Symposium Organizers
Michael (Seungju) Yu Johns Hopkins University
Seung-Wuk Lee University of California-Berkeley
Derek Woolfson University of Bristol
Ichiro Yamashita Nara Institute of Science and Technology (NAIST)
Blake Simmons Sandia National Laboratories
NN1/MM1: Joint Session:
Session Chairs
Derek Woolfson
Michael Yu
Monday PM, November 26, 2007
Room 210 (Hynes)
9:30 AM - **NN1.1/MM1.1
Non-Canonical Amino Acids in Protein Engineering.
David Tirrell 1
1 , Caltech, Pasadena, California, United States
Show Abstract10:00 AM - **NN1.2/MM1.2
Stronger and Longer Synthetic Collagen.
Ronald Raines 1 2
1 Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States, 2 Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States
Show AbstractCollagen is the most abundant protein in the human proteome. The post-translational modification of collagen by the enzyme prolyl 4-hydroxylase increases markedly the conformational stability of the collagen triple helix. We have discovered that a previously unappreciated force—stereoelectronic effects—is responsible for this increased stability. By exploiting these stereoelectronic effects (e.g., the gauche effect and n→π* interaction) and reciprocal steric effects, we have created synthetic collagen of unprecedented stability. We have also used the molecular self-assembly of triple-helical fragments to create synthetic collagen of unprecedented length. These synthetic collagens have numerous applications in biotechnology and biomedicine. [This work is supported by NIH grant AR44276.]
10:30 AM - NN1.3/MM1.3
Spatiotemporal Modification of Collagen Scaffolds Directed by Collagen Mimetic Peptide Derivatives.
Allen Wang 1 , Shirley Leong 2 , Catherine Foss 3 , Xiao Mo 1 , Martin Pomper 3 4 , Seungju Yu 1 4
1 Deptartment of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 2 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 3 Department of Radiology, The Johns Hopkins University, Baltimore, Maryland, United States, 4 Institute for NanoBiotechnology, The Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractFunctionalized collagen incorporating exogenous compounds may offer new and improved applications for collagen-based biomaterials especially in drug-delivery, multifunctional implants, and tissue engineering. We developed a specific and reversible collagen modification technique that utilizes associative chain interactions between synthetic collagen mimetic peptide (CMP), [(ProHypGly)x; Hyp:hydroxyproline] and natural type I collagen. Here we show temperature dependent collagen binding and subsequent release studies of a series of CMPs with varying chain lengths that indicate triple helical propensity driven binding mechanism similar to DNA strand invasion and exchange. The binding took place when melted, single strand CMPs were allowed to fold by cooling in contact with reconstituted natural collagens. The binding affinity is highly specific to collagen as CMP conjugated to gold nanoparticles revealed nanometer-scale repetitive binding locations along the length of type I collagen fibres and fluorescent CMPs could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, the bound CMPs discharged from the collagen at a sustained rate that correlated with CMP’s triple helical propensity suggesting that the sustainability is mediated by dynamic collagen-CMP interaction. We also report modification of collagen with linear and mutli-arm poly(ethylene glycol)-CMP conjugates. Due to the convenient nature of the modification procedure, pre-determined areas of collagen film were readily modified with PEG-CMP conjugates which exhibited temporary cell repelling activity at 37 degree C lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic application and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices.
10:45 AM - NN1.4/MM1.4
Building Tissue Engineering Scaffolds Directly from Extracellular Matrix Proteins with Microscale Spatial Control.
Adam Feinberg 1 , Sean Sheehy 1 , Kevin Parker 1
1 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractWe have developed a method for generating free-standing tissue engineering scaffolds that are spatially organized from the nanometer to millimeter length scales. These scaffolds are composed of extracellular matrix (ECM) proteins with the capability to create unique scaffold topologies that mimic in vivo structures. Integration of living cells into these ECM scaffolds should allow the generation of engineered tissues with a level of spatial control that exceeds what is possible with random mesh, sponge and gel scaffolds. Fabrication is based on microcontact printing of ECM proteins onto a transitional surface that serves as a temporary substrate during assembly. Multiple proteins are printed in a layer-by-layer process creating a microstructured, multi-component scaffold. The exact spatial structure and composition is controlled by altering the features of the polydimethylsiloxane (PDMS) stamp used for microcontact printing and/or by printing multiple proteins, multiple times at different angles. Upon dissolution of the transitional surface, the ECM scaffold is released into solution as a free-standing construct. Inherent protein-protein binding domains in the constituent ECM proteins hold the scaffold together providing structural integrity. In proof-of-concept experiments, we have fabricated “net-like” single component scaffolds composed of the ECM protein fibronectin (FN) and bi-component scaffolds composed of the ECM proteins laminin and FN. Composition and bioactivity of the ECM scaffolds has been verified by immunofluorescent staining with appropriate antibodies. This technology has potential use in a wide array tissue engineering applications. Initial cell seeding experiments have demonstrated the ability to generate highly anisotropic strands of muscle composed of neonatal rat ventricular cardiomyocytes. These myocardial fibers are typically ~20μm wide and 100’s of μm long and demonstrate uniaxial, synchronized contraction similar to papillary muscle. Future work is aimed at expanding the types of ECM proteins that can be integrated into these scaffolds and building more complex tissue constructs.
11:00 AM - NN1.5/MM1.5
Self-Assembling Hydrogels from Fibrin Coiled Coil Peptide-Polymers.
Peng Jing 1 , Joel Collier 1
1 Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio, United States
Show AbstractFibrin-based gels are clinically useful as tissue sealants and are currently being explored as matrices for regenerative medicine. However, their utility is limited by incomplete compositional definition, heterogeneity, and a potential for pathogen transmission that is inherent in biologically sourced materials. As a novel approach to address these issues, we created fully synthetic analogs of fibrin gels that self-assemble via peptides inspired by fibrin’s coiled coil domains, which are critical in the oligomerization of the six chains that comprise the full protein. We started by synthesizing peptides between 35 and 37 amino acids long from the coiled coil domain of the gamma chain of human fibrin and investigated the effect of amino acid substitutions designed to stabilize multimerization through additional interhelical electrostatic pairings and improved packing of the hydrophobic core. Through these iterations we arrived at a 37-amino acid peptide with twelve substitutions that formed stable coiled coil dimers and tetramers, as shown by circular dichroism and analytical ultracentrifugation. Stable multimers were produced from both human fibrin sequences and mouse fibrin sequences (68% homology), and the peptides were non-cytotoxic in cultures of human endothelial cells. Conjugation of the modified human fibrin peptide to mono- and difunctionalized polyethylene glycol via maleimide-thiol chemistry produced self-assembling diblock and triblock molecules, the identity and purity of which were determined by ESI mass spectrometry and HPLC. PEG conjugation had negligible impact on the secondary structure of the peptide, both for the diblock and triblock. The triblock peptide-PEG-peptide, like the unconjugated peptide, formed mixtures of dimers and tetramers at concentrations above 0.6% w/v, and above 4% w/v it formed transparent hydrogels in neutral phosphate buffer. These gels had attractive mechanical properties, as the average plateau storage modulus of 8% w/v triblock gels was 570Pa and that of 12% w/v gels was 2500Pa. These values compare favorably to reported values for fibrin-based gels. Loss moduli were about one order of magnitude below storage moduli, indicating that the gels were elastic. In contrast, rheometry indicated that the diblock did not form gels at any concentration tested, up to 12% w/v. Additionally, triblock gels had attractive degradation properties, slowly dissolving in excess phosphate buffered saline by 50% after 4 days and entirely by 8 days. Collectively, these results indicate that the triblock peptide-PEG-peptide forms hydrogels that are promising candidates for further evaluation as fully synthetic analogs of fibrin gels, sealants, and matrices.
11:30 AM - **NN1.6/MM1.6
Supramolecular Organization From Nanometers to Centimeters in Peptidic Materials.
Samuel Stupp 1 2
1 Materials Science and Engineering, Chemistry and Medicine, Northwestern University, Evanston, Illinois, United States, 2 Institute for BioNanotechnology in Medicine, Northwestern University, Evanston, Illinois, United States
Show AbstractOver the past few decades, designed peptidic materials have been of great interest as biomaterials that can interface with cells in vitro and in vivo since they bear the potential to be both bioactive and biodegradable. Our laboratory has developed an extensive familiy of peptidic biomaterials in which the primary structural element is a cylindrical nanofiber that forms by self-assembly of molecules known as peptide amphiphiles. These amphiphiles contain both a peptide segment and a non-peptidic hydrophobic segment such as the tail of a fatty acid. Under appropriate conditions these molccules aggregate to form beta sheets which in turn collapse into cylindrical nanofibers with a hydrophobic core. Very recently we have discovered that these systems can be thermally and mechanically directed to create domains of co-aligned nanofibers that reach into macroscopic dimensions. Strings of peptidic material with lengths on the order of centimeters and containing aligned nanoscale fibers can be easily formed and even populated with cells. This lecture will describe the encapusulation and differentiation of human stem cells into these macroscopic strings. These self-assembling peptidic systems offer new opportunities to create cell assays and therapies in regenerative medicine.
12:00 PM - NN1.7/MM1.7
Early Time β-Hairpin Peptide Self-Assembly into a Hydrogel Network.
Tuna Yucel 1 2 , Joel Schneider 3 , Darrin Pochan 1 2
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 2 , Delaware Biotechnology Institute, Newark, Delaware, United States, 3 Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States
Show AbstractIn dilute aqueous solution at pH=7.0 and T=22oC, MAX 1 peptide (NH2-(VK)4-VDPPT-(KV)4-CONH2) is unfolded and freely soluble. The peptide intramolecularly folds into a β-hairpin when the electrostatic interactions between charged lysine (K) amino acids are screened through an increase in the solution ionic strength. The β-hairpins consequently intermolecularly assemble via hydrophobic collapse and hydrogen bonding into a fibrillar hydrogel network. Here, we correlate a direct characterization of the temporal evolution of β-hairpin formation and intermolecular fibril formation with changes in viscoelastic properties. By combining the results of far-UV circular dichroism spectroscopy, cryogenic transmission electron microscopy, small angle neutron scattering, dynamic and static light scattering and dynamic oscillatory rheology, we observe that MAX 1 self-assembly proceeds by nucleation of semi-flexible β-sheet nanofibrils with monodisperse diameter (d~3 nm) that elongate and form branched fibril clusters. Under the assembly conditions studied here, these branched clusters had an apparent fractal dimension D~1.5 when they initially fill up the sample volume. This D value increases with peptide concentration, presumably due to increasing branching density. Clusters eventually interpenetrate and form a percolated network. Percolation leads to an ergodic to non-ergodic transition, as evidenced by a characteristic power law decay of the DLS autocorrelation function (g2(τ)~τ-0.45) followed by an increase in the frozen-in scattered intensity fluctuations due to gelation. Concurrently, the network rigidity increases significantly as observed by rheology. The self-assembly of MAX 1 was compared and contrasted with the self-assembly of biopolymer networks in literature. The potential biotechnological importance of the characterization of the early time β-hairpin self-assembly in the design of injectable hydrogels for in vivo tissue regeneration will be discussed. Ultimately, our goal is to understand possible biocompatibility-self-assembly-hydrogel material property relationships.
12:15 PM - NN1.8/MM1.8
Modification of Liposomes using α-Helical Coiled-Coil Peptides.
Hana Robson Marsden 1 , Alexander Korobko 1 , Alexander Kros 1
1 Chemistry, Leiden University, Leiden Netherlands
Show AbstractA system of active nanocapsules is investigated, using liposome capsules which are activated with interacting peptides. Lipopeptides are synthesized using a pair of peptides that form heterodimeric α-helical coiled-coils. The phospholipid tail of these hybrid molecules inserts into liposomes, resulting in vesicles with an outer surface studded with one of the two peptides. Reminiscent of cell membrane fusion, which requires the action of specific proteins, the interaction of these peptides facilitates liposome aggregation and probable fusion. The fusion of liposomes, accompanied by the mixing of liposome contents, would build up the complexity of the ‘lab in a vesicle’ concept.
12:30 PM - **NN1.9/MM1.9
Nanofibers formed by Self-assembly of Multidomain Meptides: Applications for Bioengineering to Nanotechnology.
Jeffrey Hartgerink 1 , Kerstin Galler 1 , He Dong 1 , Lorenzo Aulisa 1 , Sergey Paramonov 1
1 Chemistry & Bioengineering, Rice University, Houston, Texas, United States
Show AbstractControl over the dimension of the assemblers has been a major challenge in the self-assembly of nanostructured materials. There are few effective approaches to confine the assembled objects in a defined dimension. In this paper we report on a series of multi-domain peptide molecules (MDPs), each of which consists of three functional domains that serve to control the organization and the extent of the self-assembly through a mechanism that is mediated by “molecular frustration”. We demonstrate that when forces favoring assembly are properly balanced with forces favoring disassembly, discrete nanofibers with controlled length result. In addition, we found that the ratio of domain size determines peptides’ secondary structure, which has a dramatic effect on their supramolecular nanostructure. This observation indicates a strong correlation between peptides’ molecular secondary structures and the self-assembled nano-structures. Due to the fact that the experiments were all performed under the physiological condition, we believe this architectural motif may be utilized for novel tissue regeneration strategies and other systems which require control over chemical organization at the nanoscale. Additionally, the high solubility (up to 1 wt%) of the nanofibers formed by at neutral pH allows for the use of a variety of spectroscopic measurements to help understand and further treat various diseases associated with protein aggregation.
NN2/MM2: Joint Session
Session Chairs
Paula Hammond
Seung-Wuk Lee
Monday PM, November 26, 2007
Room 210 (Hynes)
2:30 PM - **NN2.1/MM2.1
Genetic Control of the Synthesis and Assembly of Materials for Electronics and Energy.
Angela Belcher 1 , Ki Tae Nam 1 , Yun Jung Lee 1 , Dong-Soo Yun 1 , Brian Neltner 1 , Andrew Magyar 1
1 , MIT, Cambridge, Massachusetts, United States
Show Abstract3:00 PM - **NN2.2/MM2.2
Building from Bottom Up: Fabrication of Nanomaterials Using Peptide Motifs.
Shugang Zhang 1
1 , MIT, Cambridge, Massachusetts, United States
Show AbstractMaterials science has generally been associated with metallurgy, alloy, ceramics, composites, polymer science, fiber spinning, coating, thin film, industrial surfactants and block copolymer development. That is about to change. Materials science will also expand to discovery and fabrication of biological and molecular materials with diverse structures, functionalities and utilities. The advent of nanobiotechnology and nanotechnology accelerated this trend. Similar as construction of an intricate architectural structure, diverse and numerous structural motifs are used to assemble a sophisticated complex. Nature has selected, produced and evolved numerous molecular architectural motifs over billions of years for particular functions. These molecular motifs can now be used to build materials from the bottom up. Materials science will begin to harness nature’s enormous power to benefit other disciplines and society. Zhang, S. (2002) Emerging biological materials through molecular self-assembly Biotechnology Advances 20, 321-339. Zhang, S. (2003) Fabrication of novel materials through molecular self-assembly. Nature Biotechnology 21, 1171-1178.Yokoi, H., Kinoshita, T. & Zhang, S. (2005) Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc. Natl. Acad. Sci.USA 102, 8414-8419.Zhao, X. & Zhang, S. (2006) Molecular designer self-assembling peptides. Royal Society of Chemistry 35, 1105-1110.Gelain, F., Bottai, D., Vescovi, A & Zhang, S. (2006) Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PloS ONE 1, e119, 1-11. Horii, A. Wang, X., Gelain, F. & Zhang, S. (2007) Biological designer self-assembling peptide scaffolds significantly enhance osteoblast proliferation, differentiation & 3-D migration. PloS ONE 2, e190, 1-9.
3:30 PM - NN2.3/MM2.3
Development of Novel Hard-Tissue Regenerative Materials Through Directed and Natural Evolutionary Processes.
Eddie Wang 1 , Seung-Wuk Lee 1 2
1 Bioengineering, University of California, Berkeley, Berkeley, California, United States, 2 Physical Biosciences Division, Lawerence Berkeley National Lab, Berkeley, California, United States
Show AbstractBones are natural inorganic-organic nanocomposite materials with remarkable toughness and strength. Utilizing natural and artificial evolutionary processes, we have developed novel bone-mimetic nanocomposite materials to recapitulate bone’s unique properties. A functional biopolymer, composed of an elastin-like polypeptide (ELP) fused with a hydroxyapatite binding peptide (HBP), was synthesized using bacterial biosynthetic approaches. HBP is a twelve amino acid peptide previously identified through directed evolution by phage display that binds to and promotes nucleation of hydroxyapatite, the predominant mineral component of bones and teeth. The HBP sequence has been genetically engineered as an N and/or C-terminal addition to an ELP gene. The resulting composite protein (HBP-ELP-HBP) has been expressed and purified from E. Coli then chemically cross-linked through periodically spaced lysine residues to form a thermo-responsive gel. We believe this novel protein gel will retain the favorable properties of ELPs (elasticity, fatigue resistance, biocompatibility) and will also interface with hydroxyapatite through its HBP domains. We have combined the gel with hydroxyapatite crystals or precursor ions to characterize the construct’s hydroxyapatite binding and nucleating capabilities. We are now determining and optimizing the resulting composite’s mechanical properties. The resulting composite biomaterial may be useful for bone tissue engineering/repair or in dentistry as a treatment for dental caries.
3:45 PM - NN2.4/MM2.4
Supramolecular Self-Assembly of a Metal-binding Polypeptide and Implications for Molecular Recognition.
Christopher So 1 , Emre Oren 1 , Urartu Seker 1 3 , Brandon Wilson 1 , John Kulp 2 , Candan Tamerler 1 3 , John Evans 2 , Sarikaya Mehmet 1
1 Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 3 Molecular Biology & Genetics, Istanbul Technical University, Istanbul Turkey, 2 Chemistry, New York University, New York, New York, United States
Show AbstractRecently, the utility of Genetically Engineered Peptides for Inorganics (GEPIs) has opened the prospect of achieving self-assembled hierarchical material systems due to their ability to recognize particular surfaces. One such polypeptide, 3x(MHGKTQATSGTIQS), has shown to possess properties of specificity towards gold, while being nonspecific to other noble metals (Pt), oxides (Quartz), or minerals/organics (mica, graphite), suggestive of such molecular recognition mechanisms. Here, we report novel atomic force microscopy (AFM) and molecular simulation studies that detail the formation of ordered assemblies of a gold binding protein (3r-GBP1). Simulated annealing molecular dynamics (SA/MD), based on nuclear magnetic resonance (NMR), indicates that the lowest energy structure of 3r-GBP1 features extended B-strand and random coil-like regions with repeating surface accessible side-chains identified as putative Au docking sites. Geometric models of lattice matching with the Au{111} reveal that the peptide aligns with both the <110> and <211> directions of the surface lattice. The AFM observation reveal a supramolecular assembly of the peptide forming crystallographically ordered six equivalent domains commensurate with the Au{111} surface lattice. To understand these supramolecular binding events, ex situ time-lapsed AFM experiments were carried out to quantitatively assess the kinetics of peptide assembly and to correlate the data to observed growth morphologies. Coverage trends from the concentration-varied experiment show high correlation to Langmuir fitting while approaching >90% coverage, in agreement with resulting surface plasmon resonance and quartz crystal microbalance analyses. These results provide initial insights into the molecular recognition mechanism(s) of peptide binding and self assembly on material surfaces.
4:30 PM - **NN2.5/MM2.5
Targeted Protein Cage Architectures for Biofilm Imaging and Therapeutics.
Trevor Douglas 1
1 Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, United States
Show AbstractDiagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. We have used an icosahedral viral nanoplatform to target a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding at the bacterial surface, mediated through specific protein ligand interactions, exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto these cells. The fluorescence- imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. Furthermore, the selectivity of antimicrobial photodynamic therapy (PDT) can be enhanced by coupling the photosensitizer (PS) to a targeting ligand. Nanoplatforms provide a medium for designing delivery vehicles that incorporate both functional attributes. We have used the photodynamic inactivation of Staphylococcus aureus, using targeted viral nanoplatforms conjugated to a photosensitizer (PS). Both electrostatic and targeted interactions were used to mediate PS nanoplatform delivery. Genetic constructs of a protein cage architecture allowed site specific chemical functionalization with the PS, and facilitated dual functionalization with the PS and the targeting ligand. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections.
5:00 PM - NN2.6/MM2.6
Ferritin Cage Architecture with Superparamagnetic Iron Oxide Nanoparticle for Magnetic Resonance Contrast Agent.
Masaki Uchida 1 3 , Masahiro Terashima 4 , Charles Cunningham 5 , Yoriyasu Suzuki 4 , Deborah Willits 2 3 , Ann Willis 1 3 , Philip Yang 4 , Michael McConnell 4 , Mark Young 2 3 , Trevor Douglas 1 3
1 Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, United States, 3 Center for Bio-Inspired Nanomaterials (CBIN), Montana State University, Bozeman, Montana, United States, 4 School of Medicine, Stanford University, Stanford, California, United States, 5 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, 2 Department of Plant Seicences, Montana State University, Bozeman, Montana, United States
Show AbstractProtein cage architectures are versatile nanoscale platforms amenable to both genetic and chemical modification, making them promising for cellular and molecular imaging. We have recently revealed that recombinant human H chain ferritin (rHFn), which has a 12 nm exterior diameter and an 8nm interior cavity, is a superior template for biomimetic mineralization and encapsulation of superparamagnetic iron oxide nanoparticles within its interior cavity. This nanoparticle is comparable in size to commercially available ultrasmall superparamagnetic iron oxide (USPIO) contrast agents for magnetic resonance imaging (MRI) but possess unique features not present in the commercially available USPIO contrast agents such as excellent homogeneity of particle size and demonstrated ability of further modification to impart cell-specific targeting. The aim of this research is to investigate the ability of a protein cage templated material to serve as a MRI contrast. By TEM and electron diffraction measurements, electron dense particles of magnetite (or maghemite) with narrow size distribution were formed within the rHFn after an iron oxide synthesis reaction. The average size of the particles increased form 3.6 to 5.9 nm with increasing theoretical Fe loading factor from 1000Fe to 5000Fe per cage. R2 relaxivity of the mineralized rHFn increased with increasing Fe loading factor and that of HFn5000Fe was comparable with that of a commercially available USPIO MR contrast agent. Cellular uptake of the mineralized protein cages was investigated in murine macrophage cells. The amount of Fe taken up by the cells cultured with the mineralized protein cage constructs was significantly more (9 to 39 fold) than that of the cells cultured with a commercially available USPIO, at equivalent Fe concentrations. The cells labeled with mineralized protein cages provided more intense dark MR images under a gradient echo sequence than those labeled with a commercially available USPIO. Since macrophage cells are involved in serious inflammatory diseases such as atherosclerosis, this composite magnetic cage material is expected to have great potential as a MRI contrast agent to assess inflammatory status in vivo.
5:30 PM - NN2.8/MM2.8
Protein-Mediated Assembly of Metal Nanostructures.
Silke Behrens 1 , Wilhelm Habicht 1 , Konrad Joachim Boehm 2
1 Institute for Technical Chemistry, Forschungszentrum Karlsruhe, Karlsruhe Germany, 2 , Leibnitz Institute for Age Research, Jena Germany
Show AbstractSelf-assembled nanostructures represent interesting matrices to control the organization of inorganic matter at the nanometer scale. Beside other bio macromolecules, various proteins are known to form precisely defined nanostructures by self-assembly processes. Moreover, functional groups, e.g., SH-groups or imidazole heterocycles, exposed at the surface of proteins allow further chemical modification, including metal deposition. These attributes of proteins can be combined with appropriate chemical reactions to develop reliable bottom-up strategies for the formation of nanoscale hybrid materials with novel electronic, optical, or chemical properties. In this context, tubulin is a very interesting protein able to self-assemble in the presence of GTP into protofilaments, consisting of strictly alternating αβ-dimers. Under physiological conditions, the lateral linkage of these protofilaments results in the formation of microtubules. However, in cell-free environment, depending on co-factor and additive composition, the tubulin assembly and the arrangement of the protofilaments can be controlled to form a variety of other superstructures with defined nanometer-scaled geometry such as macrotubes, sheets, spirals, or rings. Zn2+ ions, e.g., direct the assembly of tubulin into sheets or macrotubes. In our approach, these protein assemblies were used as a functionalized scaffold where noble metal is generated in situ and deposited into particle arrays, reflecting the arrangement of tubulin subunits within the assembly. As a result particle arrays of different geometry and metal nanowires were obtained. The size and structure of the materials were examined using transmission electron microscopy and scanning force microscopy. Our study demonstrates a straightforward and rapid protein-based approach to obtain metal nanoparticle arrays and nanowires, not accessible via conventional synthetic methods.
Symposium Organizers
Michael (Seungju) Yu Johns Hopkins University
Seung-Wuk Lee University of California-Berkeley
Derek Woolfson University of Bristol
Ichiro Yamashita Nara Institute of Science and Technology (NAIST)
Blake Simmons Sandia National Laboratories
NN3
Session Chairs
Ichiro Yamashita
Michael Yu
Tuesday AM, November 27, 2007
Room 201 (Hynes)
9:30 AM - **NN3.1
Silk Fibroin Assemblies for Controlled Release Biomaterials.
David Kaplan 1
1 Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Show AbstractSilks represent a versatile family of protein biomaterials due to their unique attributes of high strength and toughness, excellent biocompatibility and slow degradability. In recent years the structure and morphology of silk-based biomaterials has been controlled by regulating beta sheet crystalline content during processing, as a route to tailoring functional features of these biomaterials. In recent studies, this control has been extended to novel hydrogel systems able to entrap and maintain stem cell function, and to the regulation of small and large molecule release kinetics from films, nanolayers and vesicles formed from silks. Importantly, all aqueous processing options for the protein allows for the versatile incorporation of labile biomolecules and cells, without loss of bioactivity. This feature leads to new and versatile applications for this family of biomaterials. For example, silk fibroin multilayer structures were successfully used to regulate the release kinetics of enzymes and small molecule dyes. Similarly, vascular cell-relevant therapeutic compounds were incorporated into silk coatings and then used to control human aortic endothelial cell and human coronary artery smooth muscle cell responses to the drug-incorporated coatings. The above studies demonstrate novel utility for this protein biomaterial in a range of controlled release settings.
10:00 AM - **NN3.2
Engineering of Protein-like Molecules and Materials.
Samuel Gellman 1 , Will Pomerantz 1
1 , University of Wisconsin, Madison, Wisconsin, United States
Show Abstract10:30 AM - NN3.3
Structural and Rheological Properties of Photopolymerized Self-Assembled β-Hairpin Peptide Hydrogels.
Ronak Rughani 1 , Matthew Lamm 2 , Darrin Pochan 2 , Joel Schneider 1
1 Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware, United States, 2 Department of Material Science & Engineering, University of Delaware, Newark, Delaware, United States
Show AbstractMolecular self-assembly of rationally designed peptides is a promising approach to construct robust functional materials. Towards this goal, a 20-amino acid peptide (MAX 1) was de novo designed to undergo an intramolecular folding event to form an amphiphilic antiparallel β-hairpin that intermolecularly self-assembles to form β-sheet rich stimuli responsive hydrogels. The gel consists of a network of well-defined fibrils formed by intermolecular hydrogen bonding and hydrophobic association of the folded β-hairpin peptides. In the self-assembled state, the fibril consists of a bilayer formed in an ordered fashion due to the stacking of the hydrophobic faces of the individual hairpins directly on top of each other, minimizing exposure of the hydrophobic side chains to water. However, during the self-assembly process, irregular hydrophobic facial associations may also occur resulting in packing defects creating nucleation sites for nascent fibril growth, thus forming non-covalent interfibril junctions that serve to crosslink the gel. In addition to the interfibril crosslinks, simple fibril entanglements also help define the gel nanostructure. The number of interfibril crosslinks and fibril entanglements formed as a result of self-assembly define the material rigidity at the macroscopic level. To modulate the material rigidity, we prepared β-hairpins that display acrylate moieties so that the fibrils of the hydrogel can be covalently crosslinked via photopolymerization. Designing materials in this fashion, where the mechanical properties can be controlled, has potential applications in tissue engineering and drug delivery. In addition, incorporating covalent crosslinks into the fibril network may provide insight into the nature of crosslinks and entanglements formed as a result of self-assembly. Kinetics of folding and self-assembly, material rigidity and nanoscale structure as assessed by circular dichroism, oscillatory rheology and transmission electron microscopy will be presented.
10:45 AM - NN3.4
Generation of Bioactive Nanostructured Interfaces to Mimic Cellular Microenvironments.
Tobias Wolfram 1 2 , Ferdinand Belz 1 2 , Tobias Schoen 1 2 , Joachim Spatz 1 2
1 New Materials and Biosystems, Max/Planck Institute for Metals Research, Stuttgart Germany, 2 Biophysical Chemistry, University of Heidelberg, Heidelberg Germany
Show AbstractThere is currently great interest in material science to mimic cellular microenvironments with controlled bioactive features and biomolecule presentation at the nanometer length scale. Target cells exposed to those 2- or 3D-substrates can be induced to show a variety of cellular functions like cell adhesion, migration and differentiation of stem cells. Based on micellar nanolithography we developed an experimental setup to deposit single biomolecules on a 2D-Nanoarray over a large area on adhesive or non-adhesive cellular substrates with a nanometer resolution between 30-250 nm. To mimic a more complete cellular microenviroment with cell-cell contacts as well as cell-extracellular matrix (ecm) mediating molecular components, we used biomolecules from different cell adhesion molecule families, like Cadherins, Heparane Sulfate Proteoglycans, Immunglobulin-Superfamily, integrins and laminins. Via a Ni2+ -nitrilo triacetic acid (NTA) system, the amount and the orientation of proteins are chemically controlled and tuned to resemble native in vivo settings in the cellular microenvironment. By using different polyethylene glycol (PEG) based molecules we generated 2D-Nanoarrays with a non-cell adhesive function. By modifying a poly-l-lysine-g-PEG based approach we incorporate small bioactive peptides to create a microenvironment which allowed cell adhesion. These chemically controlled approaches resulted in substrates with specific presentation of biomolecules to mimic cell-cell contacts and simultaneously in a substrate coating to mimic cell-ecm interactions. With these substrates we investigated neural cell adhesion mediated by different biomolecules on different nanometer length scales. On cell adhesive substrates neural cell differentiation was possible. We evaluated neurite outgrowth of neuroblastoma cells as a differentiation paradigm on nanostructured peptides. Peptides contained an integrin activation sequence, the arginine-glycine-aspartic acid (RGD) motif. Cell adhesion as well as cell differentiation displayed the dependency on the nanometer length scale and on the biomolecule which is presented.Theses nanostructured substrates will provide a versatile tool to mimic cellular microenvironments.
11:00 AM - NN3.5
Cell-Adaptable Protein Scaffolds for Spinal Cord Nerve Regeneration.
Karin Straley 1 , Cheryl Wong Po Foo 2 , Sarah Heilshorn 2
1 Chemical Engineering, Stanford University, Stanford, California, United States, 2 Materials Science and Engineering, Stanford University, Stanford, California, United States
Show AbstractA key attribute missing from current state-of-the-art biomaterials is the ability to be remodeled by the host after implantation. In contrast, the natural extracellular matrix (ECM) is constantly being remodeled by proteases secreted from cells in response to local environmental changes. Mimicking this strategy, we have designed a new protein-based scaffold that can be degraded and remodeled on demand by the growth cones of regenerating neurites. Using recombinant protein techniques, we synthesized a family of biocompatible, biodegradable, and biologically active scaffold materials. The scaffolds include peptide sequences derived from the natural ECM proteins fibronectin, laminin, and elastin. Interspersed with these ECM domains are proteolytic sequences readily degraded by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), two proteases secreted by the growth cones of extending neurites. By altering the primary amino acid sequences of the protease cleavage domains, we can tune the degradation rates of five otherwise identical engineered-proteins in a controlled and predictable manner over approximately two orders of magnitude. These recombinant proteins are crosslinked to form bulk, protein-based scaffolds with tailored mechanical properties matching that of the spinal cord. The initial elastic modulus of the scaffold can be easily adjusted by specifying the extent of crosslinking within the scaffold. The crosslinked scaffolds also demonstrate sequence-specific cell spreading of PC12 cells, support PC12 differentiation into neuronal-like cells, and enhance PC12 neurite extension in response to specific ECM cell-binding domains. PC12 growth cones are known to secrete tPA and uPA enzymes, and cell populations with higher secretion levels are thought to have enhanced neurite extension rates. By tailoring the scaffold degradation rate to the tPA and uPA secretion levels of specific neuronal populations, we aim to fabricate a scaffold that will promote neurite extension through the matrix by allowing local degradation to occur specifically around the neuronal growth cone while maintaining the bulk integrity of the overall scaffold.
11:30 AM - **NN3.6
Peptide Motifs and Motif Programming.
Kiyotaka Shiba 1 2
1 Protein Engineering, Cancer Institute, Tokyo Japan, 2 CREST, JST, Tokyo Japan
Show AbstractBecause motifs are generally associated with particular biological functions, we are able to create multi-functional artificial proteins by assembling defined motifs. We named this process “motif programming.” But we now know that that simple arithmetic addition does not always work for motif programming. The association of some motifs with their functions is not absolute; instead, these motifs should be thought of as being associated with a “latent” function. Successful manifestation of the latent functions is dependent on displaying the motifs in the proper context within the polypeptide. At the moment, we do not fully understand what constitutes an appropriate context for functional expression of a motif, but recent studies have shown that in practical terms a combinatorial approach is able to solve this problem in some cases. In this presentation, I will introduce our combinatorial approach to motif programming, that is, a microgene-based method, “MolCraft”. In MolCraft, a microgene is first rationally designed so that more than two motifs are coded by different reading frames of a short DNA sequence, after which the designer microgene is polymerized to yield a library of larger genes that are combinatorial polymers of three reading frames. I will introduce applications of motif programming to medical and nano-biotechnology fields. (References) Nucl Acid Res 35(6): e38 (2007), Chem Biol 11(6): 765-773 (2004), J Mol Biol 320(4): 833-840 (2002), Proc Natl Acad Sci U S A 94(8): 3805-3810 (1997)
12:00 PM - NN3.7
Lipid Composition Governs the Activity of Cell-penetrating and Cell-permeating Peptides.
Vernita Gordon 1 , Abhijit Mishra 1 , Nathan Schmidt 1 , Lihua Yang 1 , Matthew Davis 1 , Daniel Parente 1 , Robert Coridan 1 , Abhigyan Som 2 , Gregory Tew 2 , Gerard Wong 1
1 Materials Science and Engineering, University of Illinois, Urbana-Champaign, Urbana, Illinois, United States, 2 Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts, United States
Show AbstractThe TAT protein transduction domain (PTD) of the Human Immunodeficiency Virus (HIV-1) can cross cell membranes with unusual efficiency. Since TAT PTD can deliver a variety of cargo to a wide range of cell types, it has many potential biotechnological applications. The precise nanoscopic mechanism of entry, however, is not well understood. We use confocal microscopy to systematically study the interaction of the TAT PTD with model membranes of variable composition. When Rhodamine-tagged TAT PTD (Rh-TAT-PTD) is applied to the exterior of Giant Unilamellar Vesicles (GUVs) with low PE content (0% and 20%), it remains outside the membrane. However, when Rh-TAT-PTD is applied to GUVs with 40% PE content, it crosses the membrane and equilibrates across the membrane over tens of seconds. Thus, we see that the membrane transduction activity of Rh-TAT-PTD requires the presence of PE lipid in the membrane. For another cell-penetrating peptide, ANTP, the presence of PE lipid in the membrane is even more important: Rhodamine-tagged ANTP enters GUVs with 60% PE content, but not 40% or lower.Antimicrobial peptides (AMP’s) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogs of AMP’s, such as the family of phenylene ethynylene antimicrobial oligomers (AMO’s), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. Using confocal microscopy to examine GUVs with a charge density fixed at typical bacterial values, we find that AMO causes small encapsulated, dyed molecules to leak out while larger molecules are mostly retained. This indicates that AMO creates size-defined pores in the membrane. Moreover, PG:PE GUVs release nearly all the larger molecules, while in PG:PC GUVs they are mostly retained. This indicates that the presence of PE lipid greatly enhances permeating activity of AMO in these membranes, showing the importance of specific lipid composition for the activity of cell-permeating peptides. Since bacterial cell membranes are rich in PE lipids compared with eukaryotic cell membranes, this may indicate a mechanism for antimicrobial specificity.We also use small-angle X-ray scattering (SAXS) to characterize the 3D structures formed when these cell-penetrating and antimicrobial peptides complex with concentrated lipid mixtures. When the amount of PE lipid in the system is sufficiently high, the peptides cause vesicle membranes to undergo topological changes to form other, non-lamellar phases. These phases are characterized by negative Gaussian curvature, in the case of the cell-penetrating peptides, and negative mean curvature, in the case of AMO. Induction of these phases correlates with peptide activity observed in GUV systems under the microscope. Our results show that there is a mapping between the structural tendencies manifested in such 3D systems and peptide activity in dilute, quasi-2D GUV membranes.
12:15 PM - NN3.8
Deformation and Fracture of Protein Materials: Balancing Strength, Energy Dissipation and Robustness.
Markus Buehler 1
1 Laboratory for Atomistic and Molecular Mechanics, Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractDeformation and fracture are fundamental phenomena with major implications on the stability and reliability of machines, buildings and biological systems. All deformation processes begin with erratic motion of individual atoms around flaws or defects that quickly evolve into formation of macroscopic fractures as chemical bonds rupture rapidly, eventually compromising the integrity of the entire structure. However, most existing theories of fracture treat matter as a continuum, neglecting the existence of atoms or nanoscopic features. Clearly, such a description is questionable. Here we discuss an atomistic approach to describe such processes using large-scale molecular dynamics (MD) simulation implemented on supercomputers. MD provides insight into complex atomic-scale deformation processes without relying on empirical input. We demonstrate how MD can be used within a multi-scale simulation framework to predict the elastic and fracture properties of hierarchical protein materials, marvelous examples of structural designs that balance a multitude of tasks, representing some of the most sustainable material solutions that integrate structure and function across the scales. Breaking the material into its building blocks enables us to perform systematic studies of how microscopic design features influence the mechanical behavior at larger scales. We review studies of collagen – Nature's super-glue, spider silk – a natural fiber that can reach the strength of a steel cable, as well as intermediate filaments – an important class of structural proteins responsible for the mechanical integrity of cells, which, if flawed, can cause serious diseases such as the rapid aging disease progeria. The common ground of these examples is the significance of the material properties at large deformation, its alteration under stress, presence of defects or the effect of variation of environmental conditions. Our studies elucidate intriguing material concepts that balance strength, energy dissipationand robustness by selecting nanopatterned, hierarchical features.
12:30 PM - **NN3.9
De Novo Design of Peptide-Based Hydrogels for Cell Delivery.
Joel Schneider 1 , Lisa Haines-Butterick 1 , Karthikan Rajagopal 1 , Monica Branco 1 , Daphne Salick 1 , Ronak Rughani 1 , Matthew Lamm 2 , Darrin Pochan 2
1 Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States, 2 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States
Show AbstractThe design of “smart” peptides that undergo sol-gel phase transitions in response to biological media may enable minimally invasive delivery of extracellular matrix substitutes in-vivo. Towards that goal, we have designed a family of 20 amino acid residue peptides that undergo triggered self-assembly to form a rigid hydrogel. When dissolved in aqueous solutions, these peptides exist in an ensemble of random coil conformations rendering them fully soluble. The addition of an exogenous stimulus results in peptide folding into β-hairpin conformation. This folded structure undergoes rapid assembly into a highly crosslinked hydrogel network whose nanostructure is defined and controllable. Peptides can be designed to fold and assemble in response to changes in pH or ionic strength, the addition of heat and light. In addition to these stimuli, DMEM cell culture media is able to initiate folding and consequent self-assembly. DMEM-induced gels are cytocompatible towards NIH 3T3 murine fibroblasts, mesenchymal stem cells, hepatocytes, osteoblasts and chondrocytes. As an added bonus, many of these hydrogels possess broad spectrum antibacterial activity suggesting that adventitious bacterial infections that may occur during surgical manipulations and after implantation can be greatly reduced. Lastly, when hydrogelation is triggered in the presence of cells, gels become impregnated with cells. A unique characteristic of these gels is that when an appropriate shear stress is applied, the gel will shear-thin, becoming an injectable low viscosity gel. However, after the application of shear has stopped, the material quickly self-heals producing a gel with mechanical rigidity nearly identical to the original hydrogel. This attribute allows gel/cell constructs to be delivered to target tissues via syringe where they quickly recover complementing the shape of the tissue defect.
NN4
Session Chairs
Seung-Wuk Lee
Blake Simmons
Ichiro Yamashita
Tuesday PM, November 27, 2007
Room 201 (Hynes)
2:30 PM - **NN4.1
Biomaterials for Functional Materials.
Rajesh Naik 1
1 Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Ohio, United States
Show AbstractOver the past several years, biomolecules have been used as molecular templates to stabilize, synthesize and/or assemble inorganic materials. These molecular templates potentially serve as building blocks for the assembly of hybrid structures. In addition, self-assembling properties of biomolecules can also be exploited for engineering responsive hybrid materials. In the future, one should be able to create hybrid materials using protein engineering by dialing-in the different sequence domains that could direct the synthesis and assembly of hybrid materials for a desired application. In this talk I will cover aspects of my group’s research in using biomolecular templates in the synthesis and assembly of nanomaterials for catalytic, sensing and for biotransformations.
3:00 PM - NN4.2
Directed Evolution of Bone-Associated Proteins.
Seung-Wuk Lee 1 2 , Eddie Wang 1 2 , Woo-Jae Chung 1 2 , Jin Huh 1 2 , Ki-Young Kwon 2
1 Bioengineering , University of California, Berkeley, Berkeley, California, United States, 2 Physical Bioscience Division, Lawrence Berkeley National Lab, Berkeley, California, United States
Show AbstractThe formation of natural bone is thought to occur by the templated mineralization of HA by the surrounding proteins, which include collagen and highly acidic phosphoproteins attached to the collagen scaffold. It has been proposed that the acidic groups serve as binding sites for calcium ions and align them in an orientation that matches the HA crystal lattice, but the biological mineralization process is not understood at the molecular level. Using directed evolution process of combinatorial peptide libraries, we identified specific binding peptides for single crystal hydroxyapatite in various pH ranges and study their interactions between HA binding peptides and crystal surfaces. Remarkably, the consensus HA binding peptides resulted in characteristic tripeptide repeat (Pro-X-Y) at pH 7.5 and (Ser-Ser-Asp) at pH 5. These sequences are similar to the major repeats of type I collagen and dentin phosphoproteins respectively. Using a panel of synthetic peptides, we defined the structural features required for binding and mineralizing activity of HA. We also incorporated these short HA-binding peptides to construct three-dimensional bone-like materials by designing nanofibers and responsive functional protein gels.
3:15 PM - NN4.3
Molecular Structures of Engineered Inorganic-Binding Peptides.
John Evans 1 , Won Kim Il 1 , John Kulp 1 , Sebastiano Collino 1 , Katya Delak 1 , Urartu Seker 2 3 , Chris So 3 , Emre Oren 3 , Candan Tamerler 2 3 , Mehmet Sarikaya 3
1 Center for Biomolecular Materials Spectroscopy, New York University, New York, New York, United States, 2 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey, 3 Materials Science and Engineering, University of Washington, Seattle, Washington, United States
Show AbstractWith the advent of genetic engineering techniques, materials science has now advanced to the next stage of development, where biomolecules are selected and designed for creating materials. In the case of inorganic materials, proteins and polypeptides have been synthesized and screened against a wide variety of natural and synthetic inorganic solids for the purposes of creating hybrid organic-inorganic composites that have functional significance. However, this rapidly developing field still lags in one important area: Understanding how polypeptide sequence and structure contribute to polypeptide - inorganic recognition and specificity. Using computational biology and NMR/CD spectroscopy, we have determined the structures for a number of polypeptides directed against specific inorganics, such as Au, Ag, hydroxyapatite, quartz, and platinum, and the common thread which we have uncovered is the presence of labile unfolded structures or polyproline type II structures. The significance of these structures is their ability to adapt and interact with solid surfaces, such that geometric and chemical patterns can be discerned and recognized. We believe that the exploitation of these structural features within artificial proteins will represent the next step towards developing new and useful organic-inorganic hybrid materials. Research supported by NSF-MRSEC at the UW.
3:30 PM - NN4.4
Design of Multifunctional Binding Peptides.
Ersin Emre Oren 1 2 , Ram Samudrala 2 , Deniz Sahin 1 3 , Turgay Kacar 1 3 , Marketa Hnilova 1 , Candan Tamerler 1 3 , Mehmet Sarikaya 1
1 Materials Science and Engineering Department, University of Washington, Seattle, Washington, United States, 2 Department of Microbiology, University of Washington, Seattle, Washington, United States, 3 Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul Turkey
Show AbstractIn nature, proteins control structures and functions of biological hard tissues through specific molecular interactions with inorganics. Emulating biology, recent adaptation of combinatorial biological techniques provided ways to genetically select peptides with affinities to a variety of materials leading to their utility as molecular building blocks in synthesis, nanostructural organization, and directed assembly. The selection of peptides and their molecular and functional characterization require extensive effort and resources, and successful applications thus far are due, mostly, to serendipity. Here we introduce a method that combines experimental knowledge with bioinformatics and enables in silico design of new peptides efficiently with superior binding affinities and multiple material specificities as a potential tool for molecular engineering in materials and medicine. Using our computational design techniques we have generated peptides with novel functionalities such as selective binding to given material (e.g. quartz, hydroxyapatite and gold) and designed multifunctional peptides that have binding functionality to several materials or non-binding to others. For example, we have designed peptides capable of binding to quartz, hydroxyapatite, both or neither. Experimental verifications using these computationally designed peptides confirm our predictions with high accuracy. This procedure can be generalized to any number of solid substrates as long as there is enough initial data set. The multiply-functional peptides have utility in developing surface engineering of solids including metals and oxides, combining several nanomaterials nanoparticles or nanowires, or molecular erectors when conjugated to other proteins, enzymes and DNA. Mainly supported by NSF-UW/MRSEC, and also by AFOSR-Bioinspired Materials, NSF-BioMat, NSF CAREER Award (RS), and SPO/Turkey (CT).
3:45 PM - NN4.5
Immobilization of Silaffin at GaAs Surfaces for Selective Area Silicate-biomineralization.
Okimoto Kondo 1 , Kazuhiro Matsuda 1 , Yusuke Matsuda 2 , Tadaaki Kaneko 1
1 Physics, Kwansei Gakuin University, Sannda, Hyogo, Japan, 2 Bioscience, Kwansei Gakuin University, Sannda, Hyogo, Japan
Show AbstractAs a new self-organizing methodology to fabricate higher-ordered nano-structures on semiconductor surfaces, the functionality of biomineralization is expected to be hybridized to the conventional semiconductor processing technology. From the point of view of the application to semiconductor industry, silaffin, which is a functional polypeptide obtained from the higher-ordered structural shell of the marine diatom Cylindrotheca fuciformis, has been focused on. This is because the peptide of silaffin biomineralizes dissolved silicate in water and produces bio-silica (SiO2) in the form of self-assembled nanospheres(1). This characteristic feature of the structures is considered to be the lowest-ordered product in the hierarchical structure of biomineralization. What has to be done in order to utilize the functionality of the biomineralization for semiconductor surface processing, is the immobilization of the silaffin peptide at the surface of the substrate. So far, no report has been published, as far as we know, on the direct immobilization of the silaffin at the surface without any buffer material like polymer(2). Furthermore, in order to take advantages of the biomineralization activities for the structural control of the self-assembled bio-silica nanospheres on the surface, site-control/selectivity of the immobilized silaffin is preferably required.In this report, the direct immobilization functionality of silaffin peptides at semiconductor surfaces has been investigated by employing newly constructed recombinant forms of the silaffin gene. Biomineralization activities were characterized by using single- and heptad-repeat types of silaffins at the surfaces of various kinds of GaAs substrates of different polarities, (100), (110), (111)A, (111)B, and Si(100) as a reference. In the experiments, a special treatment was conducted to stabilize the oxide layer of the surfaces by additional thermal oxidation. This is the opposite approach to expose the bare substrate surfaces for directly reflecting of the surface polarities by removing oxide. As a result, it was found that the amount of produced bio-silica nanospheres at the surfaces, which is considered to be proportional to the amount of the silaffins binding to each surface, has a significant tendency depending on the substrate used: GaAs(111)A > (100) >(110). GaAs(111)B and Si(100) did not show any evidence of the product. Those results reveal that the chemical characteristics of GaAs substrate in the order of Ga-richness play a critical role for silaffin to be immobilized at the surface. This selective binding efficiency of the silaffin suggests that selective area control of the production of the nanosphares could be possible on a patterned compound semiconductor surface.(1)Nils Kroger et al, SCIENCE, vol.286, 1129-1132, 5 November 1999.(2)Lawrence L.Brott et al, NATURE, vol.413, 20 September 2001.
4:30 PM - **NN4.6
Molecular Mechanisms of Self-assembly and Switching of the Bacterial Flagellum.
Keiichi Namba 1
1 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Show AbstractThe bacterial flagellum is made of a rotary motor and a long helical filament by means of which bacteria swim. The flagellar motor rotates at around 300 Hz and drives the rapid rotation of each flagellum to propel cell movements. The long helical filament, which is a tubular structure with a diameter of about 20 nm, is made of a single protein flagellin. The filament switches between left- and right-handed helical forms in response to the twisting force produced by reversal of the motor rotation, allowing bacteria to alternate their swimming pattern between run and tumble for taxis. The flagellum also has a short, highly curved segment called hook, which connects the motor and the helical propeller. Its bending flexibility makes it work as a nano-scale universal joint, while the filament is relatively more rigid to function as a propeller. The flagellum is constructed by self-assembly of proteins translocated from the cytoplasm through 2 nm central channel to the distal end of the growing structure, where a cap complex is attached to help efficient self-assembly of particular proteins that need to be assembled at each specific stage of assembly. Those flagellar proteins are exported from the cytoplasm into the central channel of the growing flagellum by the flagellar type III protein export apparatus, which consists of six membrane proteins and three soluble proteins. One of the proteins, FliI, is an ATPase that facilitates the export process, but the successive process of unfolding and exporting long chains of proteins though the export gate is driven by the proton motive force across the cytoplasmic membrane. A few cytosolic chaperones are also involved to facilitate the export process. We have been trying to understand how those macromolecular nanomachines assemble and work by looking at their structures by X-ray crystallography, fiber diffraction and electron cryomicroscopy. X-ray crystallography is a powerful tool to visualize atomic structures of macromolecules. However, visualization of the structures at work is difficult because many proteins involved do not form stable complexes. For example, the flagellar basal body when isolated does not contain the stator unit and most of the type III export apparatus proteins. The subunit stoichiometry may also have distributions. Electron cryomicroscopy including single particle image analysis, helical image reconstruction and tomography would allow us to visualize those structures. I will describe how we have visualized some parts of the flagellar structure at nearly atomic resolution by complementary use of the two methods, what we have learned from them, and how we will proceed further to solve the flagellar structure as a whole for ultimate understanding of the mechanisms of its protein export, self-assembly, and rotation.
5:00 PM - NN4.7
Construction of Recombinant Forms of the Silaffin Gene and their Silicate-biominerarization Activities.
Masato Horiguchi 1 , Sae Kikutani 1 , Tadaaki Kaneko 2 , Yusuke Matsuda 1
1 Bioscience, Kwansei-Gakuin University, Sanda, Hyogo, Japan, 2 Physics, Kwansei-Gakuin University, Sanda, Hyogo, Japan
Show AbstractSilaffin is a functional polypeptide obtained from the shell of the marine diatom Cylindrotheca fuciformis and this peptide biomineralizes dissolved silicate under a wide range of pHs, moderate temperature and under atmospheric pressure. Silaffin is encoded as a heptad-repeat form of functional unit sequence on a gene named sil-1. The product of each single repeat unit was obtained from the native shell of C. fuciformis and was shown to biomineralize silicate in vitro. However, the full length product of Sil-1 was not obtained from the diatom shell and it is not clear whether or not the full length product of Sil-1 is expressed and functions for biomineralization in cells or it is merely a precursor of the single repeat unit polypeptide of Sil-1 product. If the full length of Sil-1 product would possess biomineralization activity, it suggests that the heptad-repeat structure of sil-1 might confer on repeat form Silaffin a functional redundancy in vivo, which might be applied for cell-free system of silicate biomineralization. In the present study, expression systems for Silaffins of a single- and the heptad-repeat types were constructed and biomineralization activities of these products were characterized in detail. The results clearly showed that both the single- and the heptad-repeat forms of recombinant Silaffins possess biomineralization activities of silicate, and that dry weight of formed biosilica was positively correlated to the concentration of Silaffins over a range of 0 to 1000 nM. This relativity was apparently dependent upon densities of repeat unit of Silaffin in reaction mixtures rather than molality of Silaffins, that is, biominerarization activity of heptad-repeat Silaffin of unit molality was equivalent to that of single-repeat Silaffin of 7-fold concentrated molality. Given these, the single-repeat form of Silaffin is the functional unit of biomineralization activity but repeat structure of Silaffin reveals no notable function in apacity of biominerarlization activity. On the other hand, comparison of sizes of formed biosilica particles exhibited an apparent difference in between sigle- and heptad-repeat Silaffins. Namely, the averaged diameter of biosilica particles formed by 1 μM heptad-repeat Silaffin was about 350 nm whereas that by 7 μM single-repeat Silaffin was about 400 nm. This strongly suggests that sizes of biosilica particle could be regulated by repeat number of functional unit of Silaffin.
5:15 PM - NN4.8
Synthesis of Bimetallic Nanoparticles in Apo-Ferritin Cage for Rational Design of Bio-Nanoreactor.
Takafumi Ueno 1 2 , Masako Suzuki 1 , Yumio Toda 3 , Tomoki Akita 3 , Yusuke Yamada 3 , Yoshihito Watanabe 4
1 Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan, 2 PRESTO, Japan Science and Technology Agency, Kawaguchi Japan, 3 , National Institute of Advanced Industrial Science and Technology, Osaka, Osaka, Japan, 4 Research Center for Materials Science, Nagoya University, Nagoya, Aichi, Japan
Show AbstractInorganic nanomaterials are very important for magnetic devices, optical materials, and catalysts. Therefore, it will be nice if we are able to design and fabricate them. One of the promising approaches could be the employment of proteins. In fact, proteins and viruses having various structures (cage, tube, and so on) have been utilized as nanoreactors[1]. For example, zero-valent metal-, metal oxide-, and semiconductor nanoparticles (NPs) and nanowires have been prepared by deposition of metal ions and subsequent mineralization inside or on the outer surface of those proteins. Although synthesis of NPs in protein cages has been well studied, it is still challenging to rationally design NPs because there are few studies considering accommodation mechanisms of metal ions in the protein cages. We have previously reported the synthesis of a Pd nanoparticle (NP) in an apo-ferritin (apo-Fr), a cage-structured protein, as an olefin hydrogenation catalyst[2]. In order to increase the catalytic activity, we have prepared Au-Pd alloy and core/shell NPs in apo-Fr by studying the deposition mechanisms of Au(III) and Pd(II) ions in apo-Fr. It was found that metal ions in apo-Fr bind to the specific binding sites and the bound metal ions form a NP by NaBH4 reduction. In addition, Au(III) and Pd(II) ions apparent to bind different sites according to their different coordination characters to amino acid residues. Since Au(III) and Pd(II) ions are able to co-exist in an apo-Fr core due to the difference in their binding sites, alloy NP is prepared by the reduction of Au(III) and Pd(II) ions in apo-Fr at the same time. In contrast, core/shell NPs are prepared by repeated insertion of metal ions into apo-Fr and the subsequent reduction of them. The composites show different hydrogenation activities presumably due to their different NP structures. This study provides a new mechanism-based approach to prepare bimetallic NPs in protein cages. [1] Vriezema, D.M. et. al. Chem. Rev. 2005, 105, 1445-1489.[2] Ueno, T. et. al., Angew. Chem. Int. Ed., 2004, 43, 2527-2530.
5:30 PM - NN4.9
Electrostatic Nano-placement of a Single Nanoparticle by Charge-enhanced Protein Cage.
Shigeo Yoshii 1 3 , Ayako Kadotani 2 , Kazuaki Nishio 1 , Shinya Kumagai 1 , Ichiro Yamashita 1 2 3
1 ATRL, Panasonic, Kyoto Japan, 3 Material Science, NAIST, Nara Japan, 2 CREST, JST, Nara Japan
Show Abstract5:45 PM - NN4.10
Self-Assembling Peptide Nanofiber Hydrogels Targeted for Dental Tissue Regeneration.
Kerstin Galler 1 2 , Virany Yuwono 1 , Adriana Cavender 2 , He Dong 1 , Rena D'Souza 2 , Jeffrey Hartgerink 1
1 Department of Bioengineering, Rice University, Houston, Texas, United States, 2 Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M, Dallas, Texas, United States
Show AbstractPeptide based hydrogels represent a class of highly attractive synthetic biopolymers as they can be developed into multifunctional tissue engineering scaffolds. Harnessing the chemical functionality of individual amino acids or moieties attached, short peptides can be designed to self-assemble into three-dimensional networks of nanofibers, form macroscopic gels and serve as a matrix for cell encapsulation. The nanofibrillar structure resembles the extracellular matrix, and biological cell-matrix interactions can be tailored by modification of the peptide sequence or covalent linkage of bioactive molecules. This bottom-up approach allows for high control at the molecular level, which can translate into desired biological effects and be targeted towards specific cell types and applications. Bioengineering of dental tissues has become particularly interesting since postnatal stem cells have recently been isolated from various tissues such as pulp or periodontal ligament. An easily accessible source of dental stem cells are human exfoliated deciduous teeth. After transplantation into immunocompromised mice, these cells differentiate into odontoblasts and produce a dentin matrix. Combining them with a peptide hydrogel matrix specifically designed for this application may provide a suitable system for the regeneration of dental soft and mineralized tissues in vivo. In this presentation, we will describe the compatibility of dental stem cells (SHED) with different peptide hydrogels, which include cell adhesion sequences, specific enzyme-cleavable sites and bioactive molecules. Cell viability, proliferation, dispersal within the gel, and expression profiles of marker genes for odontoblast differentiation were analyzed at several time points of a four-week culture period. Histologic analysis was performed to visualize cell distribution and ECM formation. It was found that cells in the peptide nanofiber hydrogels show different proliferation rates and gene expression patterns depending on the peptide design and absence of adhesion ligands and cell differentiation factors. Histologic analysis revealed degradation of the matrix and replacement with natural ECM.
NN5: Poster Session
Session Chairs
Seung-Wuk Lee
Blake Simmons
Michael Yu
Wednesday AM, November 28, 2007
Exhibition Hall D (Hynes)
9:00 PM - NN5.1
Synthesis and Characterization of Templated Hetero-Trimeric Collagen Mimetic Peptides.
Xiao Mo 1 , Daniel Kim 1 , Allen Wang 1 , Jonathan Smits 1 , Seungju Yu 1
1 Materials Science & Engineering, The Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractCollagen mimetic peptide (CMPs) triple helices have been widely studied during the past few decades. While most efforts in the past have focused on CMP composed of homo-trimers, lately, hetero-trimeric CMP has received considerable attention because it can expand the potential library of triple helices that can be formed from a given pool of CMPs. Many natural collagens are composed of two or three different types of collagen strands; therefore hetero-trimeric CMP represents an ideal collagen model system. Recently, we reported the surprising discovery of CMP’s binding affinity to type I collagen. We expected that the templated hetero-trimer would facilitate discovery of new CMP architecture with enhanced collagen binding property. Here we present a simple approach for synthesizing templated ABB type hetero-trimeric CMPs. Tris(2-aminoethyl)amine with succinic acid spacer (TREN-(suc-OH)3) was used as a template to covalently link ABB-type hetero-trimer using two-step reactions that involve both solid phase coupling and solution phase coupling reactions. CMP hetero-trimers with varying peptide compositions were synthesized and their thermal melting behaviors were characterized by circular dichroism spectrometry. This synthetic method can be readily applied to the preparation of other hetero-trimeric peptide/macromolecule systems for collagen-targeted therapeutic and diagnostics.
9:00 PM - NN5.10
Crystal Structure of apo-Fr containing Pd Ions.
Mizue Abe 1 , Kunio Hirata 3 , Masako Suzuki 1 , Satoshi Abe 1 , Takafumi Ueno 1 2 , Nobutaka Shimizu 4 , Masaki Takata 4 , Yoshihito Watanabe 5
1 Chemistry, Nagoya Univ., Nagoya Japan, 3 , RIKEN, Sayo Japan, 2 , PRESTO/JST, Saitama Japan, 4 , JASRI/SPring-8, Sayo Japan, 5 Research Center for Material Science, Nagoya Univ., Nagoya Japan
Show Abstract Material syntheses by using biomolecules are very attractive methodology for the preparation of electronic materials, drugs and so on. It will be very nice if we could prepare metal nanoparticles having regulated particle size, composition, and morphology by employing such methodology, since it is well known that small metal nanoparticles show attractive characters in catalytic or electronic properties. Ferritin, an iron storage protein, is a spherical protein composed of 24 subunits having a cavity with inner diameter of 8 nm. The protein has been utilized to prepare nano materials.(ref:1) We have also prepared a Pd nanoparticle in apo-ferritin and the protein-Pd composite can catalyze olefin hydrogenation with very high TOF.(ref:2) The particle size is well-limited to 2.0 ± 0.3 nm. The narrow distribution in its size could be resulted by unique binding behavior of Pd ions to amino acid residues on the inner surface of the apo-ferritin subunit. We report herein the x-ray crystal structure analysis of a Pd/apo-Fr composite to elucidate accumulation mechanism of Pd ions for the preparation of Pd nanoparticles in apo-Fr. The composite was prepared by a reaction of apo-Fr and 50 equiv. of Pd ions and purified through a size exclusion column. The diffraction data were collected at the beam line BL41XU in SPring-8 (Hyogo, Japan) using synchrotron radiation with 2.15 Å resolution. The crystal structure of the Pd/apo-Fr composite shows that there are several Pd binding sites on the interior surface of the ferritin subunit. The amino acid residues utilized for the accommodation of Pd ions are Cys, His, and Arg. The residues are different from those reported for the iron nucleation sites. These results show the mechanism of metal cation incorporation into the apo-Fr cavity as well as the deposition of metal ions on the interior surface of apo-Fr.(ref:1) Douglas, T. et al., Science 1995, 268, 77-80.(ref:2) Ueno, T. et al., Angew. Chem. Int. Ed., 2004, 43, 2527-2530.
9:00 PM - NN5.11
Identification and Characterization of Montmorillonite Binding Peptides.
Lawrence Drummy 1 2 , Sharon Jones 1 2 , Richard Vaia 1 , Rajesh Naik 1
1 Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright Patterson AFB, Ohio, United States, 2 , UES Inc., Dayton, Ohio, United States
Show AbstractPeptide-inorganic interactions are of significant fundamental interest and technological relevance. Short peptide sequences have been isolated from combinatorial libraries that interact with a variety of inorganic surfaces. Using a phage display peptide library, we screened for peptide sequences that bind to montmorillonite (MMT) clay. Three different clay samples were examined, including Na+ MMT, a primary ammonium C18 functionalized MMT, and a quaternary ammonium C18 functionalized MMT. Phage binding was verified with immunofluorescence and low voltage transmission electron microscopy. X-ray diffraction of dried peptide-MMT complexes that were formed in solution indicated peptide intercalation between the layers (1.8 nm layer spacing). Competition assays were used to quantify the binding constants. FITC labelled MMT binding peptides were competed off the surface of MMT with unlabeled MMT binding peptides, and the fluorescence of the supernatant after centrifugation was measured. Fusion peptides based on MMT binding sequences and A3, a gold binding sequence, were used to produce gold functionalized MMT particles. The location of the gold nanoparticles on the surface of the MMT also provided a visual marker for direct imaging of the peptide binding sites. MMT provides a well characterized materials system for the study of peptide-inorganic interactions, and this work is expected to provide a route for the functionalization of nanomaterials.
9:00 PM - NN5.12
Gold Binding Antibody Fragment for Immobilization of Functional Proteins on Gold Substrate.
Hidenori Shiotsuka 1 , Masaru Kaieda 1 , Hideki Watanabe 2 , Satoru Hatakeyama 1 , Takahisa Ibii 1 , Mitsuo Umetsu 2 , Takeshi Imamura 1 , Izumi Kumagai 2
1 Nanobiotechnology Research Div., Canon Research Center, Canon Inc., Tokyo Japan, 2 Department of Biomolecular Engineering, Gradutate school of Engineering, Tohoku University, Miyagi Japan
Show Abstract9:00 PM - NN5.13
Using a Ring Protein to TRAP and Insert Gold Nanodots in a MOS.
Jonathan Heddle 1 2 5 , Shigeo Yoshii 2 4 , Kazuaki Nishio 4 , Christine Addy 5 , Ichiro Yamashita 2 3 4 , Jeremy Tame 5
1 Global Edge Institute, Tokyo Instititute of Technology, Yokohama, Kanagawa, Japan, 2 Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan, 5 Protein Design Laboratory, Yokohama City University, Yokohama, Kanagawa, Japan, 4 Advanced Technology Research Laboratories, Matsushita Electrical Co., Ltd, Seika, Kyoto, Japan, 3 CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
Show AbstractTRAP (trp RNA-binding attenuation protein) is a ring-shaped protein with potential uses as a component of nanoscale arrays and devices. We have modified TRAP to capture gold nanodots in solution. By engineering a titanium binding peptide onto one surface of the ring we were also able to bind it specifically and tightly to a TiO2 surface. Surface-bound TRAP was used to capture gold nanodots and attach them to prepared surfaces. Gold-protein complexes were observed using atomic force microscopy (AFM) and transmission electron microscopy (TEM). The modified TRAP was used to build gold nanodots into the SiO2 layer of a metal-oxide semiconductor (MOS). This is the first use of a ring protein, rather than the more commonly used spherical protein cages, to constrain nanodots to a surface. Our method extends the current range of bionanotechnology tools and may be the basis for future, multi-component electronic devices.
9:00 PM - NN5.14
Genetically Engineered Novel Hydroxyapatite Binding Peptides as a Utility in Guided Tissue Engineering.
Mustafa Gungormus 1 , Hanson Fong 1 , Monica Branco 4 , Joel P. Schneider 4 , Candan Tamerler 1 2 , Mehmet Sarikaya 1 2 3
1 Department of Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 4 Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States, 2 Molecular Biology, Genetics and Biotechnology, Istanbul Technical University, Istanbul Turkey, 3 Department of Chemical Engineering, University of Washington, Seattle, Washington, United States
Show AbstractBiomineralization is a complex process, in which hard tissues are generated through inorganic material formation, initiated and regulated, mainly, by proteins. Proteins control synthesis, and nano- and micro-architectures of the hard tissues at molecular and higher dimensional levels leading to tissue-specific functional properties, Hard tissue formation is an excellent process to emulate for fabricating functional materials for practical applications in technology and medicine. In this molecular biomimetic approach in the broadest sense, proteins, either isolated from hard tissues or designed theoretically have been studied extensively to understand how they influence inorganic material formation and to fabricate materials with tailored structures. Usually a large number of proteins are involved in the process of natural biomineralization and there is still a limited knowledge about their temporal and spatial distribution during tissue formation, a major drawback in these approaches. A new approach in biomimetic materials synthesis is using combinatorially selected and genetically engineered peptides that have specific affinity to desired solids. Here, as a first step towards designed material structures, we use hydroxyapatite (Hap) as a model to materialize calcium-phosphate-based nanoparticulates using biocombinatorially selected peptides. For this we first select Hap-binding heptapeptides (HABPs) through a peptide-phage or cell-based library and describe the identification of two peptides; one strong and one weak binder that are subsequently used in in vitro mineralization of calcium phosphate. The two candidate binders were identified by a combination of qualitative immunofluorescence microscopy and ELISA. The mineral formation kinetics was monitored using optical absorption and assays of calcium and phosphate, while the minerals formed by optical and electron microscopy and spectroscopy. To mimic the biological processes, alkaline phosphatase was used to control inorganic phosphate ions in solution. We found a drastic effect on mineral formation, e.g., orders of magnitude of accelerated growth and change in morphology, using the strong binder compared that of weak binder or controlled case (no binder). Based on these observations, we have incorporated the HAPBs into a beta-hairpin forming peptide that self assembles to form a hydrogel network. We have observed that addition of Hap binding peptide into the hydrogel network provides a control over the mineralization kinetics and results in a more homogeneous mineralization in the hydrogel network. These results indicate that selected Hap binding peptides might be used as active controllers for guided hard tissue engineering. Research is underway for further characterization of the morphological effects of the Hap binding peptides in peptide hydrogel tissue scaffolds. The research is supported by USA/ARO/UW-DURINT, NSF-BioMat, and NSF/UW-MRSEC.
9:00 PM - NN5.15
Targeted Photothermal Destruction of Infectious Bacteria Using Gold Nanorods.
John Stone 1 , R. Norman 2 , Anand Gole 1 , Catherine Murphy 1 , Tara Sabo-Attwood 2
1 Chemistry, University of South Carolina, Columbia, South Carolina, United States, 2 Environmental Health Sciences, University of South Carolina, Columbia, South Carolina, United States
Show AbstractPseudomonas aeruginosa is a Gram-negative bacterium which is ubiquitous in the environment and often pathogenic to individuals with already compromised immune systems. Individuals with cystic fibrosis, the most common lethal inherited disorder among Caucasians, are especially prone to P. aeruginosa infections which are responsible for most of the morbidity and mortality associated with this disease. Once in a host, these bacteria can form aggregates while simultaneously secreting a polymer-like substance resulting in a biofilm, complex structures that are highly resistant to multiple antibiotics and that evade clearance by the immune system. In this work, we test the validity of using nanotechnology-based therapeutics for the treatment of infectious bacteria. Gold nanorods were prepared having approximate lengths and widths of 70 nm and 25 nm via a wet chemical synthesis. Briefly, as-prepared gold seeds (~ 4 nm gold spheres) were added to a solution containing gold, cetyltrimethylammonium bromide (CTAB), silver nitrate, and ascorbic acid. This synthesis resulted in rods having an absorption maxima centered around 785 nm. The resulting rods were bioconjugated to primary antibodies specific to the opportunistic pathogen Pseudomonas aeruginosa via amide bonds. The bioconjugated gold nanorods enhanced targeted binding to antigens expressed on the bacterial outer membrane. The bacteria/nanorod complex was then subsequently irradiated with near-infrared radiation (785 nm) in order to produce localized photothermal destruction of the bacterial cells. Furthermore, we hypothesize that; in future work, targeted thermal destruction of bacteria will aid in the eradication of infectious biofilms and increase their susceptibility to antibiotic killing.
9:00 PM - NN5.16
Adiabatic Compressibility Variation During Amphphile Binding To Bovine Serum Albumin.
Najwa El Kadi 1 , Nicolas Taulier 1 , Wladimir Urbach 2 , Marcel Waks 1
1 CNRS LIP UMR 7623, Universite P et M Curie, Paris France, 2 LPS, Ecole Normale Superieure, Paris France
Show Abstract9:00 PM - NN5.17
Spontaneous Chirality via Long-range Electrostatic Forces.
Kevin Kohlstedt 1 , Graziano Vernizzi 1 , Francisco Solis 2 , Monica Olvera de la Cruz 1
1 Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois, United States, 2 Department of Integrated Natural Sciences, Arizona State University, Glendale, Arizona, United States
Show AbstractWe consider a model for periodic patterns of charges constrained over a cylindrical surface. In particular we focus on patterns of chiral helices, achiral rings or vertical lamellae, with the constraint of global electroneutrality. We study the dependence ofthe patterns' size and pitch angle on the radius of the cylinder andsalt concentration. We obtain a phase diagram by using numerical and analytic techniques. For pure Coulomb interactions, we find a ring phase for small radii and a chiral helical phase for large radii. At a critical salt concentration, the characteristic domain size diverges, resulting in macroscopic phase segregation of thecomponents and restoring chiral symmetry. We discuss possible consequences and generalizations of our model.
9:00 PM - NN5.18
Hierarchical Coexistence of Universality and Diversity Controls Robustness and Multi-functionality in Protein Materials.
Theodor Ackbarow 1 , Markus Buehler 1
1 Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractProteins constitute the elementary building blocks of a vast variety of biological materials such as cells, spider silk or bone, where they create extremely robust, multi-functional materials by self-organization of structures over many length- and time scales. Some of the structural features are commonly found in different tissues, that is, they are highly conserved. Examples of such universal building blocks include alpha-helices, beta-sheets or tropocollagen molecules. In contrast, other features are highly specific to tissue types, such as particular filament assemblies, beta-sheet nanocrystals in spider silk or tendon fascicles. These examples illustrate that the coexistence of universality and diversity – in the following referred to as the universality-diversity paradigm (UDP) – is an overarching feature in protein structures, and is crucial to understand how their structure and properties are linked. The most important aspect of this paradigm is that universal building blocks and highly diversified patterns are unified through formation of hierarchical structures leading to multi-functional, robust yet optimal structures. Here we apply recent insights from the system theoretical and system biological approaches to hierarchical biological materials. We illustrate the significance of the UDP in a detailed analysis of three kinds of intermediate filament proteins. First, vimentin, one of the three major components in the cell’s cytoskeleton, second, lamins, part of the nuclear envelope, and third, keratins, the main component of hair, nails or hoofs. Our analysis explains that the coexistence of disparate attributes, such as robustness and optimality, simplicity and multi-functionality, locality and non-locality is vital for the capacity of protein materials to satisfy their physiological and mechanistic roles under extreme and changing conditions. This viewpoint enables one to understand how seemingly incompatible qualities can be combined in hierarchical structures in order to create larger entities with properties that exceed those found at each hierarchical scale alone. The most significant finding is that the UDP explains the formation of structures that are not only adapted for a selection of tasks, but that are also capable of satisfying these roles robustly. The UDP also illustrates how robustness can be lost, with potentially grave consequences leading to diseases. Appreciation of this concept enables one to address important questions of protein science that gain increasing relevance for a variety of disciplines, including materials science, engineering and medicine. We provide a perspective on research opportunities and challenges in a variety of disciplines, including an outlook to structural engineering and network design.
9:00 PM - NN5.2
Synthesis and Characterization of a Novel Poly(ethylene glycol) Hydrogel with Physico-chemical Crosslinks Based on Collagen-Mimetic Peptides.
Nicole Romano 1 , Allen Wang 1 , Melissa Thompson 2 , Denis Wirtz 2 3 , Seungju Yu 1 3
1 Department of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 2 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 3 Institute for NanoBiotechnology, The Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractHydrogel systems for complex tissue scaffolding require increasingly biomimetic chemistries and mechanical properties to promote cell viability. We synthesized a hydrogel using bifunctional collagen mimetic peptide (CMP) conjugates as physico-chemical crosslinkers for star-shaped poly(ethylene glycol) (PEG). Chemical crosslinks occur between N-hydroxy-succinimide-terminated star-shaped PEG and amine-terminated bifunctional CMPs, while physical crosslinks result as three CMP strands form collagen-like triple helical complexes. CMP triple helices alone are sufficient crosslinks for the formation of a self-supporting PEG hydrogel. However, we show that tissue scaffolds with enhanced mechanical properties result from the use of both chemical and triple helical crosslinks. The viscoelastic properties of this hydrogel may be modulated by both the number of chemical crosslinks and the temperature, which affects the strength of physical crosslinks. Through the use of CMP triple helical self-assembly, this hydrogel can be readily functionalized with bioactive molecules. The system additionally offers spatio-temporal control through manipulation of physical crosslinking; this may result in improved viability of encapsulated cells compared to other hydrogels, as well as the ability to direct the formation of complex tissues.
9:00 PM - NN5.3
New Piezoelectric Material Based on Biopolymer-polymer Composites.
Dawnielle Farrar 1 4 , James West 2 , Ilene Busch-Vishniac 3 , Seungju Yu 1 5
1 Department of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 4 Applied Physics Lab, The Johns Hopkins University, Baltimore, Maryland, United States, 2 Department of Electrical and Computer Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 3 Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 5 Institute for NanoBiotecnology, The Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractPiezoelectric materials (PM) often in use today are made of ceramic crystals. Despite high piezoelectricity, they are brittle and require expensive processing conditions. An ideal PM is the one where piezoactivity and mechanical properties can be altered individually so that the mechanical stiffness of the materials can be varied for particular application or tuned to match that of the surrounding (e.g. air or water) for increased transduction sensitivity. Here we present a new class of polymer composite PM based on piezoactive biopolymer, poly(γ-benzyl α,L-glutamate) (PBLG), and a matrix polymer, poly(methylmethacrylate) (PMMA). By simultaneous poling and curing of PBLG/MMA mixture solutions via corona charging at ambient condition, we fabricated a flexible composite film with a significant portion of the PBLG molecules oriented normal to the film surface. This film exhibited high piezoelectricity (d33 = 20 pC/N), and its mechanical characteristics were similar to those of low molecular weight PMMA indicating that the piezoelectricity and mechanical strength are independently related to the two polymer components of the composite film. This piezoelectric film can be fabricated directly from solution on a substrate or in a mould. Therefore they are amenable to miniaturization for small sensors with integrated electronics or can be used as piezoelectric coating applications.
9:00 PM - NN5.5
Long Range Alignment of Peptide Amphiphile Nanofibers.
Shuming Zhang 1 , Alvaro Mata 3 , Megan Greenfield 2 , Samuel Stupp 1 2 3
1 Materials Science and Engineering, Northwestern University, Evanston, Illinois, United States, 3 Institute for Bionanotechnology in Medicine, Northwestern University, Evanston, Illinois, United States, 2 Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, United States
Show AbstractPeptide amphiphiles molecularly designed to aggregate into beta sheet structures are known to self assemble into cylindrical nanofibers. These nanofibers can be functionalized with epitopes and have shown great potential for use in regenerative medicine and drug delivery systems. An important challenge in these systems is to gain control over the length and macroscopic alignment of the nanofibers in order to create structures that can spatially guide cells, control their contacts, and possibly differentiation in the case of stem cells. Nanofiber length and alignment offers a strategy to change the physical properties of these systems. We present here a method to use thermal energy in order to create extremely long and aligned nanofibers over macroscopic dimensions, on the scale of centimeters. We also show that the alignment procedure can raise the stiffness of gels formed by these nanostructures by factors up to 4. A mechanism is proposed for the formation of large domains which involves the reorganization of molecules into assemblies with different symmetries and levels of hydration. We also demonstrate the ability of the systems to align human mesenchymal stem cells within oriented matrices. Within 2 days, cells preferential align and migrate in the highly ordered environment and within 2 weeks the matrices are able control cell differentiation as indicated by expression of specific genes.
9:00 PM - NN5.6
Reactive Microcontact Printing of Polymer Brushes for Patterning of Nanoparticles and Biomolecules.
Steve Diamanti 1 , Shafi Arifuzzaman 2 , Jan Genzer 2 , Richard Vaia 1
1 , Air Force Research Laboratories, Wright Patterson Air Force Base, Ohio, United States, 2 Chemical Engineering, North Carolina State University, Raleigh-Durham, North Carolina, United States
Show AbstractAn inexpensive and robust method for patterning of biomolecules would be of interest in medical applications such as tissue engineering. Gradient surfaces of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes have been previously shown to spatially localize biomolecule binding, while minimizing non-specific adsorption of the same biomolecule on other regions of the gradient specimen. In order to further improve the specificity and to provide latent functionality for detection of the binding events, post-polymerization modification of PHEMA with various functional groups has been investigated. Using standard N,N’-disuccinimidyl carbonate (DSC) coupling, hydroxyl pendants of PHEMA brushes were conjugated to oligo-peptides, alkanes and oligo(ethylene glycol) (OEG) through an alpha-terminus primary amine. Ellipsometry, contact angle, and X-ray photoelectron spectroscopy (XPS) indicated that coupling occurred with efficiencies ranging from 40-80%. Due to the relatively fast reaction times of the amination reaction this procedure enables surface patterning via reactive microcontact printing. For example, a 10 mM solution of amino-terminated PEG 2000 in acetone is applied to a PDMS stamp via swab application, dip-coating, or spin-coating. The latter method provided the most effective pattern transfer. The coated PDMS stamp was contacted with the DSC activated polymer brush, weighted down with approximately one pound and held in contact for 10 minutes. The stamp was then peeled off, the brush surface rinsed thoroughly with acetone, and then immersed in a 10mM solution of hexadecylamine in acetone in order to reactively backfill the stamped pattern. The transferred pattern could be visualized by optical microscopy due to the difference in refractive indices of the PEG and hexadecane functionalized regions. This process of reactively stamping activated regions can be repeated with different patterns and functionalities leading to complex surface chemical profiles. As a demonstration of the surface profile, the aforementioned patterned brush can be exposed to aqueous solutions of nanoparticles or biomolecules with different binding affinities to the two regions. For example, the patterned brush was exposed to an aqueous solution of 30 nm citrate-capped gold nanoparticles. The gold nanoparticles bind preferentially to the PEG functionalized regions and have low affinity for the alkane functionalized surfaces resulting in a replication of the brush pattern by gold nanoparticle binding visualized by SEM (Supplemental Figure 1). In a similar manner, GFP (green fluorescent protein) and collagen adhere preferentially to hydrophobic alkane modified surfaces and to resist adherence to hydrophilic OEG modified surfaces (Supplemental Figure 2).
9:00 PM - NN5.7
Cell Free Synthesis of Bioadhesive Protein mefp-1 in High Yield via a Novel DNA Hydrogel.
Jianfeng Xu 1 , Dan Luo 1
1 Biological and Environmental Engineering, Cornell University, Ithaca, New York, United States
Show AbstractMussel adhesive proteins are remarkable in that that can function over wide temperature ranges, fluctuating salinities, humidifies, and in the tide, wave and currents of marine environments. Mytilus edulis foot protein type-1 (mefp-1), consisting of about 80 repeats of a decapeptide consensus sequence AKPSYPPTYK, is considered as one of the key proteins for adhesion of the mussels underwater. Mefp-1 contains ~10-20% 3,4-dihydroxyphenyl-L-alanine (DOPA) which are primary responsible for surface adhesion and covalent cross-linking of the proteins. Attempts to produce functional and economical recombinant mefp-1 protein in several expression hosts such as Escherichia coli, yeast and plant cells are unsuccessful due to the highly biased amino acid composition of mefp-1 and different codon usage preference between mussel and other hosts, which lead to extremely low protein yields. The cell free system provides an alternative to cell-based system in recombinant protein expression. Recently we developed a novel protein producing DNA hydrogel, designed as P-gel that allows us to synthesize high-yield recombinant protein in a cell free system. Thus, we tried to express 35 and 75 repeats of the mefp-1 consensus sequence via P-gel in a wheat germ based cell free system. The yields of (mefp-1)35 and (mefp-1)75 were measured as 0.7 mg/ml and 0.6 mg/ml, respectively, which were 3-4 time higher than those expressed in conventional cell free system. The two mefp-1 proteins were then isolated via Ni-NTA column and further purified with reversed phase HPLC. Function assay shows that the adhesion force of the purified recombinant mefp-1 proteins was comparable to that of Cell-Tak, a commercial mussel extract adhesive..
9:00 PM - NN5.8
Molecular Deformation Mechanisms and Nanomechanics of Amyloid Fibrils.
Sinan Keten 1 , Xuefeng Chen 1 , Markus Buehler 1
1 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractProtein aggregation is a materials growth process that occurs spontaneously based on protein interaction properties and requires minimal biological modulation. Uncontrolled self assembly and aggregation of proteins causes amyloidogenesis; formation of well-structured insoluble fibrils that accumulate in the tissue during the course of diseases such as Alzheimer’s, Parkinson’s, and Type II diabetes. These fibrils feature well-ordered beta-sheets that run perpendicular to the axis of the fibril and exhibit regular diameter and periodicity. Recent studies with AFM have suggested that such amyloid fibrils have nanostructural features that show exceptional mechanical properties and may provide explanation to the toughness of other natural materials such as spider silk. However, little is known about the nano-scale deformation mechanisms and structure-property relationship of amyloid fibrils. Here we elucidate the nanoscale deformation mechanisms of amyloid fibrils under different loading conditions. We perform molecular modeling and simulation techniques to systematically investigate energetics and rupture mechanisms of inter-stand hydrogen bonds that partake in the self assembly process and link our findings with the structural features of these materials. Our results shed light on mechanisms recently predicted, but not readily observed by experimental methods.
9:00 PM - NN5.9
X-ray Crystal Structure Analysis of Artificial Metalloproteins: Selective Coordination of Copper Complexes with Square-Planar Structure in the Apo-Myoglobin.
Satoshi Abe 1 , Takafumi Ueno 1 2 , Seiji Okazaki 3 , Tatsuo Hikage 4 , Atsuo Suzuki 3 , Takashi Yamane 3 , Hiroshi Nakajima 1 , Yoshihito Watanabe 5
1 Department of Chemisrty, Graduste School of Science, Nagoya University, Nagoya Japan, 2 , PRESTO, Saitama Japan, 3 Department of Biotechnology, Graduate School of Engineering, Nagoya University, Nagoya Japan, 4 High Intensity X-ray Diffraction Laboratory, Nagoya University, Nagoya Japan, 5 Research Center for Materials Science, Nagoya University, Nagoya Japan
Show AbstractMolecular design of artificial metalloproteins and metalloenzymes is one of the goals of bioinorganic chemistry. There have been many reports that described protein composites containing metal catalysts, inhibitors linked to metal complexes and modified metal cofactors. However, detailed structural analyses as well as mechanistic aspects are still not clear for most of the composites. X-ray crystal structure studies of artificial metalloproteins are essential to improving catalytic activities and understanding physical properties and for further application to biotechnology. Myoglobin (Mb) is a small heme protein that functions as an O2 storage unit, and it has been used as a model for many heme enzymes. Previously, we have reported a novel method for the reconstitution of apo-Mb with synthetic metal centers and a series of crystal structures of MIII(3,3’-Me2-salophen) (salophen = N,N’-bis(salicylidene)-1,2-phenilenediamine) (M = Cr, Mn, Fe) complexes bound to the apo-Mb cavity by noncovalent interactions. Herein, we have extended our studies to other metal complexes preferring four coordinate structures. In this presentation, we report the preparation of CuII(Sal-Phe) (Sal-Phe = N-salicylidene-L-phenylalanato) (1)/apo-Mb, CuII(Sal-Leu) (Sal-Leu = N-salicylidene-L-leucinato) (2)/apo-Mb and CuII(Sal-Ala) (Sal-Ala = N-salicylidene-L-alanato) (3)/apo-Mb and the crystal structures of 1/apo-Mb and 2/apo-Mb to elucidate the coordination structures induced by noncovalent interactions in the apo-Mb cavity.1 CuII(Sal-X)/apo-Mb composites were prepared by a method reported by us with some modifications. The 1:1 composites of copper complexes with apo-Mb were confirmed by UV-visible, EPR and ESI-TOF mass spectrometry. The UV-visible spectra of these composites show a broad d-d band centered at 600 nm and a charge transfer band near 370 nm, which are commonly observed for copper (II) complexes. The EPR spectra of 1/apo-Mb, 2/apo-Mb and 3/apo-Mb (g|| = 2.25, 2.25 and 2.25, A|| = 168, 171 and 169 G, g⊥ = 2.06, 2.06 and 2.06, respectively) indicate that the coordination geometry of 1-3 are conserved with a square planar or square pyramidal structure in apo-Mb. X-ray crystal structures of 1/apo-Mb and 2/apo-Mb were determined with diffraction data of 1.65 and 1.8 Å resolution, respectively. These copper complexes are bound to the Nε atom of distal His64 while heme and MIII(salophen) bind to that of His93. The coordination geometry around the CuII atom in apo-Mb is distorted square-planar with tridentate Sal-X and a Nε atom of His64 in apo-Mb cavity and the plane of these copper complexes is perpendicular to that of heme. Several interactions such as π-π, CH-π and hydrogen bonding are observed between copper complexes and surrounding amino acid residues for fixation of the copper complexes in apo-Mb. Theses results suggest that the apo-Mb cavity can hold metal complexes with various coordination geometries. [1] S. Abe et al. Inorg. Chem. 2007, 46, 5137
Symposium Organizers
Michael (Seungju) Yu Johns Hopkins University
Seung-Wuk Lee University of California-Berkeley
Derek Woolfson University of Bristol
Ichiro Yamashita Nara Institute of Science and Technology (NAIST)
Blake Simmons Sandia National Laboratories
NN6
Session Chairs
Blake Simmons
Ichiro Yamashita
Michael Yu
Wednesday AM, November 28, 2007
Room 201 (Hynes)
9:30 AM - **NN6.1
Elastin Biopolymers for Drug Delivery.
Ashutosh Chilkoti 1
1 Biomedical Engineering, Duke University, Durham, North Carolina, United States
Show AbstractThis talk will describe thermal targeting of cancer therapeutics to solid tumors by two different classes of thermally responsive recombinant elastin-like polypeptides (ELPs) that exhibit a lower critical solution temperature transition slightly above 37 °C. The first generation of ELPs that we have designed for drug delivery are pseudorandom copolymers of the VPGXG repeat where the mole fraction of X and the polymer chain length were precisely specified so the polypeptide would undergo its phase transition between 37 and 42 °C. In vivo fluorescence videomicroscopy of human tumors implanted in nude mice demonstrated that the phase transition of this thermally responsive ELP occurs in heated tumors resulting in the formation of micron-size aggregates within the heated tumor. The phase transition results in a ~two-fold increase in tumor localization compared to the same polypeptide without hyperthermia even for heating periods as short as one hour. We have observed that thermally cycling the tumor can further increase the uptake of the ELP within the tumor. Doxorubicin was conjugated to this first generation ELP carrier via an acid labile hydrazone bond to enable release of the drug in the acidic environment of lysosomes. The ELP-doxorubicin conjugate and free drug exhibited equivalent cytoxicity in cell culture.A second generation of diblock ELPs will also be described that function as temperature triggered polymer amphiphiles. These diblock ELPs form monodisperse, ~60 nm diameter micelles in the range of 37-42 °C, a range approved for clinical hyperthermia of solid tumors, which allows the multivalent presentation of tumor specific ligands in heated tumors, thereby enhancing their accumulation in tumors.
10:00 AM - **NN6.2
Modulation of Receptor Binding and Assembly by Well-Defined Polypeptides.
Kristi Kiick 1 2
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 2 , Delaware Biotechnology Institute, Newark, Delaware, United States
Show AbstractBiosynthetic routes to protein-based polymeric materials have demonstrated their utility for the production of well-defined macromolecular materials, owing to the control of sequence and molecular weight inherent in the biosynthesis of proteins. In our work, we have utilized the biosynthesis of polypeptides with controlled presentation of functional groups in multiple positions, coupled with their subsequent chemical modification to produce well-defined, bioactive macromolecules that can mediate multivalent binding events important in toxin neutralization and inflammation. Employing the chemical versatility of non-natural amino acids in these studies has expanded the architectural control of the multivalent constructs, and affords opportunities to modulate binding via control of conformation and secondary interactions. Chemical modification of the polypeptides does not affect their well-controlled conformational behavior, and opportunities to integrate functional group placement with assembly for production of additional advanced macromolecular constructs is under exploration. Given the chemical flexibility in the design of such scaffolds, multiple opportunities exist for their application in areas where control of active side chains is important, such as in biomaterials, electronic devices, and bioinorganic structures.
10:30 AM - NN6.3
Spatio-temporal Presentation of VEGF for Microvasculature Engineering in Collagen Scaffolds Mediated by Polyanionic Collagen Mimetic Peptides.
Shirley Leong 1 , Allen Wang 2 , Yu-Chuan Liang 3 , Ru Chih Huang 3 5 , Christopher Chen 4 , Seungju Yu 2 5
1 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 2 Department of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 3 Department of Biology, The Johns Hopkins University, Baltimore, Maryland, United States, 5 Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland, United States, 4 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Show AbstractAngiogenesis is a morphogenic event that endothelial cells (ECs) undergo in response to 3D environmental triggers. Although ECs can be induced to form tube-like morphology in vitro using natural scaffolds (e.g. collagen), the cell culture needs continuous supply of growth factors, and the engineered capillaries bare no resemblance to the level of organization exemplified by the capillary blood vessels in the living tissue. Currently, no method is available for controlling the location, direction, and branching pattern of angiogenesis in macroscopic scale in 3D tissue culture. Previously, our group detailed a novel, physical technique for modifying collagen scaffolds that utilizes triple helical associative chain interactions between synthetic collagen mimetic peptide (CMP) with a -(ProHypGly)x- sequence and natural type I collagen. Here we present a new CMP architecture that displays multiple anionic charges in dendritic form at peptide’s N terminus designed to attract growth factors when applied to collagen scaffolds by charge-charge interactions. This CMPs exhibited specific binding affinity to type I collagen films and gels and at the same time attracted vascular endothelial growth factors (VEGFs) which led to enhanced tubulogenesis of ECs in defined regions of collagen gels. The results indicate that our anionic CMPs could be used for spatio-temporal presentation of growth factors in collagen scaffolds for controlled microvasculature and other complex tissue formation.
10:45 AM - NN6.4
Growth Factor Embedded Designer Self-Assembling Peptide Matrix for Tissue Engineering.
Akihiro Horii 1 2 , Xiumei Wang 1 , Shuguang Zhang 1
1 Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 , Olympus America Inc., Center Valley, Pennsylvania, United States
Show AbstractWe have been studying the designer self-assembling peptide matrix consists of peptide sequence (Ac-(RADA)4-SEQ-CONH2; SEQ: functionalized peptide motif) in tissue engineering[1]. Controlled release of growth factor from matrix and immobilization of growth factor in the matrix has also been studied to promote regeneration in tissue engineering [2]. We studied growth factor embedded functionalized self-assembling peptide matrix for tissue engineering. [Material and Method]Self-assembling peptide matrixes were formed from designer functionalized self-assembling peptides. Each self-assembling peptide matrix has different affinity to growth factor by changing charge distribution (positively and negatively charged) or specific affinity sequence such as heparin binding sequence. Heparin is known to have strong affinity to many growth factors including bFGF, VEGF and BMPs. These peptides were dissolved in water at 2% concentration that formed hydrogel scaffolds in the tissue cell culture insert (Millicell-CM, Millipore) by adding PBS. After forming scaffolds, bFGF dissolved in PBS was loaded to the inserts at 100 ng/well. These scaffolds were washed three times using PBS to wash out unbound growth factor. After washing, HUVEC (Human Umbilical Vein Endothelial Cells, Cambrex) were seeded in the inserts at 7x10^5 cells/well and cultured using EGM-2 medium (Cambrex) without growth factors for two days. Maintenance of cell adhesion to the scaffolds was evaluated using fluorescence staining of actin and nuclei. The cells cultured on peptide matrix without growth factor loading using EGM-2 medium with or without growth factors were used as positive and negative control. The growth factors retained in scaffolds were calculated by measuring bFGF in washed PBS and culture medium using ELISA. [Results]Growth factor retention rates in the scaffolds after 3-time PBS-washing varied from 0.35 to 0.85 according to the different affinity to growth factor. The growth factor retention rates were not correlated with global charge of molecule of growth factor and peptide matrix. There were no significant changes observed in trends of growth factor release profile while PBS washing and while HUVEC culturing. In some peptide scaffolds, the prolongation of cell attachment were observed compared to positive control which contained growth factor in medium, not embedded in the matrix. [Conclusion]Growth factors retention in self-assembling peptide scaffolds can be controlled by changing affinity of growth factor. bFGF embedded in the designer functionalized self-assembling peptide scaffolds can prolong HUVEC cell attachment to the scaffolds, thus they will be likely useful for tissue engineering. [Reference]1. Horii et.al, Plos One, vol.2, e190, 2007.2. Davis wt.al, PNAS, vol. 103, no. 21, 8155–8160, 2006.
11:00 AM - NN6.5
Novel Polymeric Nano-/Micro-hollow Particle ``PICsome” Based on the Self-assembly of the Block Copolymers through the Electrostatic Interaction in Aqueous Medium and Application to the Nano-/Micro-bioreactor
Akihiro Kishimura 1 , Aya Koide 1 , Kensuke Osada 1 , Yuichi Yamasaki 1 , Kazunori Kataoka 1 2 3
1 Graduate School of Engineering, The University of Tokyo, Tokyo Japan, 2 Graduate School of Medicine, The University of Tokyo, Tokyo, Tokyo, Japan, 3 Center for NanoBio Integration, The University of Tokyo, Tokyo, Tokyo, Japan
Show AbstractHollow capsules or vesicles as a nano-/micro-container have gathered great interest owing to their fundamental importance as new colloidal structures as well as to their potential utility into biomedicine including drug and gene delivery carriers, artificial cells, and bioreactors. The most versatile method for preparing hollow capsules is the approach of molecular self-assembly. Vesicles formed through this approach have attracted more attention for the lack of a template in their formation process as well as the feasibility to encapsulate a variety of guest molecules. Particularly, polymer vesicles self-assembled from amphiphilic block copolymers are characterized with the high structural stability compared to conventional lipid vesicles and the attractive chemical diversity to integrate smart functions such as stimuli sensitivity. Nevertheless, the major drawback of these amphiphilic polymer vesicles as biofunctional materials is the lack of permeability of hydrophilic solutes due to the hydrophobic nature of their membrane. The harsh preparation conditions involving organic solvents becomes problematic for the encapsulation of biologically relevant compounds, such as proteins and nucleic acids. Recently, we reported a novel polymeric vesicles with a polyion complex membrane (PICsome) to overcome these issues emerging in the conventional systems.[1] Without the use of any organic solvents, a pair of oppositely charged block ionomers consisting of polyaminoacids and biocompatible PEG segments self-assemble into a PICsome in a single aqueous medium. In this study, detailed preparation condition of the PICsome was investigated toward its structural tuning.Due to the moderate preparation in an aqueous solution, water-soluble macromolecular compounds may be readily compartmentalized in the inner aqueous core of the PICsome partitioned from the exterior by the PIC membrane sandwiched between PEG layers, which is quite beneficial for the encapsulation of the biologically active but fragile compounds, especially proteins. Moreover, the PIC membrane of the PICsome shows semipermeability based on the intrinsic property of the PIC. We also present here for the first time the successful compartmentalization of biologically relevant proteins, myoglobin, into the PICsome, demonstrating the unique function attributed to the semipermeable nature of the wall of PICsome in the physiological environment as well as the increased tolerability against protease attack, which is often to be the issue in the application of fragile proteins in the biomedical field.[2] The method provided here may lead to a general way to integrate novel carrier system platforms useful in drug delivery as well as functional nano-/micro-bioreactor systems available in diagnostic and therapeutic fields.[1] Koide, A. et al. J. Am. Chem. Soc. 2006, 127, 5988–5989.[2] Kishimura, A. et al. K. Angew. Chem. Int. Ed. in press.
11:30 AM - **NN6.6
Peptides for Desired Twisted vs. Untwisted Nanostructure for Hydrogels or Inorganic Nanoparticle Templated Assembly.
Darrin Pochan 1
1 Materials Science and Eng, University of Delaware, Newark, Delaware, United States
Show AbstractThe local nano- and overall network structure, and resultant viscoelastic and cell-level biological properties, of hydrogels that are formed via βετα-hairpin self-assembly will be presented. While discussing the properties of this peptide system, the physical and biophysical methods used to characterize these materials will be highlighted. These peptide hydrogels are potentially ideal scaffolds for tissue repair and regeneration due to their ability to mimic natural extra cellular matrix. The 20 amino acid peptide MAX1 (H2N-VKVKVKVKVDPPTKVKVKVKV-CONH2), has been shown to fold and self-assemble into a rigid hydrogel based on environmental cues such as pH, salt, and temperature including physiological conditions. The hydrogel is composed of a network of short fibrils that are 3 nm wide and up to several hundred nm long with no covalent crosslinking required for gel stiffness. In addition, slight design variations of the MAX1 sequence allow for tunability of the self-assembly/hydrogelation kinetics. In turn, by controlling hydrogel self-assembly kinetics, one dictates the ultimate stiffness of the resultant network and the kinetics through which gelation occurs. Importantly, once formed into a solid, self-supporting gel the network can be disrupted by the introduction of a shear stress. The system can shear thin but immediately reheal to preshear stiffness on the cessation of the shear stress. This shear thinning, or thixotropic, behavior of these physical networks makes them interesting candidates for injectable delivery in vivo where no post injection chemistry is required to set up the network. New peptide designs allow the control of nanoscale structure (e.g. twisted vs. untwisted) that, in turn, controllably affect bulk gel properties. In addition, assemblies have been designed to allow the templated assembly of inorganic nanoparticles into well-defined, one-dimensional nanoparticles arrays. Peptide design self-assembly, self-assembly characterization, gel and nanostructure properties will be discussed.
12:00 PM - NN6.7
Molecular Engineering of Smart Protein Scaffolds for Cardiac Stem Cell Differentiation.
Jennifer Blundo 1 , Oscar Abilez 3 , Karin Straley 3 , Joey Doll 1 , Feng Cao 4 , Joseph Wu 4 , Chris Zarins 3 , Sarah Heilshorn 2 , Beth Pruitt 1
1 Mechanical Engineering, Stanford University, Stanford, California, United States, 3 Vascular Surgery, Stanford University, Stanford, California, United States, 4 Cardiology/Radiology, Stanford University, Stanford, California, United States, 2 Materials Science and Engineering, Stanford University, Stanford, California, United States
Show Abstract“Smart scaffolds” with tunable ligand density and mechanical stiffness have been engineered to mimic the cardiac extracellular matrix (ECM) and enhance the differentiation of human embryonic stem cell derived cardiomyocytes (hESC-CMs). The protein scaffolds are synthesized in E. coli bacterial hosts, enabling molecular-level control over the material design. The engineered proteins contain RGD-binding sequences interspersed with an elastin-like sequence to promote cell adhesion and mechanical integrity, respectively. The resulting scaffolds are optically transparent and easily micro-molded in a sheet or 3D construct. Crosslinking is achieved with a water-soluble, bi-functional N-hydroxysuccinimide (NHS) ester molecule, which has excellent cell biocompatibility. By changing the degree of crosslinking, we can create scaffolds ranging in elastic moduli from 0.1 – 1.0 MPa. These protein scaffolds demonstrate several key features of the ECM, including elasticity and cellular adhesion. Unlike current natural and synthetic materials, the ligand density and elasticity can be engineered independently.Beating clusters of H9 hESC-CMs were plated on protein scaffolds (~1 x 108 ligands/cell area) and monitored for 10 days in culture. As a negative-control, the RGD sequence was scrambled (RDG) to create scaffolds with identical hydrophilicity, molecular weight, and mechanical properties. Viability of the hESC-CMs was maintained on both protein scaffolds for the length of the experiment. Phase contrast images show hESC-CMs cultured on the RGD peptide scaffold attach and are well-spread in comparison to the RDG scramble scaffolds, as well as contract rhythmically (http://microsystems.stanford.edu/~jblundo). The rate and amplitude of contraction of the hESC-CM clusters was captured using video microscopy and analyzed using edge detection software. Cell samples were fixed in 4% paraformalin and stained for DAPI nuclear marker. Immunofluorescence staining for smooth muscle actin and the cardiac specific marker, troponin, was used to analyze levels of cardiac expression in the culture. The mechanical properties of the scaffolds were measured by nanoindentation with atomic force microscopy (AFM). AFM probe data of protein scaffolds with identical RGD-ligand density and varying crosslinker density showed increasing crosslinker density yields a stiffer material.In conclusion, our results demonstrate molecular engineered RGD-elastin protein scaffolds are a viable matrix for hESC-CMs. Continued studies on the effect of scaffold stiffness and the contractility of hESC-CMs are planned.
12:15 PM - NN6.8
Micromechanical and FT-IR Spectroscopic Investigations on Films made of Recombinant Spider Silk Proteins and Silk Fibroin.
Frauke Junghans 1 , Thomas Scheibel 2 , Udo Conrad 3 , Andreas Heilmann 1 , Uwe Spohn 1
1 Biological Materials and Interfaces, Fraunhofer Institute for Mechanics of Materials, Halle (Saale) Germany, 2 Biotechnology, Technical University of Munich, Garching Germany, 3 Institute of Plant Genetics and Crop Plant Research, Leibniz Institute, Gatersleben Germany
Show AbstractFilms of recombinant spider silk proteins and native fibroins from silk worms were prepared by spincoating and casting of various solutions. The solubility of the recombinant spider silk-ELP fusion proteins isolated from transgenetic tobacco plants SO1-ELPx100 [1], of the recombinant spider silk proteins: (AQ)24NR3 and C16 produced in E.coli [2], and native fibroins from Bombyx mori and Antheraea pernyi was investigated in hexafluoroisopropanol, ionic liquids and concentrated salt solutions.The morphology and the thickness of the protein films were determined by the Atomic Force Microscopy (AFM) or by use of a profilometer. The silk protein films of (AQ)24NR3 and C16 exhibited a plain surface in contrast to SO1-ELPx100, and films of Bombyx mori silk and Antheraea pernyi silk. The aim was the preparation of films from the different silk fibroins, and the comparison of their mechanical properties. The micromechanical behavior was investigated by acoustic impedance analysis using a quartz crystal microbalance (QCMB) as well as by microindentation. Films < 350 nm showed an ideal elastic behavior in a range of 5 to 75 MHz with a linear correlation between the measured frequency shift and the thickness of the layers which simultaneously open a way to measure the density of the deposited layers. In this range of film thickness no significant increase of the half-band-half width dHBH corresponding to the dissipation of the excitation energy could be measured. At thicker films the half-band-half width increases considerably. Films with thicknesses > 350 nm show a typical viscoelastic behavior [3]. Since the essential influence of moisture on the mechanical behavior of all polyamide based materials is established, micromechanical parameters, e.g. the Martens hardness, the plastic and elastic part of the penetration work of a Vickers pyramid and the attenuation of the peak by the acoustic impedance analysis of all silk fibroin layers were measured in dependence on relative humidity and temperature. FT-IR-spectra showed no significant changes of proportion of the secondary protein structures: α- helices, β-sheets and random coils. By using the quartz microbalance the adsorption isotherm of water was measured, and it showed a langmuir isotherm up to relative humidity values of 70% followed by capillary condensation. Different water binding states can be identified. The Martens hardness decreased with increasing the relative humidity because the water acts as a plasticizer. [1] J. Scheller, U. Conrad, Biopolymers, 8, 81 (2001)[2] D. Huemmerich, U. Slotta, T. Scheibel, Applied Physics A, 82, 219 (2006)[3] F. Junghans, M. Morawietz, U. Conrad, T. Scheibel, A. Heilmann, U. Spohn, Applied Physics A, 82, 253 (2006)
12:30 PM - **NN6.9
Novel Bio-Sensors For Analyzing Substrates Of Biological Significance.
Itaru Hamachi 1 2
1 Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Katsura, Japan, 2 , PRESTO, Kyoto Japan
Show Abstract To understand complicated biological systems in molecular levels, it is important to evaluate the sort and amount of various biological substances in spatio- and time resolution manner. Useful chemo- or biosensors are greatly desirable as molecular tools to carry out detection and imaging of these analytes. In this paper, we describe our recent results on the development ofhybrid biosensors to sense substances of biological significance such as phosphorylated peptides and proteins and glucose. Artificial chemosensors consisting of Zn(II) complex that can bind and fluorescently sense phosphorylated peptides/proteins was successfully fused with a phosphoprotein binding domain to produce a new fluorescent biosensor. We also demonstrated that the engineered lectins, a sugar-binding protein, can act as a fluorescent glucose biosensor not only in test tube experiment, but also cell surface (and in cell). In addition, the immobilization of the fluorescent lectin in the self-assembled hydrogel matrix afforded a unique semi-wet lectin array for profiling saccharide derivatives. Details will be discussed in this talk. [1] Ojida, A. Mito-oka, Y. Inoue, M. Hamachi, I. J. Am. Chem. Soc., 124, 6256 (2002) [2] Ojida, A. Inoue, M. Mito-oka, Y. Hamachi, I. J. Am. Chem. Soc., 125, 10184 (2003) [3] Ojida, A. Mito-oka, Y. Sada, K. Hamachi, I. J. Am. Chem. Soc., 126, 2454 (2004) [4] Kiyonaka, S, Shinkai, S, Hamachi, I. Chem. Eur. J., 9, 976 (2003). [5] Kiyonaka, S. Sada, K. Yoshimura, I. Shinkai, S. Kato, N. Hamachi, I. Nature Materials. 3, 58 (2004) [6] Nakata, E. Koshi, Y. Katayama, Y. Hamachi, I. J. Am. Chem. Soc., 127, 13253 (2005) [7] Anai, T. Nakata, E. Koshi, Y. Ojida, A. Hamachi, I. J. Am. Chem. Soc., 129, 6232 (2007)