Alberto Saiani, University of Manchester
Dave Adams, University of Glasgow
Ayeesha Mujeeb, PeptiGelDesign
Darrin Pochan, University of Delaware
Manchester Biomaterials Network (ManBioMat)
University of Delaware
BM12.01: Session I
Monday PM, November 27, 2017
Sheraton, 2nd Floor, Constitution A
8:30 AM - BM12.01.01
Controlling the Assembly of Multicomponent Dipeptide Gelators
Dave Adams 1 , Emily Draper 1 Show Abstract
1 , University of Glasgow, Glasgow United Kingdom
Compared to single component systems, multicomponent low molecular weight gelators can be used to prepare gels with a higher degree of complexity and information content. It is however difficult to understand and control how the different components behave in the presence of one another. We have been examining the assembly of multicomponent dipeptide based gelators, focussing on using a pH trigger. This method has allowed us to prepare a range of different types of system. Here, we will discuss our work on two component systems and show examples of self-sorting, as well as co-assembly. We focus on controlling this assembly, and the effect of the different possibilities on the final properties of the gels.
8:45 AM - BM12.01.02
Using Short Peptide Hydrogels to Create Neural Networks
Adam Martin 1 2 , Yazi Ke 3 , Lars Ittner 3 , Pall Thordarson 1 Show Abstract
1 Chemistry, University of New South Wales, Sydney, New South Wales, Australia, 2 , Centre for Bio-Nano Science, Sydney, New South Wales, Australia, 3 Medicine, University of New South Wales, Sydney, New South Wales, Australia
Primary neuronal cultures are a powerful tool to understand neuronal maturation, aging and neurodegeneration. They have been used to screen the effects of drugs and misfolded proteins on neural networks in vitro. However, culturing primary neurons in vitro is notoriously difficult, owing to their high sensitivity to their environment. Currently, primary neurons are cultured on glass coverslips coated with poly-d-lysine (PdL). However, it is well known that significant differences exist in cell behaviour in a 2D versus 3D environment, which more accurately mimics in vivo conditions.
Hydrogels have significant potential biomedical applications, including in cell culture, owing to their chemical biocompatibility and physical structure, which is similar to the extracellular matrix found in the body. Short peptides capped at their N-terminus with an aromatic group have been used to form biocompatible, self-supporting hydrogels (Martin, 2016). Both capping group and amino acid sequence can be modified to yield hydrogels with tuneable stiffnesses, pore sizes and chemical functionalities.
Here, we present short peptide hydrogels which support the growth of primary neurons in a 2D or 3D environment. These peptides form self-assembled fibrillar structures when dissolved in water and can be coated onto glass coverslips or gelation triggered through addition of commonly used cell culture media. Neuronal development on peptide-coated coverslips is comparable to PdL and viable at >30 days in vitro, allowing synapse formation to be visualised. Preliminary viability of 3D neuronal cultures has been established. These hydrogels have potential in identifying neurodegenerative disease biomarkers, better screening of drug molecules, modelling CNS damage and insights into aging.
9:00 AM - *BM12.01.03
Peptide-Based Materials—The Design Challenge
Vincent Conticello 1 Show Abstract
1 , Emory University, Atlanta, Georgia, United States
Structurally defined materials on the nanometer length-scale have been historically the most challenging to rationally construct and the most difficult to structurally analyze. Sequence-specific biomolecules, i.e., proteins and nucleic acids, have advantages as design elements for construction of these types of nano-scale materials in that correlations can be drawn between sequence and higher order structure, potentially affording ordered assemblies in which functional properties can be controlled through the progression of structural hierarchy encoded at the molecular level. However, the predictable design of self-assembled structures requires precise structural control of the interfaces between peptide subunits (protomers). In contrast to the robustness of protein tertiary structure, quaternary structure has been postulated to be labile with respect to mutagenesis of residues located at the protein-protein interface. We have employed simple self-assembling peptide systems to interrogate the concept of designability of interfaces within the structural context of nanotubes and nanosheets. These peptide systems provide a framework for understanding how minor sequence changes in evolution can translate into very large changes in supramolecular structure, which provides significant evidence that the designability of protein interfaces is a critical consideration for control of supramolecular structure in self-assembling systems.
9:30 AM - BM12.01.04
Folding Driven Self-Assembly of a Stimuli-Responsive Peptide-Hyaluronan Hybrid Hydrogel
Robert Selegård 1 , Christopher Aronsson 1 , Caroline Brommesson 1 , Staffan Danmark 1 , Daniel Aili 1 Show Abstract
1 Department of Physics, Chemistry and Biology, Linköping University, Linköping Sweden
Protein-metal ion interactions are ubiquitous in nature and can be utilized for controlling the self-assembly of complex supramolecular architectures and materials. Peptides that folds as a result of metal ion coordination can be design de novo and offers a robust alternative to proteins as structural and functional components in responsive soft materials. Conjugation of a responsive peptide to a polymeric backbone can result in a hybrid material with novel properties. Numerous peptide-polymer hybrids that self-assemble into supramolecular hydrogels have been demonstrated, but typically offer limited possibilities to tune the self-assembly process.
Here, a fully tunable supramolecular hydrogel is described, obtained by conjugating Zn2+-responsive peptides to hyaluronic acid using strain promoted click chemistry. Addition of Zn2+ triggers folding of the peptides into a helix-loop-helix motif and dimerization into four-helix bundles, resulting in self-assembly of a very sticky soft hydrogel. The self-assembly process and the rheological properties of the hydrogel can be tuned by the Zn2+ concentration. The peptide-hyaluronan hybrid hydrogel show typical characteristics of supramolecular hydrogels, such as self-heling and shear-thinning properties. Addition of metal ion chelators leads to unfolding of the peptide component and rapid disassembly of the hydrogels. Moreover, hydrogel degradation can also be time-programed by encapsulation of a hydrolyzing enzyme within the gel, offering multiple possibilities for modulating material properties and release of encapsulated species. The hydrogel further shows potential antioxidant properties when evaluated using an in vitro model for reactive oxygen species.
9:45 AM - BM12.01.05
Cellulose Nanofiber-Templated Silk Fibroin Shish-kebab Nanostructure with Exceptional Mechanical Robustness and Ultrafast Water Transport
Rui Xiong 1 , Shuaidi Zhang 1 , Vladimir Tsukruk 1 Show Abstract
1 , Georgia Institute of Technology, Atlanta, Georgia, United States
The construction of multi-length scaled hierarchical nanostructures from diverse natural components is critical in the progress towards all-natural nanocomposites with structural robustness and versatile added functionalities. Here, we report a surprising spontaneous formation of peculiar shish kebab nanostructures from silk fibroin assembled in a periodic manner with nanoscale spacing along straight segments of cellulose nanofibers. We suggest that the formation of these unique organized shish-kebab nanostructures is facilitated by the preferential organization of heterogeneous silk domains along the crystalline cellulose nanofibers driven by heterogeneous axial distribution of crystalline planes, hydrogen bonding and hydrophobic interactions of cellulose nanofiber surfaces. Such shish kebab nanostructures enable the ultrathin membrane to possess open, transparent, mechanically-robust interlocked networks with high mechanical strength that facilitates their freely standing state. These nanoporous robust membranes allows for the extremely high water flux, up to 3.5 ×104 L h−1m−2bar−1 combined with good rejection rate for various organic molecules and 100% blockage of 5 nm gold nanoparticles. Moreover, these membranes have capability of capturing various heavy metal ions from solution and their further reduction into metal nanoparticles with added colorimetric sensing functionalities.
10:30 AM - BM12.01.06
Nanotape-to-Cochleate Structural Transformations in Peptide Amphiphile Assemblies
Changrui Gao 1 , Sumit Kelwaramani 1 , Honghao Li 1 , Monica Olvera de la Cruz 1 , Michael Bedzyk 1 Show Abstract
1 , Northwestern University, Evanston, Illinois, United States
Cochleate, a large bilayer sheet rolled up into spiral structure, is a drug delivery system that is being extensively studied because of its high drug capacity. However, little is known about the structural transition from flat bilayer to rolled-up cochleate. Here, we use in-situ X-ray scattering, liquid atomic force microscopy and cryogenic transmission electron microscopy to examine the case of a positively charged peptide amphiphile (monolysine palmitate, C16-K1) molecule that self-assembles into a flat crystalline bilayer of high aspect ratio (nanotapes) in salt-free aqueous solutions. Our experimental studies yield three key insights on the structural transformation of nanotapes in the presence of salts: 1. As the salt concentration (C) increases, high aspect-ratio nanotapes convert to large low-aspect ratio nanosheets prior to rolling into cochleates. 2. The inter-bilayer spacing in the cochleate structure is proportional to the Debye length (C -1/2). The tunable interlayer spacing in the cochleates could faciliate the delivery of drug particles with specific sizes. 3. The molecules in nanotapes are packed on crystal lattices with the aliphatic tails of the two leaflets interdigitated. The crystallinity decreases upon cochleate formation. Theoretical simulations indicate that the cochleate formation are connected to change of intermolecular electrostatic interaction and membrane fluidity. Up until now, cochleate structures have been studied largely in phospholipid systems. Our studies, which represent the first example of cochleate formations in PA, suggest that the cochleate formation in the presence of salts maybe a general feature of charged amphiphilic bilayer assemblies.
10:45 AM - BM12.01.07
Genetically Encoded Lipid-Polypeptide Hybrid Biomaterials that Exhibit Temperature Triggered Hierarchical Self-Assembly
Davoud Mozhdehi 1 , Kelli Luginbuhl 1 , Ashutosh Chilkoti 1 Show Abstract
1 Biomedical Engineering, Duke University, Durham, North Carolina, United States
Post-translational modification (PTM) of proteins is a common strategy employed in biological systems to expand the diversity of the proteome and to tailor the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the use of PTMs to synthesize hybrid biomaterials is still untapped. As a proof-of-concept to demonstrate the feasibility of creating a genetically encoded biohybrid material via PTM, herein we report the first example of a post-translationally modified, stimulus responsive hybrid material—fatty acid-modified elastin-like polypeptides—by a one-pot recombinant expression and post-translational lipidation methodology. These new hybrid biomaterials are thermally responsive and exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be tuned at the sequence level. The extrinsic control over self-assembly conferred by their stimulus responsiveness make them attractive for a range of applications —especially as injectable biomaterials— as it provides the ability to trigger self-assembly on demand.
11:00 AM - *BM12.01.08
Self-Assembled Peptides on Surfaces—Green and Biocompatible Coating that Resists Biofouling
Meital Reches 1 2 Show Abstract
1 Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem Israel, 2 The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem Israel
Biofouling is an undesirable process in which a surface becomes encrusted with organisms and their by-products. This unwanted colonization has a serious impact on marine devices, as it lead to deterioration of the surfaces and can alter fluid flow rates leading to significant increase in cost of marine transportation. In the healthcare system, the attachment of bacteria and biofilm formation on medical devices may lead to severe infections and consequently death. In the US alone, the Center of Disease Control and Prevention (CDC) reported that healthcare-associated infections account for an estimated 1.7 million infections and 100,000 deaths annually.
Many approaches to prevent biofouling have been suggested, however, they suffer from drawbacks such as release of toxic materials to the surroundings, low stability that limits their long-term application or complex and expensive synthesis.
We have designed a short peptide (tripeptide) that can spontaneously form a coating that resists biofilm formation. The peptide contains three elements that enable i) its self-assembly, ii) its adsorption onto any substrate and iii) its antifouling activity. Our results clearly demonstrate the formation of a coating on various surfaces (glass, titanium, silicon oxide, metals and polymers). In addition, we showed that this coating prevents the first step of antifouling, which involves the adsorption of bioorganic molecules to the substrate. Moreover, the coating significantly reduces the attachment of various organisms such as bacteria and fungi to surfaces.
11:30 AM - BM12.01.09
Towards Imaging Peptoid Nanosheets on the Atomic Scale
Xi Jiang 1 , Douglas Greer 1 2 , Joyjit Kundu 3 , David Prendergast 3 , Ronald Zuckermann 3 , Kenneth Downing 4 , Nitash Balsara 1 2 Show Abstract
1 Materials Sciences, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 College of Chemistry, University of California, Berkeley, Berkeley, California, United States, 3 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 4 Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California, United States
Most high-resolution imaging experiments are carried out on hard materials composed of heavy elements arranged on well-defined lattices with binding energies much larger than thermal energy. Obtaining electron micrographs with atomic resolution in these systems is now commonplace. There are three characteristics that help atomic resolution: the atoms are arranged on a small well-defined lattice, the atoms at a given site in different lattices superpose, and the atoms are generally heavy and stable in the electron beam. In recent years, there has been significant progress in the field of protein imaging by electron microscopy. Proteins can be thought of as well-ordered individual particles wherein the location of atoms within the particle is exactly reproduced from one particle to the next. Unlike hard crystalline materials, however, the atoms of interest are generally light and thus unstable in the electron beam. While this generally limits exposure to about 10 e/Å2 and thus produces very noisy images, the assumption that all particles in the field of view are identical enables the application of sophisticated averaging algorithms which have resulted in determining protein structure with atomic resolution. Imaging synthetic soft materials on the atomic scale is significantly more challenging than the two examples described above. In the present study, 2D crystalline monolayer nanosheets formed of polypeptoid block copolymers in water have been imaged by using cryogenic transmission electron microscopy (Cryo-EM) in their frozen hydrated state in order to preserve the native structures. While one can readily resolve stripes with a spacing of 24 Å, the steps toward obtaining atomic resolution (of order a few Å) are non-trivial but necessary in order to explore the principles for molecular packing in ordered soft materials. The merged Cryo-EM images showed that there was information in the two-dimensional (2D) density map close to 2 Å resolution so that both the backbone and side chains can be directly observed. Our preliminary three-dimensional (3D) density map revealed the orientation and the conformation of polypeptoid chains in the nanosheets. It should be evident that successful determination of atomic positions in the model peptoid nanosheets that have been chosen will immediately enable high resolution imaging of a variety of soft materials and will continue to discover a new design principles in supramolecular assembly.
11:45 AM - BM12.01.10
Designing Amphiphilic Peptoids for Bio-Inspired Synthesis of Functional Nanomaterials
Chun-Long Chen 1 Show Abstract
1 , Pacific Northwest National Laboratory, Richland, Washington, United States
In nature, assembly of functional molecules (e.g. proteins and peptides) into molecular machines that carry out the vast array of functions is a commonplace phenomenon. Inspired by nature, assembly process has been widely used for synthesis of protein- and peptide-based functional materials. However, proteins and peptides exhibit so complex folding capabilities due to the formation of both intra- and inter-sequence hydrogen bonds, the prediction of their structures and assembly is still extremely difficult. Furthermore, the application of protein- and peptide-based materials is limited because of their poor stabilities against thermal and chemical degradation. Consequently, a wide range of sequence-defined synthetic molecules (e.g., β-peptides and peptoids) have been developed as mimetics of proteins and peptides.
In this presentation, I will report my group’s studies in designing amphiphilic peptoids for bio-inspired synthesis of functional nanomaterials (e.g. membrane-mimetic 2D nanomaterials,1,2 nanotubes, hydrogels and pore-forming networks3,4). Our results show that peptoid-peptoid and peptoid-substrate interactions play critical roles in the peptoid assembly and can be tuned through the peptoid side-chain chemistry. Supramolecular interactions (e.g. hydrogen bonds, π-π stacking or coordination bonds) among peptoid side chains are the main driving forces that lead to the formation and stabilization of highly-ordered superstructures. We further demonstrated that various functional groups could be incorporated into these peptoid-based nanomaterials through the peptoid side-chain diversity, indicating that assembly of amphiphilic peptoids into hierarchical structures can be used as a robust platform to develop biomimetic materials with tunable structures and controllable functions.
1 Jin, H. et al. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids. Nat. Commun., 12252, doi:10.1038/ncomms12252 (2016).
2 Jiao, F. et al. Self-repair and patterning of 2D membrane-like peptoid materials. Adv. Funct. Mater., 8960-8967, doi:10.1002/adfm.201602365 (2016).
3 Ma, X. et al. Tuning crystallization pathways through sequence engineering of biomimetic polymers. Nat. Mater., doi:10.1038/nmat4891 (2017).
4 Chen, C. L., Zuckermann, R. N. & DeYoreo, J. J. Surface-Directed Assembly of Sequence Defined Synthetic Polymers into Networks of Hexagonally Patterned Nanoribbons with Controlled Functionalities. ACS Nano 10, 5314-5320, doi:10.1021/acsnano.6b01333 (2016).
BM12.02: Session II
Monday PM, November 27, 2017
Sheraton, 2nd Floor, Constitution A
1:30 PM - BM12.02.01
Structural Characterization of Peptide Analogs Using Solid-State NMR
Ben Hudson 1 , Alessia Battigelli 2 , Nikola Dudukovic 3 , Ronald Zuckermann 2 , Anant Paravastu 1 Show Abstract
1 , Georgia Institute of Technology, Atlanta, Georgia, United States, 2 , Lawrence Berkeley National Laboratory, Berkeley, California, United States, 3 , Lawrence Livermore National Laboratory, Livermore, California, United States
We are applying solid-state NMR based structural techniques developed for self-assembling peptides to the self-assembling peptide analogs peptoid B28 and fluorenylmethoxycarbonyl-diphenylalanine (FMOC-FF). Peptoid B28 self-assembles into nanosheets and is characterized by alternating hydrophobic and hydrophilic sidechains that, unlike peptides, are connected to backbone N atoms. FMOC-FF can reversibly self-assemble into nanocrystals or nanofibers and is composed of a dipeptide connected to a bulky aromatic group. Using solid-state NMR spectroscopy, we have discovered that peptide analogs can exhibit molecular level phenomena with interesting similarities and differences to behavior previously documented in self-assembling peptides.
We have employed 13C-13C dipolar recoupling NMR measurements to evaluate backbone conformations of peptoid B28 molecules before and after assembly into nanosheets. While the alternating hydrophobic/hydrophilic sequence of B28 sidechains was inspired by β-strand forming self-assembling peptides, our results show that B28 molecules in nanosheets are not in conformations that are analogous to β-strands. Motivated by previous theoretical predictions that peptoids are characterized by lower energy differences between trans and cis backbone amide bond conformations, we measured distances between adjacent α-carbon atoms. Our results support the theoretical predictions that suggest the trans configuration is favored in peptoids as it is in peptides. Remarkably, however, we discovered that self-assembly affects the distribution of cis and trans backbone amide bonds along the peptoid backbone. Nevertheless, we will discuss new computational modeling that shows that extended, amphiphilic peptoid conformations are still possible with cis-amide bond configurations.
We have also investigated self-assembly of FMOC-FF in different solvent environments. This system exhibits polymorphism that is reminiscent of self-assembling peptides, resulting in formation of nanocrystals or nanofibers, depending on solvent, concentration, and temperature. Reversibility of FMOC-FF self-assembly makes it possible to produce phase diagrams for nano-scale assembly. Nevertheless, solid-state NMR spectra indicate that underlying molecular structures may be kinetically trapped. NMR data for nanofibers formed in 5% DMSO (percentage volume in water) support a previous proposal that molecules are arranged into configurations that resemble anti-parallel β-sheets and are not consistent with a previously proposed disordered micelle-like structure. Taken together, these observations motivate the interpretation that FMOC-FF self-assembly behavior may present opportunities to better understand self-assembly of peptides.
1:45 PM - BM12.02.02
Hierarchical Assembly of Coiled-Coil Peptides via Click Conjugation
Chris Kloxin 1 Show Abstract
1 , University of Delaware, Newark, Delaware, United States
Protein structures are built upon simple canonical structures, such as alpha helices and beta sheets, which are formed through specific interactions dictated by the amino acid sequence. Once folded into a helical or beta sheet object, amino acid side chains are displayed at the periphery to stabilize the assembly and/or interact with other protein sequences. This precise display of chemistry from these peptidic objects can be view as functional building blocks from which complex, hierarchical structures are built. Utilizing computational modeling, non-natural amino acids are readily incorporated into the peptide sequence capable of forming specific peptide objects with unique functionality. Here, we implement computational modeling to generate non-biological peptide sequences that assemble into coiled-coil bundles. Within the amino acid sequence, substitutions of non-natural amino acids baring click functional groups are made at precise locations along the coiled-coil bundle. The synthesis and implementation of non-natural click amino acids provides control over structural formation and stability, allowing us to explore unique path-dependent macromolecular assemblies. Moreover, we demonstrate several fundamental coiled-coil assembles that enables us to create a host of unique responsive hierarchical nanostructures not possible through any other method.
2:00 PM - *BM12.02.03
Self-Assembly of Prodrugs into Supramolecular Medicine
Honggang Cui 1 Show Abstract
1 , Johns Hopkins University, Baltimore, Maryland, United States
Drugs are a special class of chemical substances that can produce a biological effect when administered to a living organism. In medical treatments, drugs have been almost exclusively considered as the functional cargo to be delivered, and their potential as molecular building blocks has been largely ignored. In this work, we report the use of drugs as effective molecular building units to create well-defined supramolecular nanostructures that contain a 100% drug loading for systemic delivery, as well as supramolecular hydrogels for local treatment of human diseases. We show that molecular design, assembly conditions and kinetic pathways are all critical factors that govern the resultant nanostructures and consequently their interfacing with cells. These findings have led us to believe that rational self-assembly of drugs into supramolecular nanostructures and materials not only offers an innovative way to craft drug-bearing nanomaterials, but also extends the functional space of molecular assembly into a new area which is specifically focused on the direct assembly of therapeutic molecules into supramolecular architectures with enhanced functionalities.
2:30 PM - BM12.02.04
Collagen-Inspired Design Rules for the Self-Assembly of Twisted Filaments
Martin Falk 1 , Michael Brenner 2 , Amy Duwel 3 , Lucy Colwell 4 Show Abstract
1 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 , Harvard University, Cambridge, Massachusetts, United States, 3 , Draper Laboratory, Cambridge, Massachusetts, United States, 4 , University of Cambridge, Cambridge United Kingdom
Despite dramatic developments in the capacity to engineer specific interactions and structures in synthetic systems, it is clear that there is more to be done in understanding the design rules by which nature forms structure from complex substrates. Here, we focus specifically on the challenge of self-assembling nanoscale twisted filaments; such structures, known as Litz wire, find engineering application in the transmission of alternating current. However, studies of self-assembly through specific interactions have primarily confined their attention to colloids, and so we instead turn to nature for inspiration on how to proceed. One of the most prominent examples of a natural triple helix is the protein collagen, which is comprised of three peptides twisted together through a periodic array of hydrogen bonds. Though the sequence and structure of collagen is known, an identification of the most important features for producing collagen as nature does has remained elusive. Through simulation, we propose a successful scheme for the self-assembly of three-twists mediated by specific interactions. We find that the handedness of the twist decays with a correlation length, the result of defects involving the switching of two filaments. We explore the effect of the material properties on this length, as well as the effect an additional of a chiral term to our scheme. With this chiral modification, we find that we are able to form twists of one handedness. Thus we identify design rules and obstacles for the self-assembly of twists, and strategies that natural collagen may be using to overcome those problems.
2:45 PM - BM12.02.05
Engineering MultiDomain Peptides for Controlled Release and Anisotropic Applications in Bioregeneration
I-Che Li 1 , Jeffrey Hartgerink 1 2 Show Abstract
1 Chemistry, Rice University, Houston, Texas, United States, 2 Bioengineering, Rice University, Houston, Texas, United States
A variety of polymeric hydrogels that mimic the native extracellular matrix have been used in tissue engineering strategies. Among all the materials, self-assembling hydrogels have shown unique physical properties, including injectability and shear recovery, as promising candidates for biomedical applications. Due to its highly compliant criteria for sequence selection, peptide hydrogels have been widely developed for controlled small molecule release, protein delivery, and cell encapsulation. As a type of peptide hydrogel, MultiDomain Peptides (MDPs) respond to external shearing forces and have reversible self-assembly under mild conditions. In addition, their high biocompatibility make them important candidates for regenerative strategies. In our recent works, we modify the MDP sequence to achieve controlled drug release and macroscopic anisotropy.
In the first part of the presentation, we design a novel “missing tooth” self-assembled material to deliver small molecules with low water solubility controllably. This is accomplished by modifying the hydrophobic interior of the MDP to construct hollow fibers for the encapsulation of anti-cancer drugs, antibiotics, or nonsteroidal anti-inflammatory drugs. Our characterization demonstrates that the gels exhibit intrafibrillar encapsulation and controlled release of hydrophobic drugs. With this design, the MDP hydrogel can be used as an effective carrier of small molecules with low water solubility.
In the second part, we modify the hydrophilic exterior of the MDP to achieve organized self-assembly into parallel aligned fiber bundles. With the help of shearing forces of syringe extrusion and the incorporation of the amino acid DOPA (3,4-dihydroxyphenylalanine), the self-assembled nanofibers form an anisotropic hydrogel string under modest shear stress. The long-range order of aligned fiber formation is revealed by the remarkable birefringence of the hydrogel string along with the highly aligned nanofibers observed by scanning electronic microscopy.
Combining the designs above, in this presentation I will describe our current work applying this novel material to bioregenerative applications that takes advantage of its ability for controlled drug release and unique anisotropy.
3:30 PM - *BM12.02.06
Modular Design of β-Peptide-Based Materials for Neuroregeneration
Mibel Aguilar 1 , Mark Del Borgo 1 , Sepideh Motamed 1 , Ketav Kulkarni 1 , Tobias Merson 1 , David Finkelstein 2 , John Forsythe 1 Show Abstract
1 , Monash University, Clayton, New South Wales, Australia, 2 , Florey Institute of Neuroscience and Mental Health, Clayton, Victoria, Australia
Peptide self-assembled systems offer significant advantages including biological compatibility, ease of synthesis, low toxicity and functionalisability. However, the control over essential features such as chemical, structural and metabolic stability and relatively slow rate of self-assembly remain significant challenges. β-amino acids contain an extra methylene in the backbone compared to α-amino acids and peptides comprised entirely of β-amino acids, called β-peptides, spontaneously form 14-helical structures. We have designed helical N-acetyl- β3-peptides that spontaneously undergo supramolecular self-assembly to form fibers. The peptide monomers self-assemble in a unique head-to-tail fashion which is driven by a 3-point H-bond motif associated with the 14-helical structure of N-acetyl- β3-peptides. In addition, the unique helical structure of the peptide monomer, irrespective of amino acid sequence, offers the opportunity to introduce a wide variety of functions to the new fibers based on straightforward modification of the side chains of the component amino acids. Furthermore, the pitch of the helix is almost exactly three amino acids, resulting in a geometrically defined position of side chain functionalities. These materials are also resistant to proteolytic degradation further adding to their potential as novel biomaterials. Application of these peptide materials to stem cell proliferation and differentiation will be discussed. In addition, we have engineered these peptides to form injectable hydrogels that facilitate migration of large numbers of neuroblasts along the length of an implant in transgenic mice in which neuroblasts are fluorescently labelled, allowing fate mapping and an assessment of the degree of integration.
4:00 PM - BM12.02.07
Tuning Biomolecular Assembly with Cation-π Interactions
Matthew Gebbie 1 , J. Herbert Waite 2 , Jacob Israelachvili 2 Show Abstract
1 , Stanford University, Stanford, California, United States, 2 , University of California, Santa Barbara, Santa Barbara, California, United States
Cation–π interactions drive the self-assembly and cohesion of many biological molecules, including the adhesion proteins of marine organisms. Cation–π interactions are also implicated in neurodegenerative protein misfolding disorders. Although the origin of cation–π bonds in isolated pairs has been extensively studied, the energetics of cation–π-driven self-assembly in biomolecular films remains uncharted. Here we use nanoscale molecular force spectroscopy in combination with solid-state NMR measurements to show that the adhesive properties of simple aromatic and lysine-rich peptides rival those of the adhesion proteins of the marine mussel. In particular, we find that peptides incorporating the amino acid phenylalanine, a functional group that is conspicuously sparing in mussel proteins, exhibit adhesion significantly exceeding that of analogous mussel-mimetic peptides. Notably, lysine and phenylalanine are implicated as structure-directing residues for the formation of amyloid fibrils, so these results suggest that strong cation-π interactions may play a role in neurodegenerative disease. More broadly, we demonstrate that interfacial confinement fundamentally alters the energetics of cation–π-mediated assembly: an insight that should prove relevant for diverse areas, from rationalizing biological assembly to engineering peptide-based biomaterials.
4:15 PM - BM12.02.08
A Computational-Experimental Approach to Understand Charge-Complementary Peptide Co-Assembly
Gregory Hudalla 1 , Dillon Seroski 1 , Kong Wong 2 , Qing Shao 3 , Carol Hall 3 , Anant Paravastu 2 Show Abstract
1 , University of Florida, Gainesville, Florida, United States, 2 , Georgia Institute of Technology, Atlanta, Georgia, United States, 3 , North Carolina State University, Raleigh, North Carolina, United States
Peptides that co-assemble into beta-sheet nanofibers yet cannot self-assemble afford new opportunities to develop supramolecular biomaterials with user-controlled assembly properties. We and others recently demonstrated that electrostatic repulsion and charge complementarity can be used to design peptide pairs that selectively co-assemble yet resist self-assembly. However, detailed understanding of the influence of peptide net charge on both the propensity for self- versus co-assembly and the structure of beta-strands in the resultant nanofibers remains limited. Here we report a computational-experimental framework to characterize relationships between peptide sequence design and peptide self- versus co-assembly propensity that combines molecular modeling, biophysical characterization, and solid-state NMR structural analysis. Specifically, we synthesized a set of peptides, referred to as CATCH(X+) or CATCH(X-), having the general primary sequence (C/Q)-Q-(C/Q)-F-(C/Q)-F-(C/Q)-F-(C/Q)-Q-(C/Q), where X denotes net charge at neutral pH and C denotes either E or K. Secondary structure prediction using PRIME20 suggested that CATCH(4-), CATCH(6-), and CATCH(6+) would be resistant to self-assembly, likely due to strong electrostatic repulsion. Interestingly, CATCH(4+) was predicted to have a weak propensity for self-assembly, despite its relatively high net charge. Mixtures of all oppositely-charged CATCH pairs were predicted to co-assemble into beta-sheet rich structures, with CATCH(6+/4-) predicted to co-assemble into a strictly alternating +/-/+/- arrangement. Consistent with these predictions, all oppositely-charged CATCH peptide pairs significantly increased the fluorescence intensity of Thioflavin T (ThT) when combined in neutral aqueous buffer, while CATCH(4+) elicited a weak increase in ThT fluorescence, and all other CATCH peptides elicited no increase above baseline. All oppositely-charged CATCH pairs adopted a secondary structure rich in beta-sheets, as determined via FTIR, although the beta-sheet content was highest for CATCH(6+/4-). Elongated fibrils with widths ~5 nm were observed for all oppositely-charged CATCH pairs visualized using transmission electron microscopy, whereas no fibrils were observed in samples of any CATCH peptide alone. At concentrations greater than or equal to 5 mM, all oppositely-charged CATCH peptide pairs assembled into self-supporting hydrogels. Solid-state NMR of CATCH(6+/4-) hydrogels suggested that the peptides co-assembled into beta-sheet nanofibers with a strictly alternating +/-/+/- arrangement, consistent with PRIME20 predictions. Together, these data demonstrate that our computational-experimental framework of molecular modeling, biophysical characterization, and solid-state NMR structural analysis can provide critical insights into peptide self- versus co-assembly propensity that will be useful for establishing sequence design criteria for this emerging class of supramolecular biomaterials.
4:30 PM - *BM12.02.09
Engineering Peptide Hydrogels—From Material Properties to Functionalisation
Aline Miller 1 Show Abstract
1 , University of Manchester, Manchester United Kingdom
The hydrogelation of self-assembling peptides involves two processes: 1. self-assembly of the peptides into fibres and 2. self-assembly of the fibres into 3-dimentional percolated networks. While significant efforts have been dedicated to elucidate the self-assembly of short peptides into fibres, the self-assembly of the fibres into networks has been little investigated despite the fact that network topology is a key factor influencing the final properties of peptide hydrogels. We have investigated how to engineering the properties of β-sheet forming peptide hydrogels by manipulating by design the topology of the fibrillar network formed. By changing the type and nature of the residues on the peptides we were able to control the topology of the network formed and fine tune the shear thinning and mechanical properties of the hydrogels. This was achieved by using a number of strategies from modifying one hydrophobic residue in the core of the fibre to design highly kinked fibres, which association and aggregation tendency are reduced resulting in weaker hydrogels, to inserting highly interacting hydrophilic residues on the surface of the fibres that promote strong fibre-fibre aggregation resulting in stronger hydrogels Here several different strategies developed in our group will be outlined for gaining fine control over the material properties, in addition to an outline of the strategies learned for the fabrication of functional, responsive and active peptide materials. Several examples of the types of functionalities that can be incorporated will be discussed, in relation to their wide range of application areas which include controlling cell culture, targeted and temporal release of therapeutics, biosensors and biocatalysis for fine chemical manufacturing.
BM12.03: Poster Session
Tuesday AM, November 28, 2017
Hynes, Level 1, Hall B
8:00 PM - BM12.03.02
Monitoring and Modulating Ion Traffic in Hybrid Lipid/Polymer Vesicles
Walter Paxton 1 2 , Sun Hae Ra Shin 1 2 , Patrick McAninch 1 2 , Komandoor Achyuthan 1 Show Abstract
1 , Sandia National Laboratories, Albuquerque, New Mexico, United States, 2 , Center for Integrated Nanotechnologies, Albuquerque, New Mexico, United States
Controlling the traffic of molecules and ions across membranes is a critical feature in a number of biologically relevant processes and highly desirable for the development of technologies based on membrane materials. In this paper, ion transport behavior of hybrid lipid/polymer membranes was studied in the absence and presence of ion transfer agents. A pH-sensitive fluorophore was used to investigate ion (H+/OH-) permeability across hybrid lipid/polymer membranes as a function of the fraction of amphiphilic block copolymer. It was observed that vesicles with intermediate lipid/polymer ratios tend to be surprisingly more permeable to ion transport than the pure lipid or pure polymer vesicles. Hybrid vesicle permeability could be further modulated with valinomycin, nigericin, or gramicidin A, which significantly expedite the dissipation of externally-imposed pH gradients by facilitating the transport of the rate-limiting co-ion (e.g. K+) ions across the membrane. For gramicidin A, ion permeability decreased with increasing polymer mole fraction, and the method of introduction of gramicidin A into the membrane played an important role. Strategies to incorporate biofunctional molecules and facilitate their activity in synthetic systems are highly desirable for developing artificial organelles or other synthetic compartmentalized structures requiring control over molecular traffic across biomimetic membranes.
8:00 PM - BM12.03.03
Flow-Induced Shape Reconfiguration, Phase Separation and Rupture of Bio-Inspired Vesicles
Xiaolei Chu 1 , Xiang Yu 1 , Joseph Greenstein 1 , Fikret Aydin 1 , Geetartha Uppaladadium 1 , Meenakshi Dutt 1 Show Abstract
1 , Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States
The structural integrity of red blood cells and drug delivery carriers through blood vessels is dependent upon their ability to adapt their shape during their transportation. Our goal is to examine the role of the composition of bioinspired multicomponent and hairy vesicles on their shape during their transport through in a channel. Via the Dissipative Particle Dynamics simulation technique, we apply Poiseuille flow in a cylindrical channel. We investigate the effect of flow conditions and concentration of key molecular components on the shape, phase separation and structural integrity of the bioinspired multicomponent and hairy vesicles. Our results show the Reynolds number and molecular composition of the vesicles to impact their flow-induced deformation, phase separation on the outer monolayer due to the Marangoni effect and rupture. The findings from this study could be used to enhance the design of drug delivery and tissue engineering systems.
8:00 PM - BM12.03.04
Sugar-Responsive Lipid Bilayer Membrane—Regarding to Response Mechanism
Yusuke Nezu 1 , Tatsuo Aikawa 1 , Takeshi Kondo 1 2 , Makoto Yuasa 1 2 Show Abstract
1 Department of Pure and Applied Chemistry, Tokyo University of Science, Noda Japan, 2 Research Institute for Science and Technology, Tokyo University of Science, Noda Japan
In this research, we propose a lipid, DPBA, having two long hydrocarbon chains, ammonium group adjacent to the hydrocarbon chain and boronic acid unit placing in the terminus. Having their large hydrophobic groups allows DPBA to form bilayer structure, which can include various types of hydrophilic and hydrophobic molecules and disperse them in water. These lipid bilayers are known to assemble to various types of higher order structures such as vesicles and lamellar sheet. Such feature should enhance applicability of DPBA in biomedical field (i.e, drug carrier). Boronic acid can form a reversible covalent bond with a diol-compound such as sugars in water. Therefore, DPBA is expected to constitute sugar-responsive lipid bilayer. The purpose of this research is to create new functional lipid bilayer, which can control the release of included molecule by changing bilayer structure through addition in sugar. In the presentation, we will discuss the changes in interaction between the lipids in response to addition of sugars.
Lipids used in this study were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-aza boronic acid (DPBA), and their equimolar mixture (DPPC:DPBA = 1:1). Behavior of the lipid molecular assembles in response to sugars, D-sorbitol, D-fructose, or D-glucose, was investigated by differential scanning calorimetry (DSC) and pressure-area (π-A) isotherm measurement. Association constants (Keq) between boronic acid and the sugars, D-sorbitol, D-fructose and D-glucose, were 370, 160, and 4.6, respectively. Gel-to-liquid crystal phase transition temperature (Tc) for the samples was determined from the obtained DSC thermograms. The intermolecular interaction between the lipids with the sugars was examined by π-A isotherm measurement.
Result and Discussion
In preliminary experiment, a cryo-transmission electron microscopy has revealed that DPBA could form vesicles in water. In DSC measurement of DPBA aqueous dispersion, observation of endothermic peak that should be derived from gel-to-liquid crystal phase transition indicates that DPBA vesicles are constituted by lipid bilayers. In the presence of sugars, Tc of lipid dispersion including DPBA changed depending on Keq. For pure DPBA system, the TC increased with increasing Keq value. Such clear dependence of the Tc in response to Keq of the sugar indicates that sugars are bound to the boronic acid headgroups and enhanced intermolecular interaction between the headgroups via hydrogen bonding of the hydroxyl groups in the sugars. From the result of π - A curve, it was revealed that intermolecular interaction occurred with a wider molecule occupied area in the group with the larger Keq. These results suggest that the recognition of lipid molecules was accelerated by the binding of sugar with a large volume to DPBA. Results in this study clearly demonstrate a sugar-response potential of lipid bilayer composed of DPBA.
8:00 PM - BM12.03.05
Cell-Instructive Biointerfaces Using Supported Lipid Bilayers
Gulistan Kocer 1 , Mark Verheijden 1 , Pascal Jonkheijm 1 Show Abstract
1 , University of Twente, Enschede Netherlands
Supported lipid bilayers (SLBs) serve as supramolecular lipid architectures with cell membrane mimetic properties that preserve the physicochemical properties of the cell membrane (i.e. phase behavior). SLB formation on hydrophilic supports using lipids with zwitterionic head groups allows to generate non-fouling surfaces and to incorporate specific ligands for molecular recognition of cell membrane components. Interestingly, the lateral dynamics of ligand presentation on SLBs can be modulated due to the phase behavior of the base lipids (i.e. gel vs. fluid SLBs presenting immobile and mobile ligands, respectively). Furthermore, SLBs could be functionalized following multiple bio-conjugation routes (e.g. using biotin-avidin interactions and lipidated peptides) giving the freedom to modulate ligand presentation on these biointerfaces. Here, SLBs were functionalized with Arg-Gly-Asp (RGD) ligands derived from the extracellular matrix (ECM) to target integrin receptors on the cell surface. These RGD-functionalized SLBs were evaluated as dynamic biomimetic stem cell microenvironments. Firstly, by using biotin-neutravidin interactions to introduce the RGD ligands (i.e. biotinylated RGD) on SLBs (DOPC (fluid) vs. DPPC (gel) SLBs), ligand density and mobility dependent human mesenchymal stem cell (hMSC) adhesion, spreading and osteogenic differentiation capacity were studied and quantified. Immunofluorescence analyses on integrin signaling showed that cell adhesion occurred specifically on RGD presenting SLBs. Furthermore, the extent of cell adhesion and spreading showed a strong correlation to ligand density and mobility with effectively spreading cells on mobile SLBs presenting high ligand densities. Interestingly, osteogenic differentiation capacity showed a similar trend to cell spreading with a higher expression of osteogenic marker alkaline phosphatase on mobile and high density ligand presenting SLBs. Secondly, to further tune the ligand presentation to cells, lipid modified RGD peptides with varying lipid length were introduced to DOPC SLBs via direct membrane insertion as a different mode of conjugation where this variation was related to cell adhesion and spreading. Lipidated peptide insertion in SLBs was studied using quartz crystal microbalance with dissipation monitoring (QCM-D) coupled to spectroscopic ellipsometry. Investigations on integrin signaling and cell spreading revealed significant regulation of cell adhesion and spreading on SLBs presenting lipidated peptides in response to lipid tail length, pointing out an optimum tail length to achieve effective cell adhesion and spreading which are critical regulators of stem cell fate. In conclusion, SLB-derived biointerfaces endow materials with more precise control over the composition, lateral mobility, density and mode of presentation of the ligands on the surface. This makes them versatile platforms to instruct cell fate and to use them as new biomaterial coatings for future studies.
8:00 PM - BM12.03.06
Tobacco Mosaic Virus-Templated Self-Assembly of Aqueous-Stable Superparamagnetic Iron Oxide Nanoparticles into Ring Structures Using Click Chemistry
Shoronia Cross 1 , Katalin Korpany 1 , Dorothy Majewski 1 , Hee Yeon Seo 1 , Amy Blum 1 Show Abstract
1 , McGill University, Montreal, Quebec, Canada
Iron oxide nanoparticles (IONPs) have been recognized as ideal materials for use in applications such as magnetic resonance imaging (MRI) contrast agents, drug targeting, and separation technologies, due to their magnetic properties, low toxicity, and low cost. The ability to selectively modify the surface coatings of the particles is essential for tuning the physical and chemical properties of IONPs for specific applications. We report a method for creating aqueous stable IONPs by performing solvent phase ligand exchange on oleic-acid capped IONPs, using a mixture of DOPAC (3,4-dihydroxyphenylacetic acid), and Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid). We further demonstrate that the surface chemistry of these DOPAC and Tiron-capped IONPs (IONP-DOPAC/Tiron), can be tuned through the use of a combination of EDC coupling and click chemistry. We first conjugate terminal alkynes to the surface using EDC coupling through the carboxyl group of the bound DOPAC ligands, which then provide a platform upon which cy5-azide dye could be bound using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The progress of the ligand exchange and conjugation reactions were monitored using a combination of Fourier transform infrared (FTIR) spectrophotometry, visible spectrophotometry, transmission electron microscopy (TEM), zeta potential analysis, and X-ray photoelectron spectroscopy (XPS). This proof of concept illustrates the versatility of small catechol derivatives as capping groups for aqueous-stable IONPs, which can be modified by simple techniques to yield clickable platforms to impart functionality to IONPs, or to promote self-assembly into ring structures using azide-functionalized tobacco mosaic virus (TMV) as a template.
8:00 PM - BM12.03.07
Chip-Scale Alignment of Long DNA Nanofibers on Patterned Self-Assembled Monolayer
Junfei Xia 1 , Ming Su 1 Show Abstract
1 , Northeastern University, Boston, Massachusetts, United States
Controlled alignment of long DNA nanofibers is challenging. This manuscript reports a method to align human genomic DNA with nearly unlimited length using lithographically produced micro-patterns of self-assembled monolayer (SAM) with positively charged terminal groups. The micro-pattern act as local DNA reservoirs to supply DNAs for nanofiber formation, and also can stretch and align DNA nanofibers to form an ordered array by controlling dewetting profile. By reducing the size and inter-patch distance of micro-patch, a nearly uniform array of long DNA nanofibers can be patterned over a large area. A controlled motion of DNA containing droplet allows for free patterning of DNA nanofibers and production of complex structures without transfer process. Bending of DNA nanofibers due to local distortion of contact line bridges more adjacent micro-patches and increases chance of producing continuous nanofibers. The inter-play between surface tension and electrostatic attraction of positively charged micro-pattern allows production of long DNA nanofibers in a simple yet powerful way.
8:00 PM - BM12.03.08
Protein-Based Silica Nanocapsules for Biomedical Applications
Kyeoung Rok Kim 1 , Chang Sup Kim 1 Show Abstract
1 , Yeungnam University, Gyeongsan Korea (the Republic of)
Silica nanocapsules have received a significant attention, especially in the field of biomedical research, because of the merits of silica and nano-sized capsular configuration, including mechanical robustness, biocompatibility, easy surface functionalization, low density, and large inner cavity. Soft or hard templates-based silica deposition strategies have been widely exploited for preparation of such nanocapsules. However, these methods have several problems, including the use of harsh condition for removal of inorganic template and the need of complicated re-filling step for encapsulation of bioactive molecules. In this study, we develop the biomimetic synthesis of silica nanocapsules using virus-like particle as template via diatom-inspired silica mineralization. A novel fusion protein, HPV16 L1-R5, is designed and produced in Escherichia coli by genetically fusing human papillomavirus (HPV) 16 L1 protein with silica-forming silaffin R5 peptide for development of silica nanocapsules. HPV16 L1-R5 protein can self-assemble into relatively globular shape with a diameter of ~60 nm, which is identical to native HPV L1 particles. Genetically encoded introduction of the R5 peptide at the loop of HPV16 L1 protein leads to the presentation of the R5 peptide on the exterior of HPV16 L1 particles, which results in controlled condensation of silica and near-monodisperse, discrete, sub-250 nm diameter silicated HPV16 L1 particles under an optimal condition. Also, this synthesis method can encapsulate bioactive materials during the self-assembly of HPV16 L1-R5 in biocompatible manner without removal of temperate and re-filling step. Thus, HPV16 L1-R5-based silica nanocapsules have a potential to be used in various biomedical applications.
8:00 PM - BM12.03.09
Entropy-Driven Formation of Protein Nanostructures and 2D Liquid-Crystals at Solid-Liquid Interface
Shuai Zhang 1 , Harley Pyles 2 3 , David Baker 2 3 4 , James De Yoreo 1 Show Abstract
1 , Pacific Northwest National Laboratory, Richland, Washington, United States, 2 Department of Biochemistry, University of Washington, Seattle, Washington, United States, 3 Institute for Protein Design, University of Washington, Seattle, Washington, United States, 4 Howard Hughes Medical Institute, University of Washington, Seattle, Washington, United States
Proteins are one of the key elements of living organisms. They nucleate into ordered structures that further self-assemble into versatile super-molecular architectures to realize high-level functions, like biomineralization of bones and teeth and transduction of biological signals. Inspired by the structural accuracy and functional specificity of natural protein super-molecular structures, fabricating similar structures from artificial proteins with spatial controllability and functional diversity represents a major challenge in biotechnology. Elucidating the dynamic behavior during assembly, as well as the mechanisms and underlying interactions that drive self-assembly addresses that challenge by filling significant gaps in our understanding of this phenomenon both from an experimental and theoretical perspective.
In this presentation, we explore the nucleation and self-assembly behavior of the designed helical repeat protein (DHR10-micaX)  via high-resolution and high-speed in-situ atomic force microscopy (AFM). Though the proper choice of background cations and the use of oriented single-crystal substrates, DHR10-MicaX with dimensions in the nanometer range can self-assemble into 2D liquid crystals with uniform orientation over at least over million-times their length. After tuning the protein head-head interaction, this class of proteins also has the ability to align into nanowire arrays with uniform orientation.
We further follow the nucleation and growth of DHR10-micaX into 2D liquid crystal and nanowire arrays in-situ with spatial resolution of ~1 nm and temporal resolution of ~ 1 frame/s. From comparison of the data to simulations, we conclude that assembly is entropy-driven at the solid-liquid interface. This work not only provides a new approach to fabricate protein super-molecular structures with uniform orientation, it also highlights the role of entropy as a driver of protein dynamics at solid-liquid interfaces.
1. Brunette, T.J., et al., Exploring the repeat protein universe through computational protein design. Nature, 2015. 528(7583): p. 580-584.
8:00 PM - BM12.03.10
Kinetics of Lipid Raft Formation at Lipid Monolayer-Bilayer Junctions Determined by Surface Plasmon Resonance
Yong-Sang Ryu 1 2 , Hansik Yun 1 , Taerin Chung 1 , Junghun Suh 1 , Nathan Wittenberg 3 , Sang-Hyun Oh 3 , Byoungho Lee 1 , Sin-Doo Lee 1 Show Abstract
1 Electrical Engineering, Seoul National University, Seoul Korea (the Republic of), 2 Sensor System Research Center, Korea Institute of Science and Technology, Seoul Korea (the Republic of), 3 Electrical Engineering, University of Minnesota, Minneapolis, Minnesota, United States
Lipid phase separation in cellular membranes results in transient microdomains formation that spatially localize a set of the particular lipids and proteins as they serve diverse membrane-associated biological activities such as signaling, cell deformation, and membrane trafficking. In particular, sphingomyelins (SPMs)- and cholesterols (CHOLs)-enriched lipid raft-phases, also known as liquid ordered (lo)-phase in vitro, have attracted considerable attention because they are functional hotspots for a signaling and a selective association of pathogenic proteins related to a number of diseases including HIV, Prion, and an Alzheimer’s disease. As efforts to explore functions and mechanisms of raft formation and to applicate on medical diagnostics and sensor applications, the supported lipid membrane (SLM) has been provided a systematic platform that enables to exploit how and why raft-phases are activated through a number of surface-sensitive techniques. However, the physicochemical interplays between raft-associated lipids and their kinetics during the raft formation have remained poorly explored. In particular, the real-time monitoring of raft formations remain a long-standing challenging issue, leaving central question behind a nature of lipid-lipid interaction in the course of domain formation.
Most techniques for investigating lo domains rely upon fluorescence (FL)-labeled reporters which can complicate an accurate interpretation due to the dye-dye interaction, the photobleaching, and partitioning artifacts resulting from the conjugation of FL moieties onto lipids. Other methods such as atomic force microscopy are slow and image only small areas of the membrane. A label-free method, such as surface plasmon resonance (SPR) which can measure real-time changes in interfacial refractive index (RI) due to changes in lipid phase, can be used to alleviate artifacts associated with lipid fluorophores and avoid drawbacks of scanning probe techniques. The majority of SPR studies using bio-membranes have focused on measuring binding interactions between a membrane-associated receptor and soluble ligands such as proteins or DNA. The SPR is limited in its utility to study the interplay between raft components because traditional instruments are incapable of accommodating the diffusive raft-units (nanorafts) for resultant assembly of lo domains at spatially-defined areas. Taken together, development of SPR platform with SLMs which enable lo domain formation in controlled time and space will facilitate label-free assays of raft organization kinetics.
Here we reported a systematic platform that enables to measure the kinetics lo domain formation along with receptor-ligand interactions. Continuous but spatially heterogeneous SLMs were achieved through combinations of a micro-contact printing technique with the microfluidics with the help of simultaneous FL imaging and SPR measurements.
8:00 PM - BM12.03.11
Shape-Specific Cellular Uptake Mechanisms of Lipid-Coated Semiconductor Nanocrystals
Minjee Kang 2 , Sung Jun Lim 1 , Mohammad Zahid 3 , Andrew Smith 3 , Cecilia Leal 2 Show Abstract
2 Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 1 , DGIST, Daegu Korea (the Republic of), 3 Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States
The use of nanocrystals as imaging probes in cells is a rapidly emerging technology that promises to enable multiplexing and molecular resolution in-vivo. The physical properties of the nanocrystals such as size and shape can be of great use as a tool to localize to specific subcellular domains. Hence, understanding how the shape of particles affects their cellular uptake is crucial to improving the particle design for cellular imaging and drug delivery applications. Previous studies suggest that the particle shape plays an important role in the kinetics of the internalization process. However, a universal relationship between the shape anisotropy and resulting kinetics remains controversial, partly due to the coupled contributions from other factors such as volume, charge, and material composition. Also, “top-down” approaches to make different shaped particles have limitations in tailoring the size and shape of the particles to be relevant for imaging, i.e. on the order of ten nanometers. To address the aforementioned problems, we have utilized “bottom-up” approaches and synthesized nanosized CdSe/CdS core/shell particles having spherical, elongated, and flat sheet shapes using colloidal synthesis methods. These inorganic nanocrystals show bright, color-tunable, and stable fluorescence that eases tracking of individual particles without the need to attach dye molecules to the particles. To make the nanocrystals suitable for cell experiments, we introduced biocompatible lipid coatings to the hydrophobic surface of the particles. The lipids were judiciously selected to stabilize the different geometry of the particles effectively with minimum coating thickness ensuring stable dispersion in biological media and most importantly, preserving the fluorescence emission efficiency. Transmission electron microscopy (TEM) imaging, small angle X-ray scattering (SAXS), dynamic light scattering (DLS) measurements were performed to elucidate dispersion stability and particle geometry.
Sheet-like nanocrystals (nanoplatelets or NPLs) show surprisingly rapid cellular entry in HeLa, HepG2 and Raw 264.7 cell lines compared to quantum dots (QDs) and quantum rods (QRs) of nearly the same volume. Confocal laser scanning microscopy (CLSM) and flow cytometry experiments showed that NPLs were internalized into intracellular regions within 10 min of incubation while QDs and QRs barely showed detectable cellular entry after one hour of incubation under the same conditions. To further investigate the uptake mechanisms of different shaped nanocrystals, cellular uptake was monitored in the presence of endocytosis inhibitors, which showed that NPLs likely enter through non-endocytic pathways. A single-particle analysis of the kinetics of a nanoparticle uptake was performed by means of total internal reflection fluorescence (TIRF) microscopy. The mechanism of rapid cellular entry of NPLs in connection with their unique morphology will be presented.
8:00 PM - BM12.03.12
Intermolecular Interaction between Lipid Headgroups Comprising Phosphocholine and Sulfobetaine Having Opposite Charge Arrangement
Yokota Keisuke 1 , Tatsuo Aikawa 1 , Takeshi Kondo 1 2 , Makoto Yuasa 1 2 Show Abstract
1 Department of Pure and Applied Chemistry, Tokyo University of Science, Noda Japan, 2 Research Institute for Science and Technology, Tokyo University of Science, Noda Japan
Phosphatidylcholine is water-insoluble amphiphilic molecule that forms lipid bilayer membranes in aqueous solutions. Lipid bilayer membranes have many potential applications such as liposomes which are carriers of drugs, models for artificial biological membranes etc. For designing new biomaterials based on lipid molecular aggregates, it is necessary to design molecules interacting within lipid molecular aggregates and analyze the interactions. Recently, we have proposed a lipid, dipalmitoyl sulfobetaine (DPSB), that has the same acyl chain groups (C16) as the typical phospholipid (dipalmitoylphosphatidylcholine: DPPC), but the charge arrangement of the headgroup (SB unit) is opposite to that of DPPC. Owing to combination of the reversely arranged headgroup charges, DPSB is expected to intermolecularly interact with DPPC. Here we will report intermolecular interaction between headgroups of DPSB and DPPC, and examine effect of the interaction on physical properties of the membrane such as thermodynamic stability and membrane fluidity.
Materials and Method
To investigate intermolecular interaction of lipids, we acquired surface pressure-area (π-A) isotherms of lipid monolayers composed of various lipid compositions (DPPC/DPSB).
Thermodynamic properties of lipid bilayer composed of DPPC and DPSB were examined by differential scanning calorimetry (DSC). Samples of DCS, multilamellar vesicles dispersed in water were used. Strength of intermolecular interaction between the lipids was estimated from gel-to-liquid crystal phase transition temperature (Tc) of the lipid dispersions.
Results and Discussion
In the π-A isothermal measurement, surface pressure of binary (DPPC/DPSB) monolayer started to raise at ~50 Å2 of area per molecule, which is a smaller than those of the single component, DPPC or DPSB. This suggests that the attractive interaction between the PC and SB should densely pack the lipid within the binary monolayer. The Tc reflects the strength of the intermolecular interaction between the lipid molecules in the bilayer membrane. The Tc of the binary lipid dispersion reached the highest value when the component of the mixture was an equimolar composition. This suggests that the interaction between PC and SB increased the van der Waals force between the acyl chains of both lipids. From these results indicates that attractive interaction between the headgroups induce close packing of the lipids in the mixed monolayer and the mixed lipids form a more stable lipid membrane.
8:00 PM - BM12.03.13
M13 Phage Based Novel Self-Assembled Laminated Nanoporous Structures
Jiye Han 1 , Vasanthan Devaraj 2 , Yujin Lee 1 , Eun-Jung Choi 1 , Jin-Woo Oh 1 Show Abstract
1 Nano Fusion Technology, Pusan National University, Busan Korea (the Republic of), 2 Research Center for Energy Convergence Technology, Pusan National University, Busan Korea (the Republic of)
In recent decade, nanotechnology had become an integral part of industry and research. In those, study of nanoscale membrane manufacturing technology utilizing nanomaterials were significantly attracted by many researchers. Generally, traditional membrane filters have a simple pore structure and can be mass-produced. However, difficulties remain in fabrication processes when treating materials according to pore size especially at nanoscale range. Fabricating nanostructures utilizing self-assembly process can solve this issue. Clues of self-assembly properties could be taken from bio-systems and fabrication of complex nanostructures could be achieved. One such interesting bio-system is M13 phage. The filamentous M13 phage has a high aspect ratio with a height of ~ 880nm and a diameter of ~ 6nm. It is composed of a single stranded DNA and helically covered by 2700 copies of major coat pVIII protein and capped with five copies of minor coat proteins pIII/pVI at one end and pVII/pIX on another end. These major coat proteins (pVIII) can be used as receptors for the target materials at regular intervals. The unique property of M13 phage as a biomaterial relies on its self-assembly nature, simple fabrication method in environmentally friendly conditions, and hence large scale commercial production could be achieved. By using genetically engineered viruses, it is possible to construct structures that are suitable for the application requirements. Thus by utilizing M13 phage, various kinds of structures through simple self-assembly method can be realized. In this work, we introduce a self-assembled nanoporous laminated structures based on a nanorod-like shaped M13 phage. The laminated nanoporous structure consists of a genetically modified 4E type M13 phage and PDDA as an alternative layers, fabricated by the pulling method. A fabrication model was proposed based on an initial rough surface layers condition followed up by porous surface on top, as number of alternative layers were deposited. The laminated nanoporous structure was successfully realized experimentally and morphology of the structure were confirmed via atomic force microscopy and scanning electron microscope. Relation between our proposed fabrication model and experimental laminated nanoporous structure was confirmed by optical experiments and three-dimensional finite-domain time-difference simulations. The final structure consists of pore diameter ~ 150nm – 500nm (±20nm) and height of ~ 15nm – 30nm (±5nm). Finally, realization of selective chloride ion extraction was observed from our M13 phage/PDDA laminated nanoporous structure when compared with M13 phage only structure and confirmed from X-ray photoelectron spectroscopy results. The major advantage of our structure relies on simple fabrication, low cost fabrication method and can be applied for large scale production. Interesting applications in field of plasmonics, sensors etc., can be realized as well from our nanostructure.
8:00 PM - BM12.03.14
Self-Assembly and Templating of Lipid-Nanorod Composites into Non-Lamellar Mesostructures
Dylan Steer 1 , You Zhai 1 , Moonsub Shim 1 , Cecilia Leal 1 Show Abstract
1 , UIUC, Urbana, Illinois, United States
In order to better take advantage of inorganic functional nanomaterials such as fluorescent quantum nanoparticles or gold nanoparticle catalysts, research efforts have been focused on assembling these materials into meso-scale periodic structures. Primarily this has been achieved by using self-assembling block copolymer amphiphiles to direct the assembly of nanoparticles. This improves the stability of the colloidal properties and of their functional properties. However, block copolymers suffer from a limited range of structural morphologies and often also require high temperatures and organic solvents to equilibriate, both of which limits their application as stimuli responsive or dynamic materials. Biological amphiphiles, such as glycerol monoolein-phospholipid mixtures, in contrast are highly responsive to temperature and humidity near physiological conditions with several accessible equilibrium mesostructures including inverse hexagonal columnar, periodic lamellar, and multiple cubic structures. Here we report the incorporation of flurorescent quantum nanorods, colloidally stabilized with trialkyl phosphate, into aqueous periodic lipid structures with periodicities between 1-15 nm. The addition of nanorods is shown to generally promote the assembly of lipids into inverse hexagonal columnar arrays. Manipulation of self-assembly parameters including lipid equilibrium curvature and membrane stiffness using lipid composition, additives (e.g. cholesterol), temperature, and hydration have an effect on the final structure and are used to develop a system with possible applications
8:00 PM - BM12.03.15
Directed Head-to-Tail Self-Assembly of Biological Janus Rods
George Bachand 1 , Adrienne Greene 1 , Marlene Bachand 1 , Mark Stevens 1 Show Abstract
1 , Sandia National Laboratories, Albuquerque, New Mexico, United States
The directed self-assembly of nanoscale building blocks (e.g., nanowires) represents an attractive approach to manufacturing complex multiscale and multidimensional materials. An understanding of the interactions involved in such assembly systems is necessary to design synthetic components with embedded molecular instructions enabling the directed self-assembly of complex structures. In the present work, we use microtubule filaments as a model system to study the directed self-assembly of biological Janus rods into extended 1D structures. Microtubules are formed through the energy-driven polymerization of αβ tubulin heterodimers, resulting hollow, anisotropic filaments that are approximately 25 nm in diameter and 10s of microns in length. Lateral aggregation and/or assembly of microtubules is limited by electrostatically repulsive forces that arise from the high linear charge density (~256 e- per micron) along their long axis. We have shown, however, that two microtubules will undergo longitudinal self-assembly when opposite ends (i.e., α-terminated and β-terminated) of the microtubules interact and anneal to form a stable junction. This directed assembly process is diffusion-limited and exhibits second order kinetics over relatively long timescales (i.e., months). Similar to Janus colloids, we demonstrate that the rate of directed self-assembly is a function of a balance between long-range electrostatic forces and short-range hydrophobic interactions, which may be modulated with the addition of monovalent counterion salts. Overall the knowledge gained from our system provides a broad understanding that may aid in the design of synthetic building blocks that are capable of directing their self-assembly into complex, multiscale structures.
*Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA-0003525.
8:00 PM - BM12.03.16
Molecular Gel Formation as a First Order Phase Transition
Nikola Dudukovic 1 , Ben Hudson 2 , Anant Paravastu 2 , Charles Zukoski 3 Show Abstract
1 , Lawrence Livermore National Laboratory, Livermore, California, United States, 2 , Georgia Institute of Technology, Atlanta, Georgia, United States, 3 , State University of New York at Buffalo, Buffalo, New York, United States
Short peptide molecules can self-assemble into ordered nanoarchitectures, offering routes to promising new gel materials for applications in tissue engineering, drug delivery, biosensors, photoelectronics, etc. However, the gelation mechanism in these types of systems remains poorly understood, limiting the development of comprehensive design guidelines for specific applications. We explore the phase behavior of the aromatic dipeptide derivative molecule fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF). The addition of water to a solution of Fmoc-FF in a solvent can result in the self-assembly of Fmoc-FF molecules into a space-filling fibrous network at low concentrations (<< 1 wt%). We provide evidence that gel formation is associated with a first order phase transition resulting in nucleation and growth of strongly anisotropic crystals with high aspect ratios. This phase transition is characterized by low energy barriers to nucleation, short induction times, and rapid one-dimensional crystal growth, stemming from solvent-solute interactions and highly specific molecular packing. We further show that in certain solvent systems Fmoc-FF exhibits polymorphism, where anisotropic crystals (nanofibers) are an initial metastable state, after which isotropic, chunky crystals are formed. Solid state NMR measurements indicate that Fmoc-FF molecular conformation is sensitive to solvent composition during assembly, and suggest the existence of multiple kinetically trapped states. The observed phase behavior indicates that Fmoc-FF and similar small self-assembling molecules can be regarded as simple model systems to study complex thermodynamic and kinetic phenomena involved in peptide self-assembly, such as pathological amyloid fibril formation by naturally occurring polypeptides.
*This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. LLNL-ABS-732725*
8:00 PM - BM12.03.17
Self-Assembly of Computationally Designed Nano-Cages Based on the Coiled Coil Bundle Motif
Nairiti Sinha 1 , Jose Villegas 2 , Dongdong Wu 1 , Kristi Kiick 1 , Jeffery Saven 2 , Darrin Pochan 1 Show Abstract
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 2 Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Computational design of coiled coil peptide bundles that undergo solution phase self-assembly presents a diverse toolbox for engineering new nanomaterials with tunable and pre-determined nanostructures that can have various applications such as in drug delivery, biomineralization and hybrid electronics. Self-assembled cages are especially useful since the cage geometry provides three distinct functional sites: the interior, the exterior and the solvent-cage interface, that can be utilized for tasks such as sequestration, containment and target recognition at the nanoscale. In this poster, syntheses and characterization of an artificial nano-cage assembly based on computationally designed homotetrameric coiled coil peptide bundles as building blocks is discussed. Results of Transmission Electron Microscopy (TEM), Small-Angle Neutron Scattering (SANS) and Dynamic Light Scattering (DLS) are presented to elucidate the size and geometry of peptide assemblies under different pH and temperature conditions. Various self-assembly pathways are shown to have a significant impact on the structure of the peptides assemblies in aqueous solutions. Comparison of characteization results with the computational design has improved the selection process of artificial cage-forming peptide sequences. These novel peptide-based nano-cages will function as robust bio-templates for both biological and non-biological applications in the near future.
8:00 PM - BM12.03.18
Probing Transcription Factor Binding Activity and Downstream Gene Silencing in Living Cells with a DNA Nanoswitch
Alessandro Bertucci 2 1 , Junling Guo 2 , Nicolas Oppmann 2 , Francesco Ricci 1 , Frank Caruso 2 3 , Francesca Cavalieri 2 1 Show Abstract
2 Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia, 1 Department of Chemistry, University of Rome Tor Vergata, Rome Italy, 3 The University of Melbourne, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Melbourne, Victoria, Australia
DNA nanotechnology has inaugurated a new era of engineering programmable molecular motion with unprecedented control and precision, affording dynamic systems that can process information through finely tuned molecular movements. In biological systems, information is achieved, processed, and signalled through complex pathways governed by dynamic non-covalent interactions. Turning to Nature for inspiration, DNA-based bio-actuators can be engineered to actively interface with biomolecules. One of the most studied intracellular dynamics is the DNA binding activity displayed by transcription factors, because of their pivotal role in cellular metabolism and their involvement in inflammatory response and cancer progression. The development of probes that could support rapid, robust, and real time analysis of transcription factor (TF) activity in response to a certain cell status would allow for finely investigating cellular dynamics at the biomolecular level. Currently, direct determination of the intracellular expression of a target transcription factor that has not been genetically tagged requires the use of Western blot on cell lysates or other antibody-based techniques. In all cases, these analyses have to be carried out on cell lysis or fixation, and besides being time- and reagent-consuming, they do not provide details on the in situ DNA binding activity of the target TF. To overcome these limitations, we developed a method based on DNA nanotechnology that enables monitoring of TF binding activity in living cells, allowing for fluorescence imaging and quantification of downstream gene silencing in real time. Our strategy is based on the use of a dynamic DNA nanodevice, a DNA nanoswitch, that features transcription factor-responsive molecular motion and acts as a molecular switch, providing a binding-induced fluorescent readout. Our DNA nanoswitch incorporates a TF-binding domain into a double stem-loop structures that interconverts between a dark non-binding off-state and a fluorescent binding-competent on-state. We showed that our technology allows for monitoring TF binding activity directly in living cells by following the binding-induced fluorescence emission of the DNA probe. We also applied stochastic optical resolution microscopy (STORM), a super resolution microscopy technique, to track the internalization route of our DNA nanodevice packaged into a lipofectamine formulation, and we provided evidence of single-vesicle endosomal escape at a resolution beyond the diffraction limit. Furthermore, we quantified in real time the siRNA-mediated knokdown of transcription factor expression by simple co-transfection of the siRNA formulation and the DNA nanodevice, which allows for rapid, simple, and cost-effective determination of downstream gene silencing. In conclusion, we have demonstrated the use of a DNA nanoswitch for probing transcription factor binding activity in vitro, which may be a powerful tool for molecular biology and life sciences.
8:00 PM - BM12.03.19
Precise Construction of Gold-Nanoparticle Architecture with Monodispersed Polyion Complex for Systemic siRNA Delivery
Hyun Jin Kim 1 , Yu Yi 2 , Kanjiro Miyata 2 , Kazunori Kataoka 3 4 Show Abstract
1 Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo Japan, 2 Department of Materials Engineering, The University of Tokyo, Tokyo Japan, 3 , Innovation Center of Nanomedicine, Institute of Industry Promotion-Kawasaki, Kawasaki Japan, 4 Policy Alternatives Research Institute, The University of Tokyo, Tokyo Japan
We developed siRNA formulation method onto sub-50 nm sized gold nanoparticle (AuNP) for systemic administration. One block copolymer, PEG-poly(L-lysine)-SH was associated with a single siRNA at molar ratio 1:1 to form unit polyion complex (unit PIC). Because degree of polymerization of poly(L-lysine) was approximately 40, the polymer had neutralized negative charged siRNA (-40 charges). The uPIC tailored to coat 20 nm-sized AuNP without aggregation and produced a 40 nm-sized uPIC-AuNP vehicle. This uPIC-AuNP has 90 ± 4 siRNAs per AuNP and enhanced blood circulation of siRNA in intravenous administration. For active targeting, cyclic RGD peptide (cRGD) was installed into alpha-terminal end of the PEG-poly(L-lysine)-SH. The cRGD is a peptide ligand for integrin αvβ3 and αvβ5 receptors, which are widely overexpressed in angiogenic sites and various tumor (e.g. ovary, lung, and glioma). cRGD-uPIC-AuNP showed higher binding affinity than control uPIC-AuNP in cultured ovarian cancer cells. The cRGD-uPIC-AuNP delivered siRNA targeting E6 oncoprotein in human papilloma virus and obtained sequence specific gene silencing in subcutaneous ovarian tumor.
8:00 PM - BM12.03.20
Soluble Peptide Bundles and 2D Peptide Lattices Designed Computationally for Solution Self-Assembly
Michael Haider 1 , Huixi Zhang 2 , Nairiti Sinha 1 , Yu Tian 1 , Kristi Kiick 1 , Jeffery Saven 2 , Darrin Pochan 1 Show Abstract
1 , University of Delaware, Newark, Delaware, United States, 2 , University of Pennsylvania, Philadelphia, Pennsylvania, United States
Peptides are ideal candidates for the design and controlled assembly of nanoscale materials due to their potential to assemble with atomistic precision as in biological systems. Unlike other work utilizing natural proteins and structural motifs, this effort is completely de novo in order to build arbitrary structures with desired size for the specific placement and separation of functional groups. We have successfully computationally designed soluble, coiled coil, peptide, tetramer bundles which are robust and stable in solution. In addition, these soluble bundles have been further modified by computational design methods to self-assemble to form specific 2D symmetries not found in nature. The designed sequences were prepared by solid phase peptide synthesis and purified. We have demonstrated the thermal stability of the soluble bundles as well as confirmed their alpha helical and coiled coil nature. The stability of the soluble bundles arises from the computational design of the coiled coil interior core residues. We established the solution behavior of the soluble bundles in solution using small angle neutron scattering. The form factor of the soluble bundles is well represented by a cylinder model and their behavior at high concentrations is modeled using a structure factor for soluble aggregates of the cylinders. The 2D symmetrical sheets prepared from the modified bundles have been characterized using TEM. The assembled structures closely match the designed spacings predicted computationally. Varying solution conditions, such as pH and ionic strength, alter the structures that are formed while maintaining the underlying symmetry of the peptide bundles. These experiments confirm that our computational designs of robust stable coiled coil bundles as well as self-assembled 2D structures were successful.
8:00 PM - BM12.03.21
Non-Fouling TEM Grid Coatings for Selective Capture of Protein Targets from Cell Lysates
David Thompson 1 Show Abstract
1 , Purdue University, West Lafayette, Indiana, United States
Single particle reconstruction of biomolecular targets is a rapidly growing method of structure determination that is capable of near-atomic resolution via cryoelectron microscopy. Recently, our lab has focused on the preparation and performance evaluation of TEM grids bearing stabilized non-fouling lipid monolayer coatings for single particle reconstruction. These films contain affinity capture ligands of controllable areal density at the distal end of a flexible long poly(ethylene glycol) spacer to avoid preferred orientation of surface-bound protein target and a dense brush layer of a shorter poly(ethylene glycol) material to suppress non-specific protein adsorption. Langmuir-Schaefer deposition of compressed mixed monolayers of these materials prior to application of clarified cell lysate yields a grid preparation that is suitable for high-resolution cryoelectron microscopy analysis. Grids modified in this manner were then used to capture T7 bacteriophage and RplL from cell lysates, as well as purified green fluorescent protein, CaMKII, and nanodisc solubilized maltose transporter, MalFGK2. Our findings indicate that TEM grids bearing supported affinity lipid monolayers are capable of specifically capturing protein targets in a manner that controls their areal densities, while efficiently blocking non-specific adsorption.
8:00 PM - BM12.03.22
Directional Gradients of Biointerfaces Based on Dual Reverse Click Reactions
Zhen-Yu Guan 1 , Hsien-Yeh Chen 1 Show Abstract
1 Department of Chemical Engineering, National Taiwan University, Taipei Taiwan
Chemical or biological gradients that are composed of multifunctional and/or multidirectional guidance cues are of fundamental importance for prospective biomaterials and biointerfaces. As a proof of concept, we herein demonstrate a general modification approach for generating multiple gradients of biomolecules. The technology utilized a vapor-based, alkyne-functionalized poly-para-xylylene, which was prepared based on chemical vapor deposition (CVD) polymerization. With the coating modification on substrates, the multifunctional gradients are created by first performing the thermoactivated thiol−yne reaction to immobilize a selected thiolterminated biomolecule with a continuous and elevated temperature in one linear direction, in which the reactivity of the thiol−yne reaction was found to correlate with the temperature gradient. After the first gradient is established, a second and reversed gradient is formed because the unreacted alkynes automatically created a reactivity gradient accordingly in the opposite direction, and is accessible for the immobilization of azide-terminated biomolecules via an alkyne and azide click reaction. The cell adhesion property of fibroblasts was guided in a gradient with an enhancement, showing that the PEG molecule and RGD peptide were countercurrently immobilized to form such reversed gradients (with negating of the cell adhesion property). Using the gradient modification protocol to also create countercurrent distributions of FGF-2 and BMP-2 gradients, the demonstration of not only multifunctional but also gradient biointerfacial properties was resolved in time latencies on one surface by showing the manipulation in gradients toward proliferation and osteogenic differentiation for adipose-derived stem cells.
Keywords: gradient, multifunctional interface, click reaction, cell proliferation, osteogenesis.
8:00 PM - BM12.03.23
Hierarchical Self-Assembly of Peptide Nanofibers via Interfibril Hydrogen Bonding
Antonietta Restuccia 1 , Gregory Hudalla 1 Show Abstract
1 , University of Florida, Gainesville, Florida, United States
Self-assembly of individual biomolecules into supramolecular structures that have exquisite order over multiple length scales is prevalent throughout nature. Despite significant progress with synthetic peptides that self-assemble into defined nanofibers, understanding how molecular interactions can direct spontaneous peptide self-organization into higher-ordered architectures remains a challenge. Here we report hierarchical self-assembly of peptides terminated with hydrogen bonding molecular motifs, such as amino acids and carbohydrates, that stabilize interfibril association. We created variants of the β-sheet fibrillizing peptide, QQKFQFQFEQQ (Q11), having different amino acids (e.g. N, Q, L) or N-linked glycans (e.g., n-acetylglucosamine (GlcNAc) or n-acetyllactosamine (LacNAc)) conjugated to an N-terminal ser-gly linker. All variants self-assembled in water and adopted a β-sheet conformation comparable to Q11, as determined by FTIR. Under dilute conditions in aqueous buffer, N-Q11 nanofibers spontaneously aligned into bundles of 30-100 nm in diameter, as observed by TEM. Bundling was dependent on the asparagine residue, as nanofibers of the control peptide terminated with the ser-gly linker did not align. Q-Q11 nanofibers formed only loose bundles, while L-Q11 formed large aggregates with indistinguishable lateral organization. At higher concentrations, N-Q11 formed a self-supporting hydrogel that was birefringent, suggestive of long-range alignment, whereas hydrogels of ser-gly-Q11 were not birefringent. Interestingly, under dilute conditions in aqueous buffer, GlcNAc-Q11 nanofibers did not spontaneously align, however introducing a macromolecular crowder induced nanofiber association into aligned bundles. At higher concentrations, GlcNAc-Q11 formed birefringent hydrogels in the absence of a macromolecular crowder, suggesting that concentration-dependent differences in GlcNAc-water interactions may impart an energetic barrier to alignment. LacNAc-Q11 nanofibers also aligned in aqueous buffer, yielding bundles with the largest diameter of ~200 nm, likely due to an increased number of hydrogen bond donor and acceptor moieties. Alignment of glycosylated nanofibers was dependent on the carbohydrate chemistry as peracetylation of the hydroxyl groups of GlcNAc moieties disrupted nanofiber alignment. Altogether, these data demonstrate that polar amino acids and carbohydrates appended onto the termini of a b-sheet fibrillizing peptide can mediate interfibril interactions and hierarchical organization. However, our data also suggest that the propensity of interfibril interactions also depend on the characteristics of the hydrogen-bond donor/acceptor and may also depend on competitive interactions between the appended moiety and water. We envision that interfibril interactions through peptide termini will be broadly applicable for programming the hierarchical self-assembly of synthetic peptides over multiple length scales.
8:00 PM - BM12.03.25
Small-Molecule Sensing Using Self-Assembly of Aptamer-Based Bioscaffolds
Natalie Hughes 1 , Nancy Nguyen 1 , Navneet Goyal 1 , Mehnaaz Ali 1 Show Abstract
1 , Xavier University of Louisiana, New Orleans, Louisiana, United States
Point-of-care systems require highly sensitive, quantitative and selective detection platforms for the real-time multiplexed monitoring of target analytes. To ensure facile development of a sensor, it is preferable for the detection assay to have minimal chemical complexity, contain no wash steps and provide a wide and easily adaptable detection range for multiple targets. Current studies involve a newly designed label-free detection strategy for relevant small-molecules and nucleic acids using aptamer based self-assembly. The designed aptamer consists of both the molecular recognition component and the signalling trigger, which is released upon target binding. In this case we describe a porphyrin selective aptamer that is temporarily bound to a redox active (suboptimal target) molecule. Upon the specific binding of a target the redox active tag is released and detected. To this end we show successful binding of the redox active trigger to the target binding aptamer. Further studies show efficacy of a secondary method involving a conjugate between the porphyrin and the signalling trigger to serve as a suboptimal target. We have explored the measurement of binding and kinetic parameters of various G-quadruplex forming aptamers, which are critical in nucleic acid research and are able to self-associate. The detection strategy is based on a decoupled approach between the signalling surface and the molecular recognition surface, which is immobilized, on a 3D nanoparticle surface to enhance reaction kinetics and reduce mass transfer limitations. Additional studies include immobilizing the detection strategy within a hydrogel matrix to provide a self-assembly based detection approach for small-molecules. Proof-of-concept work for these aptamer-polymer scaffolds was carried out using the ATP binding aptamer.
8:00 PM - BM12.03.26
Designing lysozyme-Polymer Conjugates Stable at Temperatures over 100 °C—Effect of Polymer Concentration
Chandan Choudhury 1 , Nataraja Yadavalli 2 , Nikolay Borodinov 1 , Tatiana Quinones-Ruiz 3 , Sidong Tu 1 , Igor Lednev 3 , Igor Luzinov 1 , Sergiy Minko 2 , Olga Kuksenok 1 Show Abstract
1 Material Science and Engineering, Clemson University, Clemson, South Carolina, United States, 2 , University of Georgia, Athens, Georgia, United States, 3 Department of Chemistry, University at Albany, State University of New York, Albany, New York, United States
Designing efficient enzymes that can work at high temperatures can be used for several industrial applications, such as detergent manufacturing, food and starch processing, production of high fructose corn syrup, polymerase chain reactions and enhanced oil recovery (EOR). Herein, using all atomistic molecular dynamics simulations, we demonstrate that by conjugating enzymes with copolymers at high polymer concentrations one can dramatically improve their thermal stability well beyond that of native enzymes. We conjugated the lysine residues of lysozyme with OEGMA-GMA-OEGMA oligomer (triads) forming lysozyme polymer-conjugated (LPC) and probed its thermal stability with varying polymer concentration. The system also contains free-floating triads which effectively allow us to more closely mimic the conditions in our experiments. Our studies show that LPC, unlike the native lysozyme, largely preserves its secondary structure under the same high temperature. At 50% water concentration, triads phase separate and lysozyme is pushed to the water phase, which onsets the unfolding of lysozyme. The access of water molecules to the lysozyme surface disrupts its 3-D structure at high temperature. We show that increasing copolymer concentration results in stabilization. We characterized the system by analyzing the time evolution of (a) the root mean square deviation (RMSD) of each structure with respect to its initial structure, (b) the number of intra H-bonds of the enzyme, (c) evolution of secondary structures, and (d) number of contacts within lysozyme or with triads or water. Our simulation results are in a good agreement with our experimental observations.
8:00 PM - BM12.03.28
DNA Encapsulated within Silica Nanoparticles as a Long-Term Digital Information Storage Tool
Robert Grass 1 , Wendelin Stark 1 , Reinhard Heckel 2 Show Abstract
1 , ETH Zurich, Zurich Switzerland, 2 Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, Berkeley, California, United States
DNA is nature's tool to store genetic information - by this biomolecule gigabytes of information are stored within every cell of our body, and can even be recovered from ancient fossil samples after thousands of years of storage. In this context fossilized DNA brings intrinsic properties in terms of achievable data density and data stability vastly exceeding our current data storage technologies (magnetic, optical, solid-state storage). In order to mimic these properties, we developed a method for the synthetic DNA fossilization - the encapsulation of DNA into a highly resilient inorganic material (in this case silicate glass) using sol-gel chemistry. We are able to show (1) that DNA is structurally encapsulated within such synthetic fossils are able to survive in the environment for long durations due to the protective properties of the silicate shell (non-protected DNA rapidly degrades due to hydrolysis and enzymatic attack).
Within this work we show that we can not only store natural DNA in such synthetic fossils, but that we can also utilize this technology to store DNA encoding for digital information. In a experimental proof of principle (2) we have translated the text of two ancient books to sequences of DNA, synthesized the DNA (total of ca. 5000 nucleotides) and encapsulated the DNA within the synthetic fossils. We also showed that the information could be recovered without harm after simulated storage of ~2000 years at room temperature without error by dissolving the encapsulates (utilizing fluoride buffers) and reading the DNA utilizing next generation sequencing technology (Illumina). A discussion of the future of the archival data storage using DNA is included together with details of the core properties of the technology.
(1) Paunescu et al. Nat. Protoc. 8, 2440 (2013).
(2) Grass et al. Angew. Chem. Int. Ed. 54, 2552 (2015).
8:00 PM - BM12.03.29
Solid-State NMR Structural Analysis of Binary Co-Assembling Peptides
Kong Wong 1 , Dillon Seroski 2 , Qing Shao 3 , Carol Hall 3 , Gregory Hudalla 2 , Anant Paravastu 1 Show Abstract
1 , Georgia Institute of Technology, Atlanta, Georgia, United States, 2 , University of Florida, Gainesville, Florida, United States, 3 , North Carolina State University, Raleigh, North Carolina, United States
Co-assembling peptides represent an exciting platform in supramolecular biomaterials to understand the biophysical interactions underpinning the relationship of sequence to structure. Two recently studied co-assembling pairs, CATCH(4+)/CATCH(6-) peptides developed by Hudalla and KW(+)/KW(-) peptides designed by King and Webb, have been shown to co-assemble into nanofibers upon mixing of the complementary peptide solutions, but individually, remain as random coils in solution. Prior structural analysis suggest that both peptide systems form anti-parallel β-sheets using Thioflavin T fluorimetry, circular dichroism, and FTIR spectroscopy. Though these studies provide some structural information, these techniques are unable to resolve the specific 3D arrangement of the complementary peptide components within a nanofiber necessitating further structural analysis by solid-state NMR (ssNMR) to develop an accurate all-atom model. Here, we have adapted solid-state NMR techniques, previously used to characterize self-assembling peptides, to measure specific structural constraints with high-resolution in binary co-assembling peptide systems. Specifically, we have isotopically labeled with 13C and 15N select amino acids in KingWebb nanofiber samples and performed 2D ssNMR and dipolar recoupling measurements. Our results from 2D dipolar-assisted rotational resonance (DARR) experiments confirm co-assembly and narrow down possible 3D conformations. We also present a series of PITHIRDS-CT measurements with isotopic dilution wherein we vary the isotopically labeled:unlabeled peptide ratio to evaluate the propensity for a peptide to interact with itself (self-assembly) or its complementary peptide (co-assembly) in a nanofiber sample. Preliminary PITHIRDS-CT measurements show that the CATCH(4+) and CATCH(6-) peptides interact on a molecular level indicating co-assembly and form well-mixed β-sheets i.e. A-B-A-B-A arrangement. Along with computational simulations, this structural information will refine computational models deepening our understanding of the biophysics underlying peptide co-assembly enabling the future development of novel co-assembling peptide systems as supramolecular biomaterials.
8:00 PM - BM12.03.30
Construction and Characterization of DNAzyme-Encapsulated Fibermats
Koji Mizuno 1 , Shuhei Koeda 1 , Akiko Obata 1 , Jun Sumaoka 3 , Toshihiro Kasuga 1 , Julian Jones 2 , Toshihisa Mizuno 1 Show Abstract
1 Graduate School of Engineering, Nagoya Institute of Technology, Nagoya, Aichi, Japan, 3 , Tokyo University of Technology, Hachioji Japan, 2 , Imperial College London, London United Kingdom
Here, we developed functional nucleic acid (FNA)-encapsulated electrospun fibermats. To facilitate stable FNA encapsulation in the γ-PGA/GPTMS fibermats, we used the FNA as an FNA/streptavidin complex, and as a representative FNA, we selected a DNAzyme, the DNA/hemin complex, which is composed of G-quadraplex-forming single-stranded DNA and hemin and exhibits oxidation activity with the aid of a cocatalyst, H2 O2 . Scanning electron microscopy and Fourier-transform infrared spectroscopy measurements revealed that encapsulation of the DNA/hemin complex (∼ 1 wt % against the γ-PGA/GPTMS hybrid) in the nanofibers of the γ-PGA/ GPTMS fibermats did not affect the structure of the original nanofibers. However, because a unique MW-dependent molecular permeability originated from the 3D network structure of the γ-PGA/GPTMS hybrid, low-MW substrates such as 4-aminoantipyrine, N -ethyl-N -(2-hydroxy-3-sulfopropyl)-3- methylaniline, and luminol were able to reach the encapsulated DNA/hemin complex by permeating to the inside of the nanofibers from an immersion buffer and then underwent catalytic oxidation. Conversely, nucleases, which are proteins featuring high MWs (>5 kDa), could not penetrate the γ-PGA/GPTMS nanofibers, and the encapsulated DNA/hemin complex was therefore effectively protected against nuclease digestion. Thus, encapsulating FNAs on the inside of the nanofibers of fibermats offers clear advantages for the practical application of FNAs in sensors and drugs, particularly for use in the in vivo circumstances.
8:00 PM - BM12.03.31
Biomimetic Triazine-Based Polymers with Side Chain Diversity, Defined Sequences and Protein-Like Backbone-Backbone Interactions—Synthesis, Simulation and Self Assembled Nanostructures
Jay Grate 1 , Kai-For Mo 1 , Fang Jiao 1 , Michael Daily 1 , James De Yoreo 1 Show Abstract
1 , Pacific Northwest National Laboratory, Richland, Washington, United States
Sequence-defined polymers, epitomized in nature by polypeptides and poly(nucleic acids), are polymers composed of a multiplicity of monomers, each monomer distinguished from another by having a different side chain, and sequencing of the various monomers into a polymer in a predetermined order. These are distinguished from random copolymers. In nature, sequence-defined polymers create biomaterials, encode information, perform biocatalysis, participate in molecular recognition, and shuttle species across membranes. To date the vast majority of synthetic sequence-defined polymers are either laboratory examples of the natural sequence-defined biopolymers, or close analogs based on similar structural units and bond-forming reactions. For example, peptides, pseudopeptides, and peptoids all rely on amino acid structures and peptide bonds.
We have developed a new class of synthetic biomimetic polymers we call TZPs, for triazine-based polymers. Molecular precursors derived by nucleophilic aromatic substitution reactions on cyanuric chloride provide facile approaches for the incorporation of side-chains into the monomer structures, such that biomimetic polymers with side chain diversity can be produced. These monomer structures are then assembled into polymer chains in predetermined order by an interative submonomer synthesis to create a new architecture for sequence-defined polymers that has no peptide bonds. Molecular dynamics simulations of these new types of structures have demonstrated paired hydrogen bonding motifs similar to those in peptide beta sheets, as well as nanorod configurations that are held together by both pi-pi interactions and paired hydrogen bonding patterns. Using the protein like backbone backbone interactions as motivation, we have designed sequences that form 1D nanofibers, as well as 2D and 3D superstructures, that self assemble and are observed using in situ AFM as well as TEM.
It is anticipated that side-chain functionality, self-organizing conformations, and intermolecular self-assembly of these sequence-defined polymer materials will lead to biomimetic functionality and application.
8:00 PM - BM12.03.32
Building 2D Materials One Row at a Time—Avoiding the Nucleation Barrier
Jiajun Chen 1 2 , Enbo Zhu 3 , Yu Huang 3 , James De Yoreo 1 2 Show Abstract
1 Department of Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 2 Physical Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States, 3 Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, California, United States
Crystal nucleation and growth play central roles in various materials systems. However, nucleation is currently a source of great controversy in materials science. Diverse pathways are now recognized as a common phenomenon due to the complexity of the free-energy landscapes and the reaction dynamics. Thus, nucleation control is also a challenge in materials design and synthesis systems with precise phase and morphology regulations.
Here we present an easy and straightforward approaching for building 2D materials through 1D components, where free-energy barrier can be avoided. In our study, short peptides with seven amino acid residues were selected for their ability to bind specifically to the (0001) face of MoS2. Previous works have shown that selected peptides can be highly organized. We found that our peptides form ordered hierarchical 2D structures on MoS2 (0001) with six-fold symmetry. Direct in situ observation with high resolution has revealed that peptides first join to form dimers as the smallest building blocks. These small units then pack closely to generate rows with a width of 4.1nm and aligning at 30° to the densest sulfur atom packing direction of the MoS2 lattice. Rows pointing in the same orientation further produce larger domains of parallel rows with uniform spacing running in three equivalent crystallographic directions.
In the early stages of assembly, domains aligned along lattice sites running 30° to the dominant row directions are present but disappear over time, thereby demonstrating the ability of the peptides to bind reversibly and coarsen, as well as the higher stability of domains exhibiting the orientations that eventually dominate. High-resolution structural images and comparison between different sequences imply that the existence and position of phenyl groups play an important role in surface attachment and the inter-molecular interaction leading to assembly. The longitudinal growth rate of these domains increases linearly with peptide concentration, while the lateral growth rate changes quadratically. Thus, aspect ratios of these domains can be controlled where high concentrations lead to smaller aspect ratios of the domains due to the even greater dependence of heterogeneous row nucleation rate on concentration, which also implies a preference for heterogeneous nucleation. However, although the final crystals are 2D, due to the 1D nature of the constituent rows, there is no critical size, and the nucleation rate varies linearly with concentration and is finite for all concentrations above the solubility limit.
These findings show that the structural relationship between the peptide and substrate is highly specific and argue for a true epitaxial relationship that dictates both the local order and the final macroscopic morphology. Our results also reveal the fundamental mechanism for peptide assembly on MoS2 and propose a new strategy for constructing 2D materials.
8:00 PM - BM12.03.33
Facile Fabrication of Functional Hydrogel by Rolling Circle Amplification through Rational Template Design
Leilei Tian 1 , Yishun Huang 1 , Guoyuan Liu 1 Show Abstract
1 , South University of Science and Technology of China, Shenzhen China
As a new building material, DNA has been widely used for the emerging nanoscience and nanotechnology due to its unique properties. On one side, DNA can undergo conformational changes sensitively in response to various external stimuli, such as pH, small molecules, and temperature, which has been widely applied in the construction of intelligent systems. On the other side, the predictable and precise Watson-Crick base-pairing makes DNA a fascinating molecule for self-assembly. A variety of complex and well-controlled nanostructures have been successfully fabricated by using DNA as building blocks. Recently, the DNA amplification technique, such as rolling circle amplification (RCA), are used to quantitatively amplify the functional fragments to guide programmable self-assembly, offering a more facile and efficient strategy to fabricate nanostructured or bulk DNA structures.
DNA hydrogel has attracted tremendous attentions for its promising applications in bioanalysis and biomedicine. Generally DNA hydrogel is self-assembled from branched DNA junction with sticky ends or oligonucleotide-polymer hybrids. The former method requires a large amount of oligonucleic acids; and for the latter method the synthesis of hybrid polymers is relatively complicated and the resultant hydrogel shows reduced biocompatibility. Luo and co-workers first employed the RCA method to fabricate DNA hydrogels. The RCA products are long single-stranded DNAs with periodic sequences, which are great scaffolds for programmable self-assembling; also with thousands of amplification in quantity, RCA provides a cost-friendly way to produce bulky hydrogels. This work opens a new avenue for the fabrication of DNA hydrogel; however, the related research is still at the very beginning. It is still urgent to realize the functionality of such hydrogel for broader applications. Recently, we developed a RCA method to automatically produce DNA hydrogels with high and stable catalytic functions and successfully applied it in the glucose detection. Also we realized stimuli-responsive hydrogels on the basis of the i-motif structures.
8:00 PM - BM12.03.34
Electrochemical Control of Peptide Self-Organization on Graphite Surfaces
Takakazu Seki 1 , Christopher So 2 , Tamon Page 2 , David Starkebaum 2 , Yuhei Hayamizu 1 , Mehmet Sarikaya 2 Show Abstract
1 , Tokyo Institute of Technology, Tokyo Japan, 2 , University of Washington, Seattle, Washington, United States
Aiming to construct functional interfaces for bioassay and biosensor, nano-scale self- organization of biomolecules, such as protein and peptide, on solid surface have been widely studied. One of important challenges is the control of nano-architecture of self-organized peptides. In the process of the self-organization, peptides undergo adsorption, diffusion, and organization on the surface. A question here is how we can control such surface processes by an external stimulus. Electrochemical modulation of surface charges by an applied voltage can be a promising way to control the self-organization, where the surface charge may alter the surface processes of peptides. However, the electrochemical approach has been only applied to study the process of protein adsorption on solid surface so far.
Recently we have developed biocombinatorially-selected graphite-binding peptides (GrBPs), which show a specific binding affinity to graphite surfaces.1 These peptides form long-range-ordered nano-structures on atomically flat surface. By modifying amino acid sequence, the peptides can show tailored affinity to various nanomaterials surface.2 In this work, we investigate the effect of the surface potential of graphite on the self-organization behavior of GrBPs. Under the modulation of the surface potential, peptides form various ordered structures such as well-ordered nanowires, wavy wires, and islands. Systematic investigations reveal that the peptide sequence, especially the presence and also location of charged amino acids in the sequence, affects on not only the surface coverage but also the morphological features of self-organized peptides. These results indicate that electrostatic interactions and local charge distribution in the electrochemical double layer is essential for the peptide self-organiztion. Our new approach may open a new door to control the formation of self-organized peptide layers on solid surfaces towards establishing a functional interface for biological applications.
 So, C. R. et al., ACS Nano, 6, 1648–1656, (2012)
 Hayamizu, Y. et al., Sci. Rep., 6, 33778, (2016)
8:00 PM - BM12.03.35
Controlling Self-Assembly of Solid-Binding Peptides on Atomically Flat Surfaces by pH and Ionic Strength
David Starkebaum 1 , Tamon Page 1 , Yuhei Hayamizu 2 , Mehmet Sarikaya 1 Show Abstract
1 GEMSEC, Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 2 Department of Organic and Polymeric Materials, Tokyo Institute of Technology, Tokyo, Meguro Ward, Japan
Solid-binding short peptides offer great utility as molecular building blocks in nanotechnology and nanomedicine. Some of these peptides can form self-organized nanostructures on solid surfaces due to highly specific coordination of intermolecular forces enabled by conformational changes in the peptide. These molecular interactions can be controlled via point and domain mutations of the amino acids in the sequence. For a wider practical applicability, the behavior of these peptides on the surface needs to be studied under a variety of experimental conditions. In this study, we examined how the organization of the self-assembled monolayers formed by a phage display selected “wild-type” graphite binding dodecapeptide (GrBP5-WT) changed with pH and ionic strength. The surface coverage and crystallinity of these peptide monolayers were both improved by 1mM sodium phosphate, and disrupted by 1mM sodium hydroxide. Zeta potential measurements of aqueous graphite powder dispersions showed a pH-dependent negative surface charge, which increased in magnitude when GrBP5-WT was added. A peptide mutant (GrBP5-M9) was designed by replacing two carboxylate residues with polar, but non-charged, amide residues. The non-charged mutant peptide formed crystalline nanostructures which were unaffected by changes to the ionic strength or pH, and did not contribute additional negative charge to the graphite dispersion zeta potential. We have shown, therefore, that a simple mutation to a phage-display selected solid-binding peptide can eliminate its sensitivity to buffer and pH changes. By forming more predictable bio/solid interfaces, these peptide/nanosolid hybrid systems enable the development of more robust self-organized structures that are necessary for technologies where biology is integrated with solid-state devices. The research is supported by the funds, NSF-DMR-1629071, through the MGI Program (Materials Genome Initiative).
8:00 PM - BM12.03.36
Amphiphilic Peptide Carriers for Natural Therapeutic Compounds Delivery
Yasaman Hamedani 1 , Catherine Neto 1 , Milana Vasudev 1 Show Abstract
1 , University of Massachusetts Dartmouth, Dartmouth, Massachusetts, United States
In this study, we have examined the ability of various cationic, amphiphilic peptide to self-assemble into micelles which can be utilized as drug carriers for targetted delivery in zebrafish tumor models. Recently, self-assembling peptides has attracted considerable attention due to their properties such as biocompatibility, chemical versatility, and biological recognition abilities. The self-assembly of the peptides depends on noncovalent forces including π-stacking, hydrogen bonding, and hydrophobic interactions, and based on their composition, a variety of architectures (nanotubes, nanoribbons, nanospheres) have been synthesized. In this study, electrostatic forces were used to induce the assembly of nanospheres in an electrospraying process as well as self-assembled peptide micelles were studied as potential drug carriers. The advantages of such structures in drug delivery include, responses to the external stimuli such as pH, temperature and presence of enzymes. Previous studies have found that several natural compounds found in cranberry fruit inhibit the proliferation of tumor cell lines, including colon, melanoma, breast and prostate. The compounds of interest fall into two categories with differing physical properties: polar flavonoids/polyphenols, including quercetin and pro-anthocyanidins, and nonpolar triterpenoids including ursolic acid and derivatives. As some of these compounds are likely to be metabolized and excreted from the human body preventing attainment of therapeutic concentrations, micelles or other peptide-based carriers deposited via electrospraying techniques represent a possible route to target delivery of the flavonoids to tissues and organs of interest. We have begun to explore their mechanisms of action and their bio-distribution in a zebrafish model. This study will assess the suitability of these natural compounds for micellar delivery, the extent of delivery to cells, and whether tumor cell proliferation is reduced as a result.
Alberto Saiani, University of Manchester
Dave Adams, University of Glasgow
Ayeesha Mujeeb, PeptiGelDesign
Darrin Pochan, University of Delaware
Manchester Biomaterials Network (ManBioMat)
University of Delaware
BM12.04: Session III
Tuesday AM, November 28, 2017
Sheraton, 2nd Floor, Constitution A
8:30 AM - BM12.04.01
Controlled Positioning of Enzymes within Biocatalytic Hydrogels Using Self-Assembling Protein Building Blocks
Samuel Lim 1 , Dominic Glover 2 , Francois Carruzzo 3 , Gi Ahn Jung 1 , Douglas Clark 1 Show Abstract
1 Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California, United States, 2 School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, New South Wales, Australia, 3 Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zürich, Zurich Switzerland
Hydrogels are widely used as biomaterials because of their stability under aqueous conditions, versatility in fabrication, and tunable mechanical properties. In particular, hydrogels can be functionalized to carry out sequential metabolic reactions through incorporation of multiple enzymes in combinations. Precise positioning of multiple enzymes can enhance catalytic activity through substrate channeling. However, exerting nanoscale spatial control within a functionalized hydrogel backbone remains challenging. We aim to overcome this difficulty by using protein building blocks designed to self-assemble in programmable order.
The g-prefoldin (g-PFD) is a filamentous protein isolated from the hyperthermophilic archaeon Methanocaldococcus jannaschii; its remarkable stability, unique modularity, and self-assembly into filaments with chaperone activity render it an ideal building block for the bottom-up construction of precisely patterned protein nanostructures. We verified that the g-PFD dimer could function as a template for positioning two enzymes in nanoscale proximity. Furthermore, we have engineered g-PFD subunits that enable periodic positioning of multiple enzymes along the template by replacing the assembly interface with combinations of helical coil pairs that specifically bind each other.
Precisely patterned g-PFD fragments were assembled into hydrogels through the introduction of terminal cysteine residues followed by crosslinking with PEG polymers using thiol-maleimide “click” chemistry. Hybrid g-PFD-PEG hydrogels showed rapid gelation kinetics, tunable viscoelastic properties, and good stability. We verified successful spatial organization within the hydrogel through FRET analysis of a closely positioned fluorescent protein pair. This system is being utilized to develop biocatalytic gels with spatial control over incorporated enzymes. Ultimately, the ability to position functional molecules in a controlled manner within hydrogels will considerably improve our ability to fabricate advanced multifunctional biomaterials with for enhanced performance
8:45 AM - BM12.04.02
Construction of Hybrid Optoelectronic Materials Using Robust, 2D Self-Assembling Protein Arrays
Francesca Manea 1 , Caroline Ajo-Franklin 1 Show Abstract
1 , Berkeley Lab, Berkeley, California, United States
Nature has evolved intricate, hierarchically-ordered architectures with complex self-assembly pathways. Mimicking such organizations is advantageous in order to manufacture materials with exceptional functionality, such as optoelectronic properties. However, the generation of synthetic bio-optical composites is currently limited by the inability to create densely-packed, tunable arrangements with precision at multiple length scales, thus restricting the dimensionality and complexity of patterning for these materials. Ultimately, we aim to introduce ordered-stacking of embellished protein nanosheets to fabricate lamellar structures, leading to hybrid 3D materials with unique plasmonic and optoelectronic properties.
Here we employ protein engineering approaches to modify highly robust, 2D self-organizing proteins, for the simultaneous and controlled deposition of multiple types of nanoparticles. We have exploited the triggered self-assembly pathway of surface-layer (S-layer) proteins, in particular SbsB from thermostable Geobacillus stearothermophilus, to construct molecularly diverse, highly-ordered nanostructures.
We explore a variety of embellishment avenues to selective decorate the S-layer surface with mixtures of metal, semiconductor and ceramic nanoparticles. Creating clusters of spatially-localized metal-binding amino acid residues, such as Cys and His, into SbsB allows us to bind metal nanoparticles, but with only modest 5 % sheet coverage. In contrast, we adopt SpyTag-SpyCatcher to increase particle coverage 6-fold, while maintaining the ability to bind a range of nanoparticles, including quantum dots and upconverting nanoparticles, across the array. Additionally, we can utilize two such conjugation systems orthogonally to create bimetallic arrays with tailorable nanoparticle combinations. Thus, these methodologies allow us to fabricate novel bio-inspired composites that extend the potential of hybrid optoelectronic and photonic materials.
9:00 AM - *BM12.04.03
Chemically Controlled Assembly of Dynamic and Functional Protein Assemblies
Faik Tezcan 1 Show Abstract
1 , University of California, San Diego, La Jolla, California, United States
Proteins are the preeminent synthons that living organisms utilize for constructing functional materials and molecular devices. Underlying this versatility is an immense structural and chemical heterogeneity that renders the programmable self-assembly of proteins a highly challenging design task. To circumvent this challenge, we have chosen to use strategies inspired by sythetic supramolecular and inorganic chemistry. These strategies have resulted in discrete or infinite, 1-, 2- and 3D protein architectures that display crystalline order yet are dynamic and stimuli-responsive, and possess new emergent chemical/physical properties. This presentation will highlight some of the most recent examples of such protein assemblies.
9:30 AM - BM12.04.04
Bio-Nano(Particle) Interface—A Closer Look to Protein-Nanoparticle Interactions with Novel Methods
Ahmet Bekdemir 1 , Luciano Abriata 2 , Francesco Stellacci 1 Show Abstract
1 Material Science, École Polytechnique Fédérale de Lausanne, Lausanne, Vaud, Switzerland, 2 Protein Crystallography Core Facility, École Polytechnique Fédérale de Lausanne, Switzerland, Lausanne, Vaud, Switzerland
Understanding and controlling the interactions between proteins and nanoparticles (NPs) is one of the key challenges in nanomedicine. It has been established that NPs, when immersed in biological media, are covered almost immediately by a complex layer of proteins called ‘protein corona’. It is clear that the corona form because of a binding like event, but it has been challenging to characterize the key thermodynamics parameters that are the basis for such event. All methods employed to date presented significant limitations, for example, they suffered from spurious signals produced by aggregates in solution or required additional fluorescent labelling either of NPs or of the proteins.
In this talk, I will present a new method to extract the thermodynamic parameters of NPs-protein interaction based on Analytical Ultracentrifugation (AUC). By performing AUC on a series of solutions containing a fixed concentration of NPs and varying concentration of proteins, it is possible to construct a Langmuir adsorption isotherm through a specific Hill formulation that we have developed. It will be shown that the result is a precise determination of the binding affinity (KD), the stoichiometry (Nmax) and the cooperativity constant (n). The method can be used without difficulty in the presence of aggregates and does not entail any chemical modifications of constituents. Moreover, sub-10 nm NPs do not pose any specific challenge, thus for the first time the interaction of 2 nm in diameter NPs with bovine serum albumin will be presented.
The characterization of the interaction of proteins and NPs cannot stop solely at the thermodynamic parameters. It has been extensively shown that the particles properties are determined by the way the corona proteins are presented. To address this important problem, we exploit Heteronuclear Quantum Coherence Spectroscopy NMR (HSQC-NMR). We have developed a strategy based on the analysis of the shifts (for misfolded proteins) or the changes (for folded proteins) in the signal intensity of each amino acid residues on proteins upon binding to NPs. Using this approach, we are able to generate 3D maps that illustrate the regions of interactions between the proteins and the NPs, and consequently the proteins presentation to the outside world. For the first time, we demonstrate that our model protein’s (ubiquitin) presentation depends not only on the particles’ surface chemistry but also on their size.
Finally, in this talk I will show how AUC can be used to determine competitive binding between different proteins on NPs. Overall, our methods present considerable advancement in elucidating NP-protein interactions both in thermodynamic and mechanistic sense. We believe these findings could enable the design of novel therapeutic or diagnostics nanoparticles.
9:45 AM - BM12.04.05
Fabrication of a Metamaterial Unit Cell—Plasmonic Nanorings on a Tobacco Mosaic Virus Protein Mutant
Dan Petrescu 1 , Omar Zahr 1 , Julia Del Re 1 , Serene Bayram 1 , Amy Blum 1 Show Abstract
1 , McGill University, Montreal, Quebec, Canada
Nanophotonic metamaterials can be designed to manipulate electromagnetic waves with sub-wavelength precision with applications spanning nanophotonic circuitry, photovoltaics, tumor therapy, and signal detection enhancement in analytical techniques. Previous work has predicted that a sub-wavelength array of plasmonically coupled nanoparticles arranged in a ring would exhibit a strong magnetic dipolar response along with a negative index of refraction in the visible regime. Fabrication of nanorings with such precise geometries and arrangements, however, remains limited by the current resolution, time, and cost of top-down lithographic techniques. To achieve higher precision and reproducibility, bottom-up bio-molecular self-assembly techniques present an appealing alternative. Our studies explore mutants of the Tobacco Mosaic Virus (TMV) capsid protein as scaffolds for gold nanoparticle ring-like structures. We have chemically synthesized dynamic solution-phase metamaterial unit cells by harnessing the diverse chemical functionality and self-assembling properties of the TMV viral capsid. Bioconjugation experiments confirmed site-specific functionalization of the capsid in high yield, while the growth of ring structures was followed by TEM and UV-vis spectroscopy. Varying the size and morphology of the rings afforded tunable optical properties, as evidenced by dark-field scattering spectroscopy obtained for individual ring structures.
10:30 AM - BM12.04.06
Phase Behavior of Polypeptide Based Polyelectrolyte Complex Micelles
Lorraine Leon 1 Show Abstract
1 , University of Central Florida, Orlando, Florida, United States
Polyelectrolyte complexes form by mixing oppositely charged polymers in solution. The resultant complex phase separates from solution into either irregularly shaped solids (or rather glasses), called precipitates, or micron sized liquid droplets that can coalesce into a distinct phase, called a coacervate. Using oppositely charged polypeptides, one can tune between solid and liquid complexes by manipulating the chirality of the polyelectrolyte. Homochiral complexes form precipitates with hydrogen bonded b-strand structure. In contrast, if one or more polypeptide is composed of both L and D monomers, coacervates are formed with no secondary structure. This inability to form secondary structure is attributed to steric hindrance of the racemic polypeptide impeding hydrogen bond formation. Therefore, since both types of complexes are formed using weak polyelectrolytes, the ability of the homochiral molecules to hydrogen bond causes the difference in phase. Using oppositely charged block-copolyelectrolytes that contain neutral blocks covalently linked to charged blocks allows the phase separation to be stabilized on the nanoscale, creating self-assembled micellar structures in dilute solutions. These polyelectrolyte complexes micelles can be used as drug and gene delivery vehicles for charged therapeutics like nucleic acids and proteins. Using diblock copolypeptides of varying chirality the resulting micellar structure can have both solid and liquid polyelectrolyte cores. This presentation will focus on characterization of polypeptide based polyelectrolyte complex micelles using scattering techniques (light, x-rays, neutrons), circular dichroism, and electron microscopy to reveal structural differences of solid and liquid micellar cores. In addition, the exchange kinetics of solid and liquid core micelles will be investigated by mixing solid and liquid core micelles that contain FRET pairs. Insight will be provided as to how differences in solid and liquid cores influence the design of drug delivery vehicles. If time permits, investigations into related micelles with dynamic coronas will be discussed.
10:45 AM - BM12.04.07
Bio-Inspired Dynamic Supramolecular Assembly Controlled through Molecular Conformation
Erik Spoerke 1 , Brad Jones 1 , Dominic McGrath 2 , Jeffrey Vervacke 1 , Jonathan Bollinger 1 , Mark Stevens 1 , George Bachand 1 Show Abstract
1 , Sandia National Laboratories, Albuquerque, New Mexico, United States, 2 , University of Arizona, Tucson, Arizona, United States
Biological systems employ dynamic and responsive supramolecular protein filaments, such as microtubules (MTs), to enable remarkable functional and adaptive natural phenomena. MTs, in particular, are involved in processes ranging from the transport of intracellular cargo to reconfiguring cell morphology and even facilitating separation of genetic material during cell division. Although the dynamics of MT assembly and disassembly are governed by a number of complex, cooperative phenomena, one critical driving force behind the disassembly of MTs is mechanical strain imparted by the molecular conformation of the assembled tubulin building blocks. Here, we explore synthetic self-assembling peptides, functionalized with light-responsive azobenzene derivatives, to mimic dynamic self-assembly through changes in molecular shape. Covalently modifying the tubule-forming dipeptide di(phenylalanine) (FF) with azobenzene moieties, we introduce a light-triggered mechanism to change molecular building block conformation through photoisomerization of the azobenzene constituent. Under aqueous self-assembly conditions, we demonstrate the initial self-assembly of Azo-FF into well-defined tubules, followed by the disassembly of these tubules under ultraviolet light exposure, and the subsequent (and reversible) reassembly of nanofibers upon isomer relaxation. Characterizing these materials spectroscopically and microscopically, we explore the mechanisms of this dynamic behavior and the importance of molecular shape, solvation environment, isomerization time, and molecular assembly kinetics on this bio-inspired molecular phenomenon. Continuing to distill and explore biomolecular processes, such as morphology-mediated dynamic assembly, will yield new insights into the development of adaptive and reconfigurable materials.
Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.
11:00 AM - *BM12.04.08
Peptide-Based Materials from the Bottom Up
Dek Woolfson 1 Show Abstract
1 , University of Bristol, Bristol United Kingdom
In nature a bewildering array of nanometre-to-micrometre scale materials are constructed from peptides and proteins. This requires information to be encoded in the polypeptide chains to direct and stabilise secondary and tertiary structures, and then to instruct these folded units to assemble into supramolecular structures and materials. One of the aims of our work is to understand these aspects of the so-called protein-folding problem and of protein-protein interactions. To do this, we inspect natural protein sequences and structures to uncover rules within their sequences that provide these instructions. We use this understanding to guide the de novo design of completely synthetic peptides to adopt prescribed 3D structures. In turn, we use these as building blocks to assemble more-complex surpramolecular structures and materials that span the nano-to-micron scales. One of our favoured protein-folding motifs is the a-helical coiled coil.1,2 These are simple protein-protein interfaces formed through the oligimerisation of alpha helices. Our understanding of these is sufficient to allow the design of a-helical bundles and barrels with between 2 and 8 helices.3-6 These defined structures, which we have characterised to atomic resolution and have dimensions of approximately 4 x 4 x 4 nm, can be manipulated further to direct the assembly protein cages,7 fibres8,9 and nanotubes.8,9 This talk will describe our research in this area over the past 5 years, and touch on possible applications for these assemblies and materials in nanoscience, synthetic biology and medicine.
1. Coiled-coil design: updated and upgraded.
DN Woolfson. Subcellular Biochemistry 82, 35-61 (2017)
2. De novo protein design: how do we expand into the universe of possible protein structures?
DN Woolfson et al. Curr Opin Struct Biol 33, 16-26 (2015)
3. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology.
JM Fletcher et al. ACS Synth Biol 1, 240-250 (2012)
4. A set of de novo designed parallel heterodimeric coiled coils with quantified dissociation constants in the micromolar to sub-nanomolar regime.
F Thomas et al. J Am Chem Soc 135, 5161-5166 (2013)
5. A de novo peptide hexamer with a mutable channel.
NR Zaccai et al. Nature Chem Biol 7, 935-941 (2011)
6. Computational design of water-soluble a-helical barrels.
AR Thomson et al., Science 346, 485-488 (2014)
7. Self-assembling cages from coiled-coil peptide modules.
JM Fletcher et al. Science 340, 595-599 (2013)
8. Modular design of self-assembling peptide-based nanotubes.
NC Burgess et al. J Am Chem Soc 137, 10554-10562 (2015)
9. Controlling the assembly of coiled-coil peptide nanotubes.
F Thomas et al. Angew Chem Int Ed 55, 987-991 (2016)
11:30 AM - BM12.04.09
Characterization and Biocompatibility Studies of Linear and Cyclic Self-Assembled Peptide Nanotubes
Prathyushakrishna Macha 1 , Milana Vasudev 1 Show Abstract
1 , University of Massachusetts Dartmouth, Dartmouth, Massachusetts, United States
Self-assembly, a natural process by which organization of molecules into ordered structures occurs, is the foundation for the synthesis of several nanostructures. Biomolecules like DNA, peptides, and proteins have been a part of an extensive research for the fabrication of novel nanostructures that are not only functional but also, biocompatible. In this study, we have utilized aromatic dipeptides tryptophan-tyrosine and dityrosine to form nanotubes through solution-phase self-assembly. The structural, chemical, thermal characteristics, and biological interactions will be studied in detail. In addition to solution-based assembly methods, vapor deposition of the peptides will also be studied. Plasma-enhanced chemical vapor deposition, a solvent-free, eco-friendly bottom-up synthesis method will be used to deposit dipeptides into nanotubes. These tubes will be analyzed too, any differences between the characteristics of former and latter tubes will be reported.
Fourier transform infrared spectroscopy, Raman scattering, liquid chromatography-mass spectroscopy, and circular dichroism spectroscopy will be used to gain insights into the chemical composition while thermogravimetric analysis and differential scanning calorimetry will be conducted to understand their thermal characteristics. Scanning Electron Microscopy and Confocal microscopy will be used to study the morphological features. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and dopamine assays will be studied in vitro rat adrenal pheochromocytoma cells (PC 12) to assess the cytotoxicity of the tubes and physiological effect on the PC 12 cells as well as any changes in dopamine release. Also, computational studies will be performed to simulate self-assembly and know the possible intramolecular and intermolecular interactions leading to the process.
11:45 AM - BM12.04.10
Kinetic Control of Computationally Designed Peptide Self-Assembly and Templated Synthesis of Gold Nanomaterials
Yu Tian 2 , Frank Polzer 2 , Huixi Zhang 1 , Kristi Kiick 2 , Jeffery Saven 1 , Darrin Pochan 2 Show Abstract
2 Materials Science and Engineering Department, University of Delaware, Newark, Delaware, United States, 1 Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Peptides with well-defined secondary-structures have the ability to exhibit specific, local shapes that enables the design of complex supramolecular structures through intermolecular assembly. Our computationally designed peptides can fold into α-helical secondary-structure and further assemble into anti-parallel, homotetrameric coiled-coil bundles with a hydrophobic internal core. Based on the same parent sequence, derivative sequences can be generated with only modification of exterior coiled coil residues to install directional interactions and can self-assemble into supramolecular structures with predetermined local symmetries. Due to the robust design, the bundle structures can tolerate different solution conditions. Therefore, solution parameters such as pH and temperature can be used to kinetically control the morphologies of inter-bundle quaternary structures. In this work, we explored the kinetic control of peptide assembly and their capability as templates to direct the synthesis of gold nanomaterials. Solution pH influences peptide assembly by affecting the external charged state of peptide bundles. This can lead the bundles to be either repulsive or attractive to each other. Significant aggregation occurs with peptides assembled at pH values near their respective iso-electric points. These kinetically trapped, disordered aggregates greatly slowed down further assembly and regular 2D lattice structure development for assembly times greater than a month. The solution temperature can largely eliminate the formation of disordered aggregates and accelerate the assembly of mature, desired 2D nanomaterial plates by providing extra energy for the organization process of assembly building blocks and preventing kinetic traps. With other peptide sequences, the solution pH affected the charged state of peptides similarly, but created more specific supramolecular structures with distinct diverse morphologies, varying from nanotubes, to 2D platelets, to 1D needle-like structures at acidic, neutral and basic conditions, respectively. The formation of these different quaternary structures is proposed to be triggered by certain highly charged residues at pH values away from the peptide pI. In the end, we used these self-assembled peptide structures with proper chemical modification as templates to direct the growth of gold nanomaterials into specific 1D or 2D organizations.
Funding acknowledged from NSF DMREF program under awards DMR-1234161 and DMR-1235084.
BM12.05: Session IV
Tuesday PM, November 28, 2017
Sheraton, 2nd Floor, Constitution A
1:45 PM - BM12.05.01
‘miRacle’—An Injectable Hydrogel for Long-Term microRNA Therapy in Malignant Pleural Mesothelioma
Poulami Majumder 1 , Anand Singh 2 , Chuong Hoang 2 , Joel Schneider 1 Show Abstract
1 Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland, United States, 2 Thoracic and Gastrointestinal Oncology Branch, National Cancer Institute, Bethesda, Maryland, United States
Malignant Pleural Mesothelioma (MPM) is a rare but highly aggressive asbestos-related tumor that develops within the chest cavity. Due to long period of latency, insensitivity towards radiotherapy and extremely challenging surgical procedures, finding treatments for MPM has been challenging. Surgical resection of MPM leaves a large surface area of tissue that can possibly be directly treated with therapy to prevent recurrence. However, this necessitates the use of a drug delivery vehicle that can cover large areas of tissue having complex surface topology. We have developed ‘miRacle’, a biodegradable hydrogel that can encapsulate therapeutics and be delivered directly to the pleural cavity to allow uniform coverage of the tissue surface with drug. The gel is prepared from self-assembling peptides that allow for direct encapsulation of nanoparticles containing microRNA (miRNA) known to induce p53 positive feedback loop causing apoptosis in MPM cells. Fabrication of the delivery platform begins by first condensing miRNA with a positively charged amphiphilic peptide affording nanoparticles capable of trafficking the miRNA into cells after being delivered by hydrogel to the tissue. We show that these nanoparticles ferry miRNA into human MPM cells via endocytosis and endosomal escape, resulting in coordinated downregulation of multiple cell cycle transcripts. These miRNA-nanoparticles can be stably encapsulated into peptide-based hydrogels to achieve sustained miRNA release for about a month. Appropriate control of release rate is possible by varying the net charge of the peptide matrix in ‘miRacle’. Based on these results, the efficacy of these local miRNA depot is now being evaluated under pre-clinical settings. Together, our work describes a simple and effective delivery platform that could be extended for a diverse array of locoregional therapies.
2:00 PM - *BM12.05.03
Energy Transport Processes Enabled through Peptide-Based Nanomaterials
John Tovar 1 Show Abstract
1 , Johns Hopkins University, Baltimore, Maryland, United States
The development of robust and predictive structure-function relationships is a current challenge in peptide-based materials science. This contribution will describe several examples from our group’s recent work seeking to understand the impact of rational peptide sequence variations in peptide-pi electron-peptide triblock molecules on the emergent energy transport properties that arise after molecular self-assembly. Among the properties we have explored are the photophysical nature of the electronic excited states, the electrical conductivity and field-effect behavior, and how these observables can be impacted by the kinetic nature of the peptide assembly process. More recently, we have explored photonic creation of electric fields and are now examining how these fields might be able to impact cell physiological processes in tissue engineering applications.
2:30 PM - BM12.05.04
Macroscopic Anisotropic Actuation in Muscle-Inspired Polymer-Supramolecular Hybrid Materials
Stacey Chin 1 , Christopher Synatschke 1 , Samuel Stupp 1 Show Abstract
1 , Northwestern University, Evanston, Illinois, United States
Nature creates mechanically useful materials through the bottom-up hierarchical ordering of small building blocks to provide macroscopic responses, such as the sarcomeres within skeletal muscles. Inspired by these materials, we have designed macroscopic tubes that exhibit anisotropic actuation driven by a thermal stimulus. The hybrid tube is composed of high aspect-ratio supramolecular peptide amphiphile nanofibers which are aligned using weak shear forces, followed by growth of thermoresponsive covalent polymers radially from the fiber surface. The hierarchically ordered tube exhibits reversible anisotropic actuation upon changes in temperature, with greater actuation observed in directions perpendicular to the nanofiber alignment. Simulations of both the bulk and nanostructure show that the hierarchical ordering within the material is necessary to achieve maximum actuation. This work suggests a new strategy to create soft matter with the molecularly encoded capacity to perform complex motions.
2:45 PM - BM12.05.05
Nanoparticles for Compaction and Wrapping of DNA and RNA—Strategies for Rational Design
Matthew Manning 1 , Jessica Nash 1 , Yaroslava Yingling 1 Show Abstract
1 , North Carolina State University, Raleigh, North Carolina, United States
Condensation, bending, and wrapping of nucleic acids is of significant biological and clinical importance. DNA wrapping histone proteins control gene expression via transcription factor accessibility. Nucleic acid therapeutics, such as RNA vaccines and gene therapy, show great promise in treating and preventing a variety of diseases, from Ebola to melanoma. Current delivery method rely on viral particles, which can elicit undesirable immune responses and must be manufactured by cell culture. Cationic nanoparticles offer a level of programmability and immuno-compatibility not available with viral vectors. Using atomistic molecular dynamics, we have designed cation-functionalized gold nanoparticles which act as histone mimics to form synthetic nucleosomes or facilitate the packaging of DNA and RNA by wrapping. From this, we have determined the design factors controlling nucleic acid compaction and characterized the associated forces and structural changes.
While DNA is easily precipitated by electrostatic screening and inter-strand attraction with polycations, tight wrapping requires a nanostructure, such as the histone octamer. Nanoparticles of similar charge and shape as the histone assembly are shown to induce the same superhelical turns. We have found nanoparticle charge and solvent ionic strength as key factors to induce wrapping and preserve helical integrity and present a model for investigating the effects of sequence and structure on dynamical nucleosome behavior.
Duplex RNA (dsRNA) fails to condense in the presence of polyamines, which act as powerful condensation agents in DNA. This has been attributed to dsRNA’s deeply buried phosphate groups. By controlling the flexibility and excess free volume of a nanoparticle’s cationic ligands and their distribution in relation to the helical axis, we show that dsRNA can be bent well below its persistence length and without significant helical distortion. This change results from a periodic bending along the long axis of base-pairs and identifies the favored mode of bending in dsRNA.
3:30 PM - *BM12.05.06
How Does a Simple Virus Self-Assemble?
Aaron Goldfain 1 , Rees Garmann 1 , Vinothan Manoharan 2 Show Abstract
1 Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 2 Harvard John A. Paulson School of Engineering and Applied Sciences and Department of Physics, Harvard University, Cambridge, Massachusetts, United States
Many simple RNA viruses, which consist of proteins that form a highly-ordered protective shell (called a capsid) around the viral RNA, are self-assembled structures: infectious viruses can be spontaneously formed simply by mixing the RNA and the capsid proteins in a buffer, in the absence of any host factors. We do not yet understand how this self-assembly process results in such highly ordered shells. The yield and fidelity of the assembly is particularly remarkable in viruses with a triangulation number of 3 or higher, in which case some of the proteins must find their way to 5-fold coordinated sites and others to 6-fold coordinated sites on the same shell. To better understand how such systems assemble, we do experiments on a much larger, model system: attractive colloidal particles on the surface of a sphere. On the curved droplet surface, the particles form branched networks of slender domains with no 5-fold defects. The growth and shape of these domains -- and the absence of topological defects -- are the consequence of an elastic instability arising from the Gaussian curvature of the underlying surface and the short-ranged attraction between the particles. These results illustrate that closed structures are difficult to assemble when the range of attraction between the building blocks is short, which suggests that the viral assembly pathway may be anything but trivial. I will conclude by showing the results of recent optical experiments that attempt to resolve the kinetics of assembly of a single viral capsid. These experiments may inform methods to design synthetic systems that assemble robustly into ordered shells.
4:00 PM - BM12.05.07
Wet Adhesive Nanomaterials Inspired by the Barnacle Adhesive
Christopher So 2 , Elizabeth Yates 1 3 , Luis Estrella 2 , Ashley Schenck 1 , Catherine Yip 1 , Kathy Wahl 3 Show Abstract
2 Chemistry, U.S. Naval Research Laboratory, Washington, District of Columbia, United States, 1 sited in Chemistry Division, Naval Research Laboratory, Washington, District of Columbia, United States, 3 Chemistry, US Naval Academy, Washington, District of Columbia, United States
Barnacles produce a micron-thick layer of ordered amyloid-like nanofibers from proteins that function as a permanent wet adhesive. Recent proteomic work from our lab shows that barnacles achieve this largely through display of complex charged chemistries using small and flexible side-chains, folded in a manner similar to adhesive silks used by spiders and insects.1 Their well-defined, modular, nature result in bulk materials that serve many purposes: adhesion, durability, bacterial resistance, and even potent enzymatic activity2. Fibers are shaped by a highly conserved domain alternating between short 20-residue low complexity sequences (Gly/Ser/Thr/Ala residues) and regions with charged and aromatic side chains, with more than 40 such domains in just five proteins. The adhesive properties of these unique sequences and their function in an amyloid-like structure remain unclear. To study this, we produce miniaturized synthetic peptides from consensus barnacle cement sequences and also insert sequences into a host amyloid system of bacterial fimbriae to produce abundant and engineered wet adhesive mimics. Multiple design strategies will be discussed, including chimeric proteins where the curli subunit protein csgA is fused with whole barnacle cement proteins, csgA mutants that display glue-like chemistries, as well as csgA displaying short 20 residue cement consensus tags. We characterize these materials by AFM-based nanomechanical measurements and compare them to the Wild-Type barnacle adhesive. Synethetic and recombinant adhesive materials provide a route to scale up and study a scarce but potent class of multifunctional adhesive nanostructures produced by one of the most tenacious marine fouling organisms in the ocean.
 So, C. R., Fears, K. P., Leary, D. H., Scancella, J. M., Wang, Z., Liu, J. L., Orihuela, B., Rittschof, D., Spillmann, C.M., Wahl, K. J. Sequence Basis of Barnacle Cement Nanostructure is Defined by Proteins with Silk Homology. Scientific Reports, 6, 36219, 2016.
 So, C. R., Scancella, J. M., Fears, K. P., , Essock-Burns, T., Haynes, S. E., Leary, D. H., Diana, Z., Wang, C., North, S., Oh, C. S., Wang, Z., Orihuela, B., Rittschof, D., Spillmann, C.M., and Wahl, K.J. Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes, ACS Applied Materials and Interfaces, 2017, DOI: 10.1021/acsami.7b01185.
4:15 PM - BM12.05.08
Modular Self-Assembly of Protein-Based Nanoreactor Superlattices for Multistep Catalysis and Material Regeneration
Masaki Uchida 1 , Kimberly McCoy 1 , Masafumi Fukuto 2 , Lin Yang 2 , Hideyuki Yoshimura 3 , Heini Miettinen 4 , Ben LaFrance 4 , Dustin Patterson 5 , Benjamin Schwarz 1 , Jonathan Karty 1 , Peter Prevelige 6 , Byeongdu Lee 7 , Trevor Douglas 1 Show Abstract
1 , Indiana University, Bloomington, Indiana, United States, 2 , Brookhaven National Laboratory, Upton, New York, United States, 3 , Meiji University, Kawasaki Japan, 4 , Montana State University, Bozeman, Montana, United States, 5 , University of Texas at Tyler, Tyler, Texas, United States, 6 , University of Alabama at Birmingham, Birmingham, Alabama, United States, 7 , Argonne National Laboratory, Lemont, Illinois, United States
The assembly of individual molecules into hierarchical structures is a promising strategy for developing novel three-dimensional materials with collective behavior and properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular structures hierarchically assembled from a limited number of protein subunits. Here we demonstrated the bio-inspired modular construction of materials with two levels of hierarchy. The first level of hierarchy is the formation of catalytically active virus-like particles (VLPs) through directed subunit self-assembly with enzyme encapsulation. This was achieved by exploiting a virus capsid formation process assembled from coat proteins (CPs) and scaffolding proteins (SPs) that direct the assembly of CPs into capsid structure. Cargo proteins such as enzymes can be directed to encapsulate inside of VLPs through genetic fusion with SP. The second level of hierarchy is the self-assembly of these VLP building blocks into ordered three-dimensional lattices. This was realized through electrostatic interaction between negatively charged VLPs and positively charged macromolecule PAMAM dendrimer as a linker molecule. Proper balance of attractive and repulsive interaction between the building blocks, which can be controlled through modulating surface charge of VLP and ionic strength of solution, resulted in formation of VLP arrays with long-range order. These superlattice materials, which were composed of two populations of enzyme packaged VLP modules, exhibited a coupled two-step catalytic conversion for isobutanol synthesis from α-ketoisovalerate via intermediate isobutyraldehyde. The selective and reversible assembly of the two populations of VLP modules into superlattice materials was achieved by rational modification of the VLP surfaces. The ability to remove and incorporate individual components in a selective and controlled manner highlights the utility of this modular approach and leads to a capability to regenerate the materials via selective replacement of individual VLP module upon catalyst senescence. Importantly, the interactions which govern assembly of the VLPs into higher order structures depend on the exterior surface of VLPs and are independent of cargo molecules encapsulated on the interior, thus the approach to construct higher order assemblies presented here is widely applicable.
4:30 PM - *BM12.05.09
Recombinant Biomaterials for Treatment of Spinal Cord Injuries
Sarah Heilshorn 1 Show Abstract
1 , Stanford University, Stanford, California, United States
Approximately 15,000 new spinal cord injuries (SCI) occur in the US each year, primarily affecting young adults. Schwann cells are a promising therapy for SCI and are currently being explored in clinical trials; however, significant limitations in cell delivery and long-term survival decrease their therapeutic potential. The low cell retention post-transplantation is partly attributed to (i) mechanical forces during injection that damage the cell membrane and (ii) the lack of a three-dimensional (3D) matrix to support cell viability post-injection. We hypothesized that the development of a shear thinning, injectable hydrogel would improve cell viability, engraftment, and regenerative capacity after transplantation. This hydrogel, produced from a blend of engineered recombinant proteins and peptide-modified synthetic polymer is termed SHIELD: Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery. SHIELD formulations with storage moduli, G', spanning 10-500 Pa all showed excellent viability of encapsulated Schwann cells (>98%) and significant mechanical protection from membrane damage when exposed to syringe needle flow. After 7 days in vitro, 3D cultures of within all SHIELD formulations showed positive immunostaining for Schwann cell markers (p75, S100), but cultures in formulations of intermediate stiffness showed higher proliferation rates and decreased caspase activity. In a rat cervical contusion model of SCI, Schwann cells delivered in SHIELD resulted in smaller lesion volumes and improved functional outcomes compared to cells delivered in saline and injury control groups. Since even a mild functional recovery would mean a vast quality-of-life improvement for SCI patients, developing a regenerative therapy for SCI would be extremely significant clinically.