as collagen. Variation of the cRGD-P11-4/P11-4 mixing ratio manifests as differences in scaffold effectiveness. With too low an RGD concentration (1:200), no enhancement was gained over collagen. With the highest proportion of RGD (1:50), scaffolds performed better than collagen at early stages but not at later stages. This is potentially explained either by cell overcrowding on the 1:50 scaffold, or perhaps by cells on collagen beginning to generate their own ECM. By contrast, 1:100 cRGD-P11-4/P11-4 scaffolds outperformed collagen throughout the 28 day study. These RGD-modified scaffolds are now being applied to a range of soft tissue engineering applications. Furthermore, these findings show that modifying self-assembling peptides in this fashion is a promising route to optimising these materials for tissue engineering. [1] Haubner et al., JACS 1996, 118, p7461-7472

Organophosphates are some of the most acutely toxic compounds synthesized on an industrial scale, and organophosphorus hydrolase (OPH) has the ability to hydrolyze and inactivate a number of these chemicals. However, OPH activity is vulnerable to harsh environmental conditions that would accompany its practical utility in the field; a limitation that can also be extended to conditions required for incorporation of OPH into useful materials. Here we present evidence that entrapment of OPH in silk fibroin leads to stabilization of OPH activity under a variety of conditions that would otherwise reduce free enzyme activity, such as elevated temperature, UV light exposure and the presence of detergent. However, modulation of silk fibroin secondary structure through annealing of films under different conditions did not correlate with the resulting specific activity of entrapped OPH. Silk fibroin entrapment of OPH also allowed for its dispersal into a polyurethane-based coating that retained organophosphate hydrolysis activity after formulation, application and drying. Together, the data presented here demonstrate the utility of silk fibroin entrapment for the protection of OPH activity under a variety of environmental conditions.

The elastin-like peptide (ELP) is temperature sensitive biopolymer which undergoes inverse transition at critical temperature. The design of elastin-like peptides is motivated by their ability to self-assemble in response to clinically relevant intrinsic stimuli; however the understanding of how sequence and length of the peptide controls self-assembly processes is unknown. The hydrophobic peptide with repeating Val-Pro-Gly-Val-Gly sequence, (VPGVG)n, is a widely used model of peptides with elastin like properties. While this peptide is soluble in water at cold temperatures, it can undergo a self-assembly into fibril like structures at physiological temperatures. We performed molecular dynamics simulations of (VPGVG)n peptides with different length at temperatures above and below critical temperature. A comprehensive analysis of the temperature dependence of the shape, size, hydration and dynamics of (VPGVG)n with varying lengths has been performed. Simulations suggest that single (VPGVG)n show compactions at all temperatures, with more β-type structures for higher temperature. With increasing temperature, the peptides are found to transition from rigid to flexible states. A detailed molecular study involving thermodynamics parameters and reaction coordinate for this hydrated peptide system is presented along with the hydrogen-bond analysis within peptide as well as between the peptide and solvating water molecules. The results from our study provide an atomic-level description of the thermo-responsive conformational properties of VPGVG elastin-like peptides. Support for this research was provided by the NSF's Research Triangle MRSEC (DMR-1121107).

Over the last decade, self-assembling peptide like materials have proved their potential for biomedical applications such as drug delivery , in vivo imaging, tissue regeneration, bio-inspired materials and vaccine delivery. Self-assembling peptide nanostructures are versatile in terms of physicochemical characteristics, which can be fine tuned by varying the amino acid sequence or composition. Recently we have developed a new class of polypeptide-b-peptide based amphiphiles (PbP-As), which has a hydrophilic block comprising a coiled coil-forming peptide and a hydrophobic block of poly-γ-benzyl-L-glutamate (PBLG). In this work we have synthesized and characterized a new TAT conjugated PBLG (PbP-A). Using the solvent evaporation method, we produced assemblies and successively formulated them with CpG, a TLR9 ligand known to enhance Th1 type immune responses, and with an influenza sub-unit vaccine. Physico-chemical characterization of these formulations have been achieved and their potency as vaccine delivery system was tested in vitro and in vivo. In conclusion, in this work we succeeded to develop self-assembled peptide NPs (based on the a new TAT PbP-A), loaded with CpG, which enhanced the immunogenicity of a NP-associated antigen, HA. These results are very encouraging in terms of vaccination, as the optimal vaccine delivery system differs for each vaccine, and the optimal effect required specific changes. Therefore, further functionalization of the PbP-NP surface can be achieved and should be investigated in more detail.

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role during bone regeneration and repair. This protein has seen clinical application as a bone-graft substitute in combination with an absorbable collagen sponge; however, there is still a great need for improved strategies to localize the cytokine within the defect. We report here on supramolecular nanofiber gels that promote bone regeneration through BMP-2 signaling. These nanofibers are constructed from building blocks of peptide amphiphile (PA) molecules that present specific sequences on the surface of the nanofibers. We evaluated a PA that forms nanofibers with surfaces that display strands of heparan sulfate because of the affinity of one of their peptide segments for the polysaccharide, thus binding BMP-2 through its heparin-binding domains. Another BMP-2-binding PA was designed using a peptide that specifically binds BMP-2 in order to create a PA gel that could localize the growth factor. Both supramolecular nanofibers were allowed to form gel networks within the pores of a collagen scaffold by infiltrating dilute solutions of the components. The hybrid biomaterials exhibited prolonged retention of BMP-2 in vitro and enhanced bone regeneration in vivo using a rat critical-size femoral defect model using BMP-2 amounts that are one order of magnitude lower than required for healing in this model. Our findings demonstrate the possibility of designing materials that enhance the regenerative capacity of growth factors.

Anisotropic structures with distinct micro- and nanoscaled compartments have been of great interest in various biomedical applications including advanced drug delivery system, molecular imaging and particle-based diagnostics. Recently, electrohydrodynamic (EHD) co-jetting of multiple polymer solutions with a side-by-side geometry has been used for synthesis of anisotropic structures. While anisotropic structures of synthetic polymer-based biomaterials have been burgeoning, there have been very limited studies on anisotropic polypeptide nanostructures for in vivo applications. We are specifically interested in environmentally-responsive elastin-like polypeptides (ELPs) derived from the tropoelastin because they undergo an inverse phase transition in aqueous conditions at a specific temperature (Tt) called as lower critical solution temperature (LCST). ELPs are highly soluble in an aqueous solution below Tt while being desolvated and becoming insoluble above Tt in a completely reversible way. In this study, we first report a facile method to prepare a new class of anisotropic ELP nanoparticles with environmental responsiveness by spontaneous structure transformation of biphasic ELP nanofiber architecture. Rationally designed ELP diblock copolymers composed of crosslinkable- and uncrosslinkable block were genetically engineered by recursive directional ligation for concatemerization, over-expressed by E. coli with isopropyl β-D-thiogalactopyranoside induction for 24 hours, and purified by inverse transition cycling. Acrylic moieties were incorporated into the crosslinkable block of ELP diblock copolymers by using methacrylic anhydride for selective chemical crosslinking. The biphasic ELP nanofibers composed of acrylated ELP diblock copolymers were synthesized by organic solvent-based EHD co-jetting to prevent aggregation of ELP blocks during solvent evaporation, and then chemically stabilized via UV irradiation in the presence of a photoinitiator. Finally, when incubated in aqueous conditions, the biphasic ELP nanofibers were spontaneously disintegrated and then stabilized into the anisotropic ELP nanoparticles, potentially due to dissolution of the uncrosslinked block and equilibrium of the selectively crosslinked block. Therefore, anisotropic ELP nanoparticles formed by spontaneous structure transformation of biphasic ELP nanofibers were stable in dry and swollen state while maintaining distinct two compartments with environmental responsiveness, determined by scanning electron microscopy and confocal laser scanning microscopy. It suggests that rationally designed ELP block architecture and composition precisely controlled at a genetic level could be useful to create multifunctional polypeptide nanostructures formed by spontaneous structure transformation. Conceptually, this study could establish a facile method to prepare anisotropic polypeptide nanoparticles for smart drug delivery system and molecular imaging.

In recent years, self-assembling peptides have been widely investigated due to their potential in a wide range of applications, including nanotechnology and biomedicine. Amongst these systems are “ionic-complementary” peptides, which are designed to induce β-sheet fibrillar self-assembly due to their constitutive alternating charged, non-charged, hydrophobic and hydrophilic amino acids. Under specific conditions these fibrils typically self-associate further into thicker fibres that entangle to form 3D networks and hydrogels. One of the key challenges is to control the characteristics of these materials, in particular the mechanical properties. The properties of peptidic hydrogels will not only depend on the intrinsic characteristics of the fibres but also on the way they assemble to form 3D networks. Here we have investigated the use of arginine residues to control the mechanical properties of peptidic hydrogels. FEFEFKFK has previously been shown to adopt an antiparallel β-sheet conformation at a supramolecular level and to self-assemble into a branched network of semi-flexible fibres at a macroscopic level [Saiani, A. et al. Soft Matter, 2009, 5, 193-202]. Replacing both lysine (K) by arginine (R) residues in FEFEFRFR induced the formation of fibres that had the propensity to laterally associate, enhancing their rigidity and favouring the formation of a highly entangled network. This topological difference was found to impact on the mechanical properties of the hydrogels formed. In order to control the network topology of the self-assembling systems two options were subsequently considered. The first one consisted in mixing physically both FEFEFKFK and FEFEFRFR peptides. The second lied in modifying the peptide design by incorporating K and R amino acids within one same peptide, FEFEFRFK. The effect of these combinations was investigated using rheometry, TEM and SANS.

Biomimetric artificial light harvesting systems are an attractive approach to the development of new, boundless energy sources. Self assembling β-sheet peptides, may serve as a scaffold for the presentation of Light Harvesting Complexes (LHC&’s) similar to those often seen in plants and bacteria. Taking advantage of high peptide expression levels and efficient purification methods, novel β-sheet forming peptides GH6[(GA)3GK(GA)3GW(GA)3GE(GA)3GW]8C1 and GD5[(GA)3GK(GA)3GW(GA)3GE(GA)3GW]8H6 were prepared based on previously well characterized model peptides. Our single-molecule approach offers three immediate advantages. First, the necessity of using two separate peptides tagged with appropriate donor and acceptors molecules is eliminated. Secondly, acceptor emission is enhanced as a result of high donor to acceptor molar ratios per molecule. Finally, resonance energy transfer (RET) is optimized by the tight control of distance and orientation of donor and acceptor molecules on the β-sheet scaffold. Tryptophan, an intrinsic chromophore found in peptides, can be an excellent donor species when utilized with the appropriate acceptor dye. Fluorescence characterization of tryptophan with respect to environment, i.e. free tryptophan, disordered tryptophan-bearing peptide, β-sheet-bearing tryptophan, and finally fibrillar peptide assemblies bearing tryptophan, will be reported. Chemical modification of a terminal cysteine residue, acceptor dye attachment via maleimide-thiol coupling, places both donor and acceptor species within a single molecule. Moreover, selective folding to form β-sheet assemblages, by modulation of solution properties such as pH, leads to β-strand-turn formation where solvent exposed tryptophan moieties, are now in conformationally constrained alignment with acceptor dye molecules. The influence of the spatial alignment of donor and acceptor molecules, subsequent to β-sheet formation, provides a productive environment for resonance energy transfer, and will be discussed in detail. In conclusion, we plan to report on the preparation of a novel single-peptide based LHC, capable of resonance energy transfer while possessing biotechnological utility at the nanoscale level.

Bio-molecular integrated electronic devices that exploit peptide based self assembly are the focus of increasing interest. To be useful in practical applications the optoelectronic properties of protein based nanostructures in ambient conditions must be understood at a fundamental level. Toward this goal, a peptide molecule incorporating porphyrin-chromophore molecules is designed and patterned on electrode substrates (HOPG), and is characterized by Torsional Resonance NanoImpedence Microscopy. We show that the peptides align on the substrate, retain the structure in ambient (i.e. non liquid) conditions, and furthermore exhibit interesting opto elecronic properties. The ground state and excited state polarizability are quantatively compared. These results have implications to energy harvesting and sensing applications.

It is well known that the extracellular environment that cells experience influences numerous biological processes. However, in vitro cell biology studies, such as drug screening and stem cell engineering, often use polystyrene-based labware that is not chemically and physically similar to what cells would experience in vivo. Here, we are examining the design of an appropriate in vitro environment for retinal pigment epithelial (RPE) cells that mimics the in vivo niche. The use of stem cell-derived RPE cells to repair and cure Age-Related Macular Degeneration (AMD) is of great interest. Previously, we have described a model extracellular matrix comprised of collagen type 1 that self-assembles into thin films of fibrils. Type 1 collagen is the prominent component of the acellular intercollagenous layer of the Bruch&’s membrane, which resides adjacent to the RPE cell layer within the choroid. We demonstrate that by exploiting the biochemical and mechanical control we have over these robust fibril films we are able to quantitatively elucidate the relationship between the ECM niche and RPE morphology, tight junction formation, and proliferation rates. These insights will be useful for applications in forming transplantable complexes of ECM and induced pluripotent stem cell-derived RPE cell sheets to treat AMD.

Inorganic binding peptides are of importance in nanobiotechnology since they can be exploited to tailor peptide based hybrid systems and novel materials for practical applications in a wide range of areas including materials science, engineering, environment and medicine. In our group, we have identified and characterized peptides that can bind a variety of inorganic materials specifically and with high affinity using biocombinatorial techniques. As previously reported, we have selected two gold binding peptide (AuBP1 and AuBP2) sequences using a FliTrx random peptide display library. Here, we employed a single step peptide based controlled patterning where gold binding peptide, AuBP2, was used for the directed immobilization of a multifunctional protein on gold substrates. Multifunctional protein which includes green fluorescent protein (GFPuv) and Maltose Binding Protein (MBP) was successfully produced through genetic conjugation of combinatorially selected AuBP2 to GFPuv. Fusion DNA fragment was cloned into pMALc4x expression vector encoding N-terminal MBP tag. Following expression and purification, the excitation, emission wavelengths and fluorescence intensity of the multifunctional protein MBP-GFPuv-AuBP2 were compared with a negative control MBP-GFPuv. It has been shown that MBP-GFPuv and MBP-GFPuv-AuBP2 proteins have equivalent excitation and emission wavelengths with similar intensities. Protein patterns were prepared by directed stamping of MBP-GFPuv-AuBP and control MBP-GFPuv fusion proteins on flat gold surface. Fluorescence microscopy images of the protein-arrayed gold surfaces showed that the multifunctional MBP-GFPuv-AuBP protein immobilized on gold surface displays higher affinity compared to the negative control. Site-specific protein immobilization and patterning is important for the fabrication of efficient biotechnological tools such as biosensors and microarrays. Using AuBP2 as a specific molecular linker, we demonstrated for the controlled immobilization of a fluorescent heterofunctional protein on metal surface.

Recent applications of heterofunctional hybrid nanosystems that combine nano-scale inorganic materials and functional biological molecules in diagnostics and therapeutics is now emerging biomedical research field with high potentials for development of new bio-imaging, delivery and -targeting platforms. In order to generate hybrid targeting and delivery systems, the paradigm of combining organic and inorganic interfaces should be carefully considered. To overcome difficulties related to conventional synthesis and cross-linking methods, we have biocombinatorially selected genetically engineered peptides for inorganics, GEPIs, that have nanomolar binding affinities to inorganic surfaces. GEPIs can be further modified, engineered through genetic engineering methods and synthesized to utilize as molecular tools with controlled functionalities. In order to be able to provide new functionality to GEPI, new recombinant bi-functional proteins can be generated using maltose binding protein (MBP). Various types of GEPIs such as gold binding peptide (AuBP), quartz binding peptide (QBP), and silver binding peptide (AgBP) can be designed and expressed genetically. In conjugation of GEPI to MBP, numerous spacers such as S(G)3 or (PG)3 between peptide and protein play an important role in functionality of novel fusion proteins. By using molecular dynamic methods S(G)3 spacer is used in this work. And functionality of designed proteins is demonstrated by self-assembly and soft lithography processes in various nanostructures and biomolecules onto solid surfaces. The peptide-based molecular tools are an alternative to the conventional chemical coupling for specific assembly of inorganic and organic entities onto patterned inorganic surfaces for the assembly of hybrid nanostructures, nanobiophotonic platforms and biosensing. The research is supported by NSF-MRSEC Program through the University of Washington GEMSEC and ITU BAP-Istanbul Technical University.

Stem cell transplantation is a promising therapy for many diseases and injuries; however, current delivery protocols are inadequate. Transplantation by direct injection, which is clinically preferred for its minimal invasiveness, commonly results in less than 5% cell viability. To overcome this limitation, we demonstrate that cell encapsulation within shear-thinning, physical hydrogels can significantly improve viability by preventing cell membrane disruption. We have designed an injectable, bio-resorbable, customizable hydrogel using protein-engineering technology. In our Mixing-Induced Two-Component Hydrogel (MITCH), network assembly is driven by stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with polymer physics models, we manipulate the polypeptide chain interactions and demonstrate the direct ability to tune the network crosslinking density, sol-gel phase behavior, and gel mechanics. This is in contrast to many other physical hydrogels, where predictable tuning of bulk mechanics from the molecular level remains elusive due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. These MITCH materials enable stem cell and growth factor encapsulation at constant physiological conditions - a significant advantage over other commonly used hydrogels. Through a series of in vitro and in vivo studies, we demonstrate that these materials may significantly improve transplanted stem cell retention.

In recent years, self-assembly and stimuli-responsive materials have received considerable attention due to their potential applications in drug delivery, bio-sensing or regenerative medicine. In this context the choice of building block is crucial to allow the design of structures with controlled geometry and properties. While such supramolecular structures are traditionally fabricated from high molecular weight amphiphilic polymers, peptides have emerged as alternative building blocks due to their propensity to self-assemble into ordered supramolecular architectures. Being able to trigger the self-assembly of these molecules by an external stimuli such as pH, ionic strength, light or enzyme is attracting increasing attention as a route to control the fabrication of novel soft-solid biomaterials. Such fabrication route is particularly attractive for 3D tissue engineering applications offering the opportunity to incorporate cells into a 3D network by triggering the sol-gel transition. Herein we focus on the enzymatic triggered gelation of ionic oligopeptides containing the alternating hydrophobic/hydrophilic and charged/non-charged amino acids F, E and K. Octapeptides containing these amino acids are known to self-assemble into β-sheet rich fibrillar networks. Our aim was to generate self-assembling oligopeptides from soluble, short precursor peptides (namely FEFK) simply by adding enzyme, thus inducing a sol-gel transition. Thermolysin, a protease enzyme known to specifically catalyze the hydrolysis of peptide bonds containing hydrophobic amino acids (F), was selected as it has been shown that proteases can be encouraged to work in reverse hydrolysis when the reaction product is thermodynamically stabilized relative to its precursors. Solutions of FEFK were prepared by dissolving the tetrapeptide in water and adjusting the pH to 7 prior to the addition of thermolysin. At low tetrapeptide concentrations (le; 50 mg mLminus;1), the sample remained in liquid state. At high concentrations (ge; 60 mg mLminus;1), clear, self-supporting gels formed. The resulting systems have been characterised using MALDI-TOF MS, HPLC, TEM, oscillatory rheometry, FTIR and SAXS/SANS. This revealed that a dynamic library of peptides formed, with octapeptides forming preferentially. Once formed the octapeptide sequences assembled into β-sheet rich nanofibers that subsequently entangled triggering the sol-gel transition. We also showed that the enzyme concentration (Cenz) does not affect the self-assembly of these peptides at a molecular level nor the structure of fibrillar network formed at the nanometer scale. On the other hand the mechanical properties were found to be affected by Cenz and the hydrogels shear modulii were found to increase with increasing Cenz. These results suggest that morphological differences between the hydrogels at the micro-scale are at the origin of the difference in mechanical properties. We propose a morphological model in which denser network regions are found around the enzymes, resulting in the creation of heterogeneous networks. The existence of these denser network regions result in the reinforcement of the hydrogels and explain the high shear modulii obtained.

Protein aggregates involving amyloid fibrils have recently received enormous attention in a wide variety of research areas such as medicine, nanotechnology, food, and soft matter science. The most widely known example of amyloid fibrils is the one frequently associated with neurodegenerative diseases such as prion, Alzheimer&’s and Parkinson&’s diseases. On the other hand, amyloid fibrils, induced by the hydrogen bonds between parallel or anti-parallel beta sheets, have great potentials as functional nano-biomaterials due to their highly ordered structure, robust mechanical properties, and biodegradability. In this study, we introduce a novel platform for in-situ nanocomsposites based on the fibrillation of proteins within Layer-by-Layer (LbL) films by controlling intermolecular interactions between precursor proteins of amyloid fibrils and charged polyelectrolytes. κ-Casein, which is one kind of proteins from bovine milk without any connection to diseases, and poly(acrylic acid) (PAA) were used to build up multilayered thin films. The intermolecular interactions within the multilayered films, assembled by a combination of hydrogen bonding and electrostatic interaction, were tuned by external pH change. When the interactions between κ-casein precursors and PAA are weaker than the initial interactions of an assembled film by the change in pH, the thermal treatment of the weakly bound film leads to long fibrils of κ-casein precursors within the film. The fibrillation-driven in-situ external stimuli within the LbL films could serve as useful advanced platforms for reinforced polymeric systems as well as biomedical applications.

Aggregation of proteins is central to many aspects of daily life, ranging from food technology and pharmaceutical science, to blood coagulation and health disorders, such as sickle-cell disease, arterial thrombosis, or eye cataract formation. In particular, association of proteins into amyloid fibrils is a highly specific process occurring both in-vivo, such as in the Alzheimer, Parkinson or prion-related neurodegenerative diseases, and in-vitro, as in the case of processed food proteins. In this talk I will discuss the recent developments in the understanding of the association processes converting globular proteins into amyloid fibrils, with emphasis on β-lactoglobulin and lysozyme, which have both fundamental and practical relevance. I will first illustrate how the unique combination of experimental techniques (light, neutron and x-rays scattering, AFM and cryoTEM), with polymer and colloidal physics concepts, can reveal important structural features from the nanometer to micron lengthscales and how these information can be used to understand the main mechanisms ruling aggregation. I will then touch on how these mechanisms can be engineered to produce highly ordered multistranded amyloid fibrils with tunable nanostructures, in which the number of protofilaments forming a single fibril, their topology, and the twisted ribbon, helical ribbon and nanotube polymorphism can be efficiently controlled. Finally, I will discuss how these protein fibrils can be exploited as unique building blocks for complex and functional fluids, serving fields as diverse as food science, biomaterials, biosensors and optoelectronics, and I will illustrate this via relevant examples for each specific category. References 1. “Understanding amyloid aggregation by statistical analysis of atomic force microscopy images” Adamcik et al., Nature nanotechnology, 5, 423 (2010). 2. “General Self-Assembly Mechanism Converting Hydrolyzed Globular Proteins Into Giant Multistranded Amyloid Ribbons”, Lara et al. Biomacromolecules, 12, 1868 (2011). 3. “New biocompatible thermo-reversible hydrogels from PNiPAM-decorated amyloid fibrils”. Li et al. Chem. Comm. 47, 2913 (2011). 4. “Sub-persistence Length Complex Scaling Behavior in Lysozyme Amyloid Fibrils”. Lara et al. Phys. Rev. Lett. 107, 238101 (2011). 5. “Protein Fibrils from Polymer Physics Perspective” Adamcik & Mezzenga Macromolecules, 45, 1137 (2012). 6. Li et al. "Biodegradable nanocomposites of amyloid fibrils and graphene with shape-memory and enzyme-sensing properties" Nature nanotechnology, in press (2012).

Spider silk is a natural protein fibre with outstanding mechanical properties, and its high strength combined with large extensibility outshines many synthetic materials. Recently, the thermal conductivity, shape memory, and humidity-driven changes in mechanical performances have raised new interest in this promising future material. Another remarkable property of spider dragline silk is “super-contraction” [1]: when dragline silk is in contact with liquid water, it contracts instantaneously and often substantially depending on the species of the spider and silk composition and morphology. More interestingly, when the contracted silk is restretched back to its original length, it retains its ability to super-contract. The contraction-stretching cycles can be reproduced many times. Instead of exploring the phenomenon itself, we are pursuing the fundamental mechanisms: what are the structural components causing “super-contraction” in spider silk? We define the structural components in spider silk as permanent order (PO), meta-order (MO), meta-disorder (MD) and permanent disorder (PD), loosely correlating to beta-sheet, proline-containing beta or helical structure, oriented random coil, and random coil. PO or PD is not susceptible to structural shrinkage, while meta-order and meta-disorder are both contributors, but each operating with different mechanisms. The key experiment to separate the contribution from MO or MD and to quantify one contribution was the observation of the fraction of the total supercontraction - glass transition, Tg, of the MD component, which is temperature- and stress-dependent. The maximum Tg-Contraction for Nephila spider dragline turned out to be 13% out of 28% for full supercontraction, so that a faction of 16% for MD component and 18% for MO component was estimated. A simple linear series model for the composition and organization of the four components was proposed for the structure-property relations of this spider silk. The two mechanisms for “super-contraction” in spider silk were attributed to MD oriented random coil for Tg-Contraction, and MO stretched helical for the water-contraction that is more novel. This could offer insights into designing new responsive materials to environmental stimuli, such as humidity, temperature and mechanical stress. We also demonstrate that the new approach of using dynamic mechanical thermal analysis, DMTA, to understand the structure-property relations of micron-thick natural silks is a powerful analytical tool that is potentially useful for other natural materials, particularly with temperature-humidity scans to augment the standard temperature scans. References: 1. J. Guan, F. Vollrath and D. Porter. Biomacromolecules 2011, 12, 4030-4035.

pH-Responsive properties of soft microcapsules are demonstrated using the layer-by-layer (LbL) shells fabricated from poly-amino acid modified silk fibroin. In contrast to most commonly studied synthetic polyelectrolytes, the silk ionomers are biodegradable and biocompatible. Mono-dispersed microcapsules showed dramatic swelling when exposed to extremes of acidic and basic conditions, but without noticeable disintegration. These changes were accompanied by pH-induced shell permeability at acidic (pH below 2.5) and basic (pH above 11.0) conditions with two orders of magnitude increase in the diffusion coefficient. Associated with the swelling and increase in porosity, the silk ionomeric shells undergo remarkabl softening, with a drop in Young&’s modulus by more than an order of magnitude. Moreover, the stiffness of the capsules can be further tuned by chemical cross-linking as needed. Engineering cell surfaces such as with E. coli, B. subtilis and S. cerevisiae with inomeric silk shells provides advantages due to biocompatibility, enhanced stability via cell protection to harsh environments. Viability rates depend on the type of cell, where yeast cells coated with 3 and 4 bilayers showed significantly higher survivability (86% and 90%, respectively) compared to the bacterial cells.

Protein design opens new ways to study proteins and arrive at novel molecules, materials and nanostructures. Theoretical methods for identifying the properties of amino acid sequences consistent with a desired structure and functional properties will be discussed. Such methods leverage concepts from statistical mechanics and address the structural complexity of proteins and their many possible amino acid sequences. We present the application of these methods to the design of nonbiological protein assemblies, including protein crystals where space group and multiscale ordering with subnanometer precision are specified a priori. The exterior of a protein is designed de novo, and the experimentally determined structure of one such designed crystal is in close agreement with the computationally designed structure.

Self-assembly represents a simple and efficient route to the construction of large, complex structures. Protein and peptide self-assembly in particular offers the possibility to design new functional bio-materials that are finding application in drug delivery, tissue engineering, energy harvesting and bioelectronics to name but a few. A number of design rules are beginning to emerge in this area, where secondary structure of the self-assembled fibre, the fibre thickness, and hydrogel porosity and mechanical properties (modulus, viscosity and compression) can all be varied predictably simply by changing the amino acid sequence and its concentration. Such self-assembled structures are, however, essentially bare, so here we will focus on the design rules for introducing bioactivity and responsiveness into the gelled state. The general design opportunities offered by these peptide based systems will be presented here with different applications within the regenerative medical area.

Chirality can be used as a design tool to control the mechanical rigidity of hydrogels formed from self-assembling peptides. Hydrogels prepared from enantiomeric mixtures of self-assembling β-hairpins show non-additive, synergistic, enhancement in material rigidity compared to gels prepared from either pure enantiomer, with the racemic hydrogel showing the greatest effect. CD spectroscopy, TEM, AFM, molecular modeling, and isotope edited FTIR studies indicate that this enhancement is defined by nanoscale interactions between enantiomers in the self-assembled state.

Hydrophobic collapse of amphiphilic b-hairpin peptides (e.g. MAX1 VKVKVKVKVDPPTKVKVKVKV-NH2) into fibrils and their hierarchical assembly into branched, hydrogel networks has been extensively studied. A physically crosslinked hydrogel network is formed due to fibrillar entanglement and branched defects in hydrophobic collapse during fibril formation. Alternating valine residues with side chains of the same size are responsible for the hydrophobic collapse of the molecule into a b-hairpin and fibril nanostructure with branching. In a new sequence LNK1 (LNK1 (Nal)K(Nal)KAKAKVDPPTKAKAK(Nal)K(Nal)-NH2) the non-beta turn valines were replaced with Napthylalanine and alanine amino acid residues, with hydrophobic side chains of larger and smaller volume, respectively, than valine. Thus, formation of a ‘lock and key&’ type structure was attempted in the hydrophobic core of the peptide fibrils that would eliminate fibril branching. The folding and network formation of LNK1 has been studied by Circular Dichroism spectroscopy (CD), Transmission Electron Microscopy (TEM), Oscillatory Rheology and in-situ rheology coupled with Small Angle Neutron scattering (rheo-SANS). Preliminary rheological characterization suggests the elimination of branching in the fibrils and also a possibility that LNK1 networks, unlike MAX1, are just nanofibrillar suspensions rather than permanently physically crosslinked hydrogels.

Peptides and proteins are attractive medicines for a wide variety of diseases. Judiciously combining these biologics with synthetic polymers provides opportunities to overcome problems related to their (limited) stability and plasma half-life, allows targeted delivery and release and may be used to the augment the activity of peptide and protein therapeutics. This contribution will discuss the preparation and properties of peptide - polymer conjugates that are designed to fight HIV-1 infection. Specifically, this presentation will cover 2 examples, which will successively discuss: (i) polymer-modified HIV fusion inhibitors that show increased stabilities as compared to the unmodified peptides while maintaining activity and (ii) multivalent HIV entry inhibitors, which allow augmenting the activity of the peptide. These two selected case studies serve to highlight the importance of controlling polymer molecular weight, site-selective of polymer conjugation as well as the influence of polymer architecture on the final biological properties of peptide - polymer conjugates.

In many ways collagen epitomizes multi-hierarchical self-assembly. The first step of self-assembly is the winding of three protein alpha chains around one another to form a right handed super helix. These helices continue to assemble into fibrils, fibers and eventually form the primary scaffolding of the viscoelastic extracellular matrix. While triple helix formation has been studied for decades, only recently have serious efforts been made to control the composition and register of heterotrimeric assemblies. I will describe our efforts to control collagen triple helix assembly using rationally designed charge pair interactions and how our design rules can be translated into an automated approach to residue selection. The next step of self-assembly, fibrillogenesis, has had even fewer successes and, until recently, no reported synthetic system had demonstrated triple helix, nanofiber and hydrogel formation simultaneously. I will present our work on designed systems to control triple helix self-assembly, composition and register and how our improved molecular understanding of these assembling units led to successful fibrillogenesis and hydrogel formation.

The past decade has seen growing interest in the investigation of self-assembling nanostructures, particularly in aqueous solution. In this context, polypeptide-based copolymers show considerable promise as building blocks that allow enhanced control over intra- and intermolecular interactions, in concert with stable, yet modifiable, secondary and tertiary structures. In this context, polypeptide-based vesicles are certainly the most interesting structure and may offer many advantages compared to low molar mass lipid vesicles, in particular for applications in drug delivery (1). We report here different series of polypeptide-based block copolymers that we are currently studying in our group, combining a large range of experimental analysis in order to describe in great details the self-assembly properties of such systems. One of our aim is to exploit the pH-sensitivity of polypeptides&’ secondary structure to manipulate the size and shape of the supramolecular structures formed by self-organization of these block copolymers in aqueous media. In contrast to most other amphiphilic polyelectrolyte (block) copolymers, the use of polypeptide based diblock copolymers for the preparation of such nanostructures is more precise and is offering great promise in biomaterials design (2). Indeed, the hydrophilic peptide block has the ability to fold into well-defined secondary structures such as α-helix, β-sheet or coil depending on environmental changes (temperature, pH, ionic strength). The last development—and certainly one of the most promising for future research—involves the use of protein-like polymers. Recombinant DNA techniques allow fine-tuning of amino acids within the polymer chain, thereby determining specific biological functions (including properties such as degradation), in addition to imparting intrinsic biocompatibility (3). (1) Carlsen, A.; Lecommandoux, S. Current Opinion in Colloid & Interface Science 2009, 329-339. (2) (a) Sanson, C. ; Schatz, C. ; Le Meins, JF. ; Soum, A. ; Thevenot, J. ; Garanger, E. Lecommandoux, S. J. Control. Release (2010), 147, 428. (b) Sanson, C. ; Schatz, C. ; Le Meins, JF. ; Brulet, A. ; Soum, A. ; Lecommandoux, S. Langmuir (2010), 26, 7953. (c) Sanson, C. ; Le Meins, JF. ; Schatz,. ; Soum, A. ; Lecommandoux, S. Soft Matter (2010), 6, 1722. (d) Schatz, C. ; Louguet, S. ; Le Meins, JF. ; Lecommandoux, S. Angew. Chem. Int. Ed. (2009) 48, 2572. (e) Upadhyay, K.K.; Le Meins, JF.; Misra, A.; Voisin, P.; Bouchaud, V.; Ibarboure, E.; Schatz, C.; Lecommandoux, S. Biomacromol. (2009), 10, 2802. (3) (a) Garanger, E.; Lecommandoux, S. Angew. Chem. Int. Ed. 2012, 51, 3060-3062. (b) W. Kim, J. Thévenot, E. Ibarboure, S. Lecommandoux, E. L. Chaikof, Angew. Chem. Int. Ed. (2010) 49, 4257-4260. (c) R.E. Sallach, M. Wei, N. Biswas, V. P. Conticello, S. Lecommandoux, R. A. Dluhy, E. Chaikof, JACS 2006 128, 12014-12019.

We prepared mono- and difunctional derivatives of low molecular-weight, soft, amorphous and hydrophobic polymers comprising one or two terminal, monodisperse oligopeptide hard segments. The chiral hard segments have a strong preference to form well-ordered antiparallel β-sheet fibrilar aggregates via hydrogen-bonding that serve as reversible, non-convalent cross-links of the resulting supramolecular network. Since β-sheet-forming oligopeptides are self-complementary and geometrically well-defined ditopic hydrogen-bonded ligands, and their aggregation and superstructure formation is well-understood, these compounds and their blends can be rationally tailored (based on their molecular, supramolecular, nanoscopic and macroscopic structures) to give rise to materials with a broad range of mechanical properties, such as nanofiber-reinforced elastomers with good processing properties, or high performance damping materials. The aggregation behavior and supramolecular structures of these polymer-oligopeptide derivatives were investigated by infrared spectroscopy (IR) in solution and in bulk. Their nanoscopic morphologies were characterized by atomic force microscopy (AFM). Mechanical properties of single component materials or blends were investigated by rheology; their damping properties were studied by forced vibration tests on sandwich structures.

Molecular self-assembly—the spontaneous, noncovalent association of molecules to give well-defined aggregates—is ubiquitous in biology. Emerging evidence from several unrelated fields over the last decade has highlighted the significance of the self-assembly of small molecules in biology and medicine. This talk will introduces the development of hydrogelators consisting of nucleobases, amino acids, and glycosides (i.e., molecular trinity), or nucleobases and amino acids (i.e., nucleopeptides). These novel small molecule hydrogelators self-assemble in water to form stable supramolecular nanofibers/hydrogels and exhibit useful biological properties (e.g., biocompatibility, biostability, and the ability to bind and transport DNA into live cells). By discussing the results that prove the self-assembly of small molecules are able to serve as functional molecular entities in cellular environment, we suggest that the approach discussed here not only provides a new strategy to develop soft biomaterials as a form of nanomedicines, but also contributes to the understanding of molecular self-assembly in water by modulating the non-covalent interactions derived from the three basic building blocks used in living organisms.

Depsipeptides are a unique set of materials that incorporate esters within a peptide backbone. Ester substitutions are commonly used to investigate protein folding, however little work has focused on the synthesis of chemical structures with alternating esters. Materials with such a pattern may produce a new class of materials with unique mechanical and biological properties for tissue engineering applications. We have developed a family of charged depsipeptides based on Fmoc-Lys(Boc)-Lac-OH amd Fmoc-Asp(OtBu)-Lac units, using both solution and solid phase peptide methods. Synthesis in solution utilized two protection strategies: activation of the peptide ester with pentafluorophenol or carbobenzyl protection of lactic acid. Coupling strategies were investigated with DIPEA, DIC, or DCC and/or DMAP or Oxymapure. The Fmoc-depsidipeptide was purified on silica gel with hexanes:ethyl acetate to yield white crystals (80-95%) and were characterized with NMR, MS-ESI, and LCMS. The Fmoc-depsipeptides were used as the growing unit for SPPS, which proceeded with general Fmoc strategies and monitored with ninhydrin staining. Trityl chloride resin was swelled in DCM and coupled with Fmoc-Alanine-OH and DIPEA. The Fmoc group was removed with pyridine in DMF and Fmoc-peptide-OH was coupled to the resin with DIC and DMAP. The Fmoc group was removed with pyridine in DMF. The Fmoc-depsidipeptide was dissolved in DCM with Oxymapure and was added to the reaction vessel upon a 5 minute preactivation time with DIC. The Fmoc-removal, coupling, and washing steps were repeated with the Fmoc-depsidipeptide until the desired length was achieved. The cleaving cocktails were investigated to study the stability of the ester bonds under the acidic concentrations needed to remove the Boc or OtBu protecting groups. MADLI of crude samples show little or no hydrolysis of the ester bonds with all cocktails. Following purification with HPLC, our goal is to investigate the assembly process of Fmoc-oligodepsipeptide gels. Detailed analysis of the assembly of depsipeptide gels will be reported with rheology, CD, AFM, SAXS, and TEM. The gels will be formed by dissolving the ionic pairs in aqueous solution and varying ionic concentration, depsipeptide concentration, and depsipeptide length. Our control studies were conducted with oligopeptides Fmoc-(Lys-Ala)4-OH and Fmoc-(Asp-Ala)4-OH. The controls were synthesized under standard Fmoc-SPPS methods and purified with HPLC. The equivalence points for Fmoc-(Lys-Ala)4-OH) and Fmoc-(Asp-Ala)4-OH were found to be pH 5.17 and pH 6.47 respectively via titration tests. Fmoc-(Lys-Ala)4-OH (20.60-5.2 mg/ml) and Fmoc-(Asp-Ala)4-OH (1.6 mg/ml) were dissolved in water and mixed. Gel formation was determined with the upside-down vial method, and our results yield gels with total peptide concentrations of 3.75-11.10 mg/ml. Our initial studies suggest that Fmoc-oligodepsispetide gels may form under similar conditions.

Synthetic inorganic model compounds of the active site of the hydrogenase enzymes (H₂-ases) have attracted much scientific attention as a result of their ability to catalyse the reversible reduction of protons to form dihydrogen. It is anticipated that these systems will ultimately offer a more economic and renewable alternative to the platinum-based approach currently used in fuel cells. These mimics are however somewhat limited by oxygen sensitivity and a lower catalytic rate in comparison to the native enzyme. In an effort to address some of these issues we have incorporated hydrogenase model compounds into low molecular weight, peptide based, hydrogels that are able to modify both chemical stability and photochemistry offering potential new routes to exploitation of these systems. (1) Low molecular weight (LMW) hydrogelators, in particular self-assembled oligopeptide-based gels, have shown great potential in encapsulating enzymes with retention, or even improvement, of catalytic activity. (2) These materials are generally low-cost, biocompatible and highly tuneable for assembly in particular conditions (pH, temperature, presence of enzymes etc.). We have successfully incorporated a range of [FeFe]-H2-ase active site mimics into Fmoc-dipeptide hydrogels (Fmoc = 9-fluorenylmethoxycarbonyl) containing only 1% gelator in a 9:1 water:methanol (w/v) mixture. This process improved the stability of the mimics in an aqueous environment from minutes to up to two weeks. Fourier transform infrared spectroscopy (FTIR) and UVpump-IRprobe time-resolved infrared spectroscopy (TRIR) indicate significant changes in the photochemistry of the model compounds relative to the solution environment. Both FTIR and TRIR experiments show results that are consistent with an immobile, but strong hydrogen bonding environment that restricts dynamic processes such as photo-induced isomerisation while maintaining the fast vibrational relaxation rates observed in solution (3), confirming the gel&’s unique properties. Further, evidence for prolonged existence (> 1 ns) of unsaturated diiron centres was observed after UV excitation suggesting stabilisation of these reactive intermediates by the gel. In addition, the peptide-based nature of the gels offers the potential for coordination of the diiron site analogous to the natural enzyme, to optimise its efficiency towards catalytic hydrogen formation. In summary, encapsulating hydrogenase active site mimics in LMW hydrogels induces significant changes in their photochemistry and chemical stability. Since understanding and ultimately controlling the mechanistic role of ligands near Fe centres is likely to be crucial in exploiting artificial hydrogenases, these gels may offer a new option for future materials design involving catalysts. References (1) Frederix et al., Dalton Transactions, 2012, accepted, doi:10.1039/C2DT30307H (2) Gao et al., Chem. Soc. Rev., 2010, 39, 3425 (3) Kania et al., J. Chem. Phys., 2012, 136, 044521

Low molar mass gelators self-assemble in solution to form gels. When two different gelators self-assemble, they may co-assemble. Alternatively, the molecules may self-sort, where the two molecules discriminate between self and non-self. In the latter case, pure assemblies of one molecule co-exist with pure assemblies of the other. The generation of more complex, higher order self-sorted systems is of interest in areas including catalysis, as well as in the formation of p-n bulk heterojunctions. However, designing self-sorting systems is difficult, requiring detailed knowledge of the specific interactions that drive gelation or molecules where the self-association constants are very different. Here, we describe a new method for the controlled self-sorting of two low molecular weight gelators. We show how the order of the assembly can be controlled and demonstrate that such systems are truly self-sorted at the molecular level. The properties of gels formed using such mixed, self-sorted systems will be described.