Bionanomaterials Lab - UC Berkeley and Lawrence Berkeley National Laboratory

Research at Bio-Inspired Nanomaterial Lab

Brief Introduction

The primary goal of our research program is to create precisely defined, bioinspired nanomaterials that can be used for studying complex interfacial phenomena and as functional materials, devices and therapeutics. In my research group, we are particularly inspired by how nature manages complex interactions at interfaces and self-assembles functional bionanomaterials. Therefore, we use bioengineering approaches to both study and mimic these processes. Specifically, we use genetic engineering techniques to design novel peptides/proteins or virus particles and then use them to create functional nanostructures for regenerative medicine, therapeutics, biosensing, and energy generation. Through our research, we would like to address the following questions:

  • Protein-Protein Interfaces: How do proteins interact to create exquisite, hierarchical structures with diverse functions in a spatially and temporally controlled manner from simple nanofibrous building blocks?
  • Protein-Cellular Interfaces: How can protein-based material interfaces be tuned to create physical, chemical, and mechanical structures that can direct cell behavior for regenerative medicine and therapeutics?
  • Protein-Organic/Inorganic Interfaces: How can proteins be engineered to recognize specific target organic/inorganic molecules for the development of biosensors or to template the growth of inorganic materials?
  • Protein-Electronic Interfaces: How can the dipole of protein sequences and structures be engineered to interchange between electric and mechanical forces for the generation of clean protein-based piezoelectric energy?

Jump to section:

A. Protein-Protein Interfaces
B. Protein-Cellular Interfaces
C. Protein-Organic Material Biointerfaces
D. Protein-Inorganic Interfaces
E. Protein-Electric Interfaces


In our research, we developed a novel, bio-inspired, self-assembly process termed 'self-templating assembly'. Self-templating assembly of phage virus-based helical nanofibers results in the formation of structures with tunable optical, biological and electric properties (Fig. 1 and 2; Nature 2011). The structures mimic many of the hierarchically organized structures formed by collagens, chitins and celluloses in nature. Through genetic engineering of the phage, we applied self-templating to the design of hard and soft tissue engineering materials (Nature 2011, Nano Lett. 2009), biosensors (Nature Comm. 2014), and electrical energy generating materials (Nature Nanotech. 2012).

Figure 1. Schematic illustration of our biomimetic self-templating assembly process using phage as a collagen-like helical nanofibers. The work is based on our on-going research and chosen as one of twelve highlights in President Obama's National Science Foundation Report for US Congress (2014) entitled 'Manufacturing Goes Viral'.

Our research has also touched on various aspects of organic-inorganic biointerfaces. We used genetic engineering and directed evolution of viruses to understand how bone proteins evolved to create bone minerals (Langmuir 2011) and investigated how environmental factors control remodeling processes of bone minerals in various conditions (Langmuir 2009, 2011 & JPC-C 2010). We also used the acquired knowledge to design novel nanocomposites, including injectable bone materials (Biomacromol. 2011) and light-controlled soft-robot like hydrogels (Nano Lett. 2013), by controlling protein-nanomaterial interfaces.

My group's research endeavors have thus far resulted in 50 published papers and eight patents. Our research was recognized by many honors, including being chosen as one of twelve research highlights in President Obama's National Science Foundation Report for US Congress (2014). In addition, our energy research was chosen as one of the top five nanomanufacturing processes by Scientific American (2013). Our research on virus-piezoelectricity was chosen as one of 17 Breakthrough Discoveries in 2012 by iO9. My research efforts were recognized with multiple awards including an NSF CAREER Award (2008-2013), the Hellman Faculty Award (2008), Best Paper Awards in conferences (MRS 2007 and IEEE NEMS 2007 meeting) and an R&D 100 award (2013) and etc. Our ongoing research was featured by many news media, including TV (e.g., ABC7 news, AP news, NTV (Russian national TV), CCTV (Chinese national TV), Euronews (European major news channel), Al Jazeera (Arab major news channel), Korean News (YTN)); radio (i.e., BBC radio, BR (German public radio), WBAL radio (Baltimore, MD based radio channel), and newspapers and magazines (e.g., New York Times, Washington Times, LA Times, USA Today, Forbes, Scientific American, Discovery, The Scientist).


Detailed Description of Research:

The primary goal of our research program is to create precisely defined bioinspired nanomaterials and utilize them to study complex interfacial phenomena, and to develop novel, functional materials, devices and therapeutics. In our research group, we have used bioengineering approaches to create these biomimetic nanomaterials and investigated the complex interactions at their interfaces at the molecular level. An understanding of these interactions is critically important in the biosciences and bioengineering fields. We utilize phage or bacteria as bioengineering toolkits to interrogate the biointerfaces between protein-protein, protein-inorganic/organic and cellular materials. We apply the knowledge we gain from interfacial phenomenon to address various scientific and engineering challenges in energy, biosensors and bionanomedicine.

A. PROTEIN-PROTEIN INTERFACES

Jump to section:

B. Protein-Cellular Interfaces
C. Protein-Organic Material Biointerfaces
D. Protein-Inorganic Interfaces
E. Protein-Electric Interfaces
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The primary goal of this research is to understand the mechanisms nature uses to create diverse hierarchical structures and exquisite functions in a spatially and temporally controlled manner from a simple nanofibrous building block through protein-protein assembly processes.

Biomimetic self-templating supramolecular structures: In nature, helical macromolecules such as collagen, chitin and cellulose are critical to the morphogenesis and functionality of various hierarchically structured materials. During tissue formation, these chiral fibrous macromolecules are secreted and undergo self-templating assembly, a process whereby multiple kinetic factors influence the assembly of the incoming building blocks to produce non-equilibrium structures. A single macromolecule can form diverse functional structures when self-templated under different conditions. Collagen type I, for instance, forms transparent corneal tissues from orthogonally aligned nematic fibers, distinctively colored skin tissues from cholesteric phase fiber bundles, and mineralized tissues from hierarchically organized fibers (Fig. 1). Nature's self-templated materials surpass the functional and structural complexity achievable by current top-down and bottom-up fabrication methods. However, self-templating has not been thoroughly explored for engineering synthetic materials. In our recent paper (Chung et al, Nature 2011), we used phage-based nanofibers to determine the mechanism by which the hierarchical organization of self-assembled structures occurs. We used the M13 phage as a model system because it possesses collagen-like structural features with genetic flexibility (a helical nanofiber-like shape, monodispersity, and the ability to display functional peptides on its surfaces). A single-step process, which we called 'self-templating assembly', produces long-range-ordered, supramolecular structures showing multiple levels of hierarchical organization and helical twists (Fig. 2). Using our novel self-templating material assembly process, we could create many biologically induced hierarchical structures in a controlled manner. More importantly, we could create structures never observed in nature nor produced by other engineering approaches. Three examples of those distinctly newly created phases of supramolecular structures are reported in our work (Nature 2011): nematic orthogonal twists, cholesteric helical ribbons and smectic helicoidal nanofilaments. Both chiral liquid crystalline phase transitions and competing interfacial forces at the interface are found to be critical factors in determining the morphology of the templated structures during assembly. The resulting materials show distinctive optical and photonic properties, functioning as chiral reflector/filters and structural color matrices. In addition, M13 phages with genetically incorporated bioactive peptide ligands direct both soft and hard tissue growth in a hierarchically organized manner (See 'Protein-Cellular Interfaces' section for further discussion). Our newly developed phage-based material approach may provide a means for understanding the helical self-assembly of the hierarchical structures of biomaterials and give insight into hierarchical structure-function relationships in nature. Currently, we are applying our self-assembly techniques to other biomacromolecules (collagen, chitin and cellulose) and investigating their molecular assembly processes for the development of biomimetic hierarchical structures for tissue regeneration.

Figure 2. Schematic diagram of the phage-based self-templating process. (a) Schematic illustration of the phage structure covered by ~2700 a-helical major coat (pVIII) protein subunits. It also has five copies of pIII (bottom) and pIX(top) minor coat proteins. (b) Schematic illustration of the helical self-templating assembly of phage particles controlled by competing interfacial forces at the meniscus where LC phase transitions occur. (inset) The POM image shows iridescent colors originating from LC phase formation at the air-liquid-solid interface. (Chung et al, Nature 2011)

Our newly developed phage-based material system was highlighted in President Obama's US Congress report for the National Science Foundation (NSF) Budget entitled "Manufacturing goes viral" this year (2014) (Fig. 1). NSF produced a 30-min TV interview program based on this research and broadcasted it for general public education.

B. PROTEIN-CELLULAR INTERFACES

Jump to section:

A. Protein-Protein Interfaces
C. Protein-Organic Material Biointerfaces
D. Protein-Inorganic Interfaces
E. Protein-Electric Interfaces
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Based on our understanding of the protein-protein interfaces to form a controllable nanostructure to tune biochemical, chemical, mechanical cues, we apply this system to interrogate the protein-cellular interface. Protein-cellular interfaces play a critical role in the development of regenerative medicines and novel therapeutics.

Phage-based nanofiber matrices for regulating cell behavior: The primary goal of this research is to understand how chemical and physical structures affect the interactions between proteins and cells in guiding cellular behavior to develop novel regenerative tissue matrices. Designing biomimetic materials with precisely controlled structural organization that closely mimics the natural tissue environment is critical for the development of regenerative medicines. Many attempts have been made to mimic natural tissue microenvironments in vivo by using an extracted extracellular matrix (ECM) or synthetic ECM-mimetic materials to manipulate the ECM's biological, chemical and mechanical properties. Although the resulting structures provide great promise for improving cell behavior, emulating complex tissue microenvironments and precisely controlling biochemical and physical cues using conventional approaches are still challenging. Any modification of chemical structures requires labor-intensive chemical synthesis, and the resulting chemical structures affect other important physical and mechanical parameters. A material that mimics the nanofibrous structure of the ECM and whose biochemical structure can be conveniently modified with little change in physical structure and assembly processes would be very desirable for investigating various biochemical cues and their effects on cellular growth processes. Recently, we developed novel phage-based biomimetic nanofiber matrices that could easily be engineered to display various biochemical cues and form self-assembled nanostructures for regulating cellular behaviors (Fig. 3). Using this phage-based tissue matrix system, we investigated various protein and cellular interfaces and discovered new findings on the interactions between protein and cells. In our recent work (Yoo et al, Biomacromolecules 2011), we examined the effects of a collagen-derived biochemical cue (DGEA) on bone cells. We engineered the M13 phage to display the DGEA-peptide in high densities on their major coat proteins and studied their effects on mouse derived bone stem cells (preosteoblasts, MC3T3-E1). Through our study, we verified that the DGEA-peptides could stimulate bone stem cells to outgrow, an event that is linked to osteogenic differentiation. We expanded our protein-cell interface investigation by using other cell types and biochemical cues. Our papers (Merzlyak et al, Nano Letters 2009 and Chung et al, Langmuir 2010; Chung et al, Nature 2011) reported that phages engineered to express RGD (integrin binding peptide) and IKVAV (neural cell stimulating peptide) peptides formed two- and three-dimensional matrices that regulate the directional growth of neural cells in a chemical and physical cue specific manner. In addition, our paper (Yoo et al, Soft Matter 2010) presented a facile strategy for immobilizing growth factors on genetically engineered phage matrices for tissue regeneration. We modified M13 phages to express streptavidin-binding peptides (HPQ) and/or integrin binding peptides (RGD) on their major and minor coat proteins. The resulting phages formed nanofibrous matrices that could easily immobilize the streptavidin-conjugated growth factors FGF-b and NGF for neural cell proliferation and differentiation. We verified that the immobilized growth factors possessed a prolonged ability to stimulate the target cells. We demonstrated the synergistic roles of the growth factors and integrin binding peptides in controlling cell morphologies and the growth of neural cells. Our phage matrices, which could be easily functionalized with various ligands and growth factors, can be used as convenient test beds for investigating the functions of various biochemical stimulants on numerous cell types.

Figure 3. Schematic diagram of the development of novel biomedical engineering materials using genetically engineered phage. After creating the functional motif-displaying phage, we can construct nanofibrous matrices that can control and guide cell behavior. These matrix materials can be used for the development of topical therapeutics. (Merzlyak et al, Nano Lett. 2009)

Novel phage-based therapeutics: The primary goal of this research is to understand how specific sequences of protein/peptide and their display can affect the interaction between proteins and cells to develop novel phage-based therapeutics. An M13 phage possesses the unique ability to present multiple peptides or proteins on its surfaces, which makes it useful for various therapeutic purposes. We observed that phage displaying RGD peptides could be internalized into cells through integrin-mediated endocytosis. Furthermore, we enhanced the internalization process of the phage particles by engineering the phage with a high copy number (up to ~140 copies) of cyclic RGD peptides on their major coat proteins (Choi et al, Bioconj. Chem. 2014). This suggests that the phage could be used as therapeutic delivery vehicles. For example, we collaborated with Dr. Deborah Dean (Oakland Children's Hospital, UC San Francisco) to develop a novel phage to use as a topical mucosal treatment or as a microbicide to prevent and ameliorate Chlamydia trachomatis (Ct) urogenital infections. No vaccines or microbicides are currently available to prevent sexually transmitted infections (STI) due to Ct. Ct is a Gram-negative obligate intracellular bacterium and the most common cause of bacterial sexually transmitted diseases (STD) worldwide. In our recent work (Bhattarai et al, Biomaterials 2012), we constructed phages that were engineered to express RGD (integrin binding) peptides and Ct polymorphic membrane protein D (PmpD), which is known to interfere with Ct propagation. The resulting phage significantly decreased Ct infections in HeLa cells and primary endocervical cells compared with those cells exposed to Ct alone (Fig. 4).

Figure 4. Effect of RGD8-PmpD3 on Chlamydia infection on primary endocervical cells. Primary endocervical cells were treated with RGD8-PmpD3 (1011 pfu/mL) with and without Ct (L2, MOI of 1) at 36 h. (A) Ct infection alone; (B) RGD8-PmpD3 alone; C) Pre-treated for 2h with RGD8-PmpD3 and then with Ct; and D) Co-treated. Blue, DAPI; Green, RGD8-PmpD3; Red/orange, Ct. Bar-diagram; Effects of RGD8-PmpD3 on Ct infection in HeLa and primary endocervical cells. (Bhattarai et al, Biomaterials 2012)

Recently, we expanded our therapeutic research to cancer targeting based on collagen dystrophy. Collagens are over-expressed in various human cancers and subsequently degraded and denatured by proteolytic enzymes, which makes them a novel target for diagnostics and therapeutics. Genetically engineered bacteriophage (phage) is a promising candidate for the development of imaging or therapeutic materials for cancer collagen targeting due to its promising structural features. In our recent work (Biomaterials 2014), we genetically engineered M13 phages with two functional peptides, collagen mimetic peptide and streptavidin binding peptide, on their minor and major coat proteins, respectively. The resulting engineered phage functions as a therapeutic or imaging material to target degraded and denatured collagens in cancerous tissues. We demonstrated that the engineered phages are able to target and label denatured or cancerous collagens expressed on A549 human lung adenocarcinoma cells after conjugation with streptavidin-linked fluorescent agents. Our engineered collagen binding phage could be a useful platform for cancerous collagen imaging and drug delivery in various collagen-related diseases. Furthermore, we constructed the collagen-mimetic peptide library on the phage and screened against cancerous collagen tissues. We plan on further enhancing their specificity depending on collagen type and structures in future research.

C. PROTEIN-ORGANIC MATERIAL BIOINTERFACES

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A. Protein-Protein Interfaces
B. Protein-Cellular Interfaces
D. Protein-Inorganic Interfaces
E. Protein-Electric Interfaces
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The primary goal of this research is to interrogate protein-organic material interfaces to understand how proteins bind desired chemicals through molecular recognition and to utilize it for the development of sensitive and selective biosensors.

Development of peptide based recognition element for biosensor: Miniaturized smart sensors that can perform sensitive, selective, and real-time monitoring of explosives and biological toxins are tremendously valuable to our nation's ability to deploy effective homeland security measures and protect civilians and our military forces throughout the world. Current sensing devices are still far from being able to offer selective, sensitive, and real-time point-detection. They are also lacking in multi-analyte assessment, ease-of-use, and low manufacturing costs. To address these critical issues, we have developed a new approach, whereby the principles of molecular recognition in biology are mimicked to achieve highly selective binding to small molecular targets such as explosives and biotoxins. We discovered molecular recognition elements (MREs) against explosives (TNT and DNT) and biological and environmental toxins (Cholera toxin, PBDEs, and pesticides) by using directed evolution of phage peptide libraries. Using these MREs, we developed multiple nanocoatings for cantilever and quartz crystal microbalance sensing platforms which showed highly selective and sensitive multimodal detection (Jaworski et al, Langmuir 2008 and Anal. Chem. 2009) through the collaboration with Professors Arun Majumdar and Roya Maboudian (UC Berkeley). Using nuclear magnetic resonance spectroscopy, we studied the molecular level binding mechanism between the identified peptide (Trp-His-Trp) and desired target TNT chemicals (Jaworski et al, Langmuir 2011). In addition, we have recently developed a novel selective and sensitive biomimetic nanocoating by combining TNT receptors bound to conjugated polydiacetylene (PDA) polymers. PDA is a lipid-like polymer comprised of a conjugated polymer backbone with carboxylic acid and alkyl side-chains. The amphiphilic nature of PDA monomers facilitates its formation into supramolecular assemblies such as vesicles and membranes. PDA's conjugated polymer backbone can serve as a stable and sensitive colorimetric sensor due to changes in its conjugated electronic band structure resulting from interactions between target analytes and specific functional motifs on PDA's head-groups. In our recent work (Jaworski et al, Langmuir 2011), we described our colorimetric PDA-based TNT sensor development. Furthermore, we applied these PDA-TNT receptor coupled nanocoating materials into CNT-FET devices through collaboration with Professor Seunghun Hong (Physics, Seoul National University). Our recent paper (Kim et al, ACS Nano 2011) reported that selective binding events between the TNT molecules and phage display-derived TNT receptors were effectively transduced to sensitive SWNT-FET conductance sensors through the PDA coating layers. The resulting sensors exhibited unprecedented 1 fM sensitivity toward TNT in real time, with excellent selectivity over various similar aromatic compounds. Our biomimetic receptor coating approach may be useful for the development of sensitive and selective micro- and nanoelectronic sensor devices for various other target analytes.

Turkey skin collagen inspired colorimetric biosensors: The primary goal of this research is to combine our understanding of protein-organic and protein-protein interfaces and develop a bio-inspired novel biosensor to detect desired chemicals in a sensitive and selective manner. Many animals change their skin colors to communicate, to express mood, for camouflage, or to respond to environmental changes. In the tissues of these animals, various nano and microscale components play roles in generating distinct colors and achieving rapid color changes. Inspired by nature, sensors that change color in response to target chemicals are being developed by employing biomimetic structures and mechanisms. In particular, structurally colored biomaterials, such as butterfly wings, beetle exocuticles, cephalopod skins, mammalian skins, and avian skins/feathers provide insight into developing colorimetric sensors. These materials exhibit brilliant colors that are derived from their hierarchically organized structures and are resistant to photobleaching. Furthermore, they can rapidly shift colors upon exposure to chemical vapors due to structural and/or refractive index changes. Therefore, both structurally colored materials in nature and their synthetic analogues are being explored as simple and portable colorimetric sensor platforms.

A significant drawback of previous structural color sensors is their limited intrinsic affinity for specific targets of interest (e.g., explosives and pathogens) and resulting poor selectivity against analytes with similar chemical structures. Current methods to promote target specificity by either chemically incorporating specific recognition motifs or by synthesizing arrays of cross responsive platforms for "artificial nose" type pattern recognition are promising, but incorporating analyte-responsive elements into the sensing devices is still challenging because it requires complex designs and multistep synthetic pathways. Furthermore, many structurally colored sensors exhibit viewing-angle dependent color changes (iridescence) that may complicate analysis.

In our recent work (Oh et al, Nature Comm. 2014), we demonstrated a novel biomimetic colorimetric sensing material composed of filamentous bacterial viruses (M13 phage) as depicted in Figure 5. Tunable colorimetric phage-based structures are fabricated using the self-templating assembly process we previously developed (Chung et al, Nature 2011). The resulting films are composed of quasi-ordered phage bundle nanostructures and exhibit viewing-angle independent colors due to the isotropic deposition of the phage nanobundles. These films mimic the structure of turkey skins (M. gallopavo), which are structurally colored blue due to the coherent scattering of light from collagen bundle-based nanostructures (Fig. 5a-c). Arrays of differently colored phage matrices, termed Phage litmus (Fig. 5d), rapidly swell or shrink upon exposure to external chemicals, resulting in color changes similar to those seen on turkeys when they get flustered (Fig. 5a). The chemicals are identifiable through color pattern analyses in a quantitative manner. To enhance selectivity, a trinitrotoluene (TNT)-binding motif identified by phage display is incorporated onto the phage coats. The TNT-binding phage litmus detects TNT down to 300 ppb with the aid of a common handheld device (iPhone) and can distinguish between similar nitroaromatic molecules (i.e. TNT, dinitrotoluene (DNT), and mononitrotoluene (MNT)). The facile synthesis, ease of use, portability, and successful introduction of tunable receptors suggest that Phage litmus colorimetric sensors can be useful for the detection of a wide variety of harmful toxicants and pathogens to protect human health and national security. Currently, we are developing the phage-litmus to enhance their sensitivity and selectivity for the environmental toxicants (PBDE, flame retardant). In addition, we also developed a phage to detect the acetone metabolite to monitor glucose levels for diabetic patients.

This foundational biosensing technology received first place in the "Berkeley Big Idea" business competition (2014). This technology was spun-off to the company 'Bioinspira'.

Figure 5. Schematic of biomimetic colorimetric sensor system. (a) Turkeys autonomously change their red skin to white and/or blue when excited. The blue color is associated with structural colorization of collagen nanostructures, although their color change mechanism is not known with molecular detail. As a result, turkeys are known as "seven-faced birds" in Korea and Japan. (b) A histological section of turkey skin shows that turkey skin consists mainly of collagen and highly vascularized tissues (50 μm scale bar). (c) Transmission electron micrograph of perpendicularly aligned collagen bundled fibers in the dermis (red square) (200 nm scale bar). (d) Bioinspired phage-based colorimetric sensors, termed Phage litmus, are composed of hierarchical bundles like the collagen fibers in turkey skins. Application of target molecules (chemical stimuli) causes color shifts due to structural changes, such as bundle spacing (d1 and d2), and coherent scattering. Using a handheld device's camera (iPhone) and home-built software (iColor Analyzer), we can identify target molecules in a selective and sensitive manner (Oh et al, Nature Comm. 2014).

D. PROTEIN-INORGANIC INTERFACES

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A. Protein-Protein Interfaces
B. Protein-Cellular Interfaces
C. Protein-Organic Material Interfaces
E. Protein-Electric Interfaces
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The primary goal of this research is to interrogate protein-inorganic material interface to study the biomineralization process of bones and teeth in the human body.

Molecular level investigation of biomolecular interfaces on bone: In our bodies, protein-inorganic crystal interfaces play a critical role in developing our bones and maintaining the mechanical integrity of the body. In order to interrogate protein-inorganic interfaces, we developed bone model surfaces whose structures were defined at the molecular level and investigated their structure-dependent characteristics using in situ atomic force microscopy techniques. Mature mammalian hard tissues are composed mainly of calcium phosphates in the form of mineralized biological apatites. Biological apatites vary in impurity content but are most closely related to hydroxyapatite (HAP), Ca10(PO4)6(OH)2. Tremendous efforts have been made to determine how various molecules interact with biologically derived and synthetic apatites in order to better understand their roles in hard tissue biomineralization and integrity. However, the interactions between bone crystal and other biological molecules are not understood at the molecular level due to the complexity of bone structures, the lack of a well-defined model system, and the lack of tools to investigate bone mineral's complex interfaces. Therefore, we used two crystal surfaces [(100) and (001) HAP] as models to study events at the bone interface: The former was used to model the bone surface at which proteins interact, and the latter was used to model the surface at which bone crystal growth dominantly occurs, when interfaced with collagen. Using in situ atomic force microscopy techniques, we investigated these HAP crystals under precisely controlled fluid conditions and demonstrated the first detailed characterization of hydroxyapatite interface properties at the molecular level (Fig.6). Based on this crystal structure dependent (100) HAP model system, we investigated bone mineral interface properties under precisely controlled conditions and found multiple new discoveries related to bone and tooth health:

Figure 6. Structure dependent demineralization morphology of the (100) HAP bone model surfaces. (A) Defect-induced hexagonal shaped etch pit. (B) Fluoride-induced triangle morphology. Schematic inset shows that fluoride ions can bind to steps in a structure dependent manner. The triangle exhibited mirror symmetry through the horizontal plane. (C) Diamond shaped etch pit morphology induced by L-aspartic acid that exhibited symmetry breaking through the mirror axis. (Kwon et al, Langmuir 2009, 2011)

  1. High ionic concentrations of NaCl suppress bone demineralization: Na+ and Cl- ions are the major contributors to the ionic concentration in our bodies. Our publication (Kwon et al, JPC 2009) showed that high ionic concentrations of NaCl play a critical role in stabilizing bone crystals. Our findings suggest that typical Na+ and Cl- ion concentrations (140 mM) in extracellular body fluids may play a significant role in bone mineral stability.
  2. Bone defects accelerate the demineralization of bone crystal: Defects in bone minerals play a critical role in the bone remodeling processes. Our paper (Kwon et al, Langmuir, 2008) demonstrated that local structural defects on the model bone surfaces significantly accelerated demineralization (Fig. 6A). We characterized demineralization both in the presence and absence of defects. Our characterization showed that defects in bone could be easily removed by exposure to acidic buffers secreted by osteoclasts. Crystal surfaces with defects are dissolved ~100 times faster than non-defected surfaces.
  3. The anti-cariogenic property of fluoride originates from the specific binding of fluoride to specific HAP steps: Fluoride ions play a critical role in preventing tooth decay. The work reported in our paper (Kwon et al, Langmuir 2011) verified the molecular mechanism behind the anti-cariogenic properties of fluoride. Through in situ dynamic characterization of steps on the (100) HAP surfaces under medically relevant fluoride concentrations (ranging from those found in tap water and mouth rinses), we measured the dynamic evolution of the crystal morphology. Changes to individual steps across a wide range of fluoride concentrations suggested that the anti-cariogenic property of fluoride ions originates from their strong interactions with the molecular steps in a structure dependent manner (Fig. 6B).
  4. Chiral interaction with L-aspartic acid induces chiral dissolution of bone surfaces: Organic modifiers (i.e., amino acids, peptides, and proteins) play critical roles in bone remodeling. Our work (Kwon et al, in preparation) presented the first detailed experimental investigation of specific structure dependent chiral interactions between L-aspartic acid (Asp) and HAP crystal surfaces (Fig. 6C). Aspartic acid residues are enriched in bone-associated proteins. Bone mineralization and demineralization are believed to be highly influenced by aspartic acid. We verified specific aspartic acid interactions with individual HAP molecular steps during the HAP dissolution process. Our study provides fundamental insight into the interactions between organic modifiers and bone crystals and expands our understanding of the morphogenesis of bone and tooth crystals.

Discovery of collagen-like peptide that nucleated HAP crystals: Based on our understanding of the bone model surfaces, we investigated the molecular level interactions between well-defined model bone surfaces (HAP (100) surfaces) and HAP-binding peptides discovered through phage display. The formation of natural bone is thought to occur by the templated mineralization of HAP by surrounding proteins, which include collagen and highly acidic phosphoproteins associated with the collagen scaffold. It has been proposed that the acidic groups serve as binding sites for calcium ions and align them in an orientation that matches the HAP crystal lattice. However, the biological mineralization process is not understood at the molecular level. An appealing strategy for biomimetic bone synthesis would be to employ HAP-binding peptides to facilitate HAP crystal growth. Unfortunately, the rational design of such peptides is deterred by a lack of knowledge regarding the sequences that guide HAP mineralization in vivo.

Using phage display, we identified a 12-residue peptide that bound to single crystal (100) HAP surfaces under physiological pH conditions (pH 7.5) (Langmuir 2011). This peptide was able to template the nucleation and growth of crystalline HAP minerals in a sequence- and composition-dependent manner (Fig. 7). The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp; Hyp: Hydroxyproline) of type I collagen, a major component of the bone extracellular matrix. Using a panel of synthetic peptides, we defined the structural features required for mineralizing activity. The results suggest a model for the cooperative non-covalent interaction of the peptide with HAP and support the theory that native collagen has a mineral-templating function in vivo.

Figure 7. Transmission electron micrograph of mineralized HAP crystal templated by collagen like peptide identified from phage display (CLP12) peptide (Langmuir 2011).

Microscopic level interactions at bone biointerfaces: We investigated microscopic bone protein-crystal interactions to develop biomimetic, organic-inorganic nanocomposite materials. The organic component of bone materials is comprised of collagenous and non-collagenous protein matrices. Hierarchical collagenous protein matrices are known to provide a structural framework for templating mineralized bone crystals, whereas non-collagenous proteins in bone bind the mineral phase, transfer strain, and dissipate energy through the breaking of 'sacrificial bonds'. These proteins form various bonds through non-covalent intra-chain, inter-chain, and interfacial interactions. Because of their importance in nature, it would be extremely beneficial to further explore the rational design of synthetic organic phase analogues. Because conventional approaches of utilizing off-the-shelf polymers and naturally derived biological materials are very limited and fail to emulate the highly tuned sequences of natural proteins, we designed sequence specific biomacromolecules that closely emulate the function of bone protein matrices using biological approaches. Specifically, we developed sequence specific elastin-based protein matrices and phage-based collagen-mimetic nanofiber matrices.

Bioinspired elastin-based bone nanocomposite materials: Non-collagenous protein matrices play multiple critical roles in bone mechanical properties. We presented our approach of emulating the mechanical enhancements observed in nature by designing sequence specific protein-based polymers and incorporating them into calcium phosphate bone cement materials (Wang et al, Biomacromolecules 2011) . We developed novel elastin-like polymers (ELP) to construct mechanically enhanced calcium phosphate cement (CPC)-protein nanocomposites. By controlling ELP primary structures at the molecular level through genetic engineering, we could synthesize protein matrices to mimic the adhesive proteins of bone. We incorporated octaglutamic acid, a previously characterized HAP binding motif, at the protein termini and positively, negatively, or neutrally charged amino acids along the polymer backbone to control intramolecular, intermolecular, and interfacial interactions with HAP. By imparting ELPs with varying modes of inter/intra molecular bonding and interfacial bonding, we were able to demonstrate sequence specific property changes in ELP-HAP mixtures (Fig. 8A). Our results showed that interfacial binding to HAP through octaglutamic acid motifs was critical for imparting functionalities such as nanoparticle dispersion and mechanical property improvements in ELP-CPC composites (Fig. 8B). In the absence of binding, the physical properties of the protein had no effect. We also demonstrated the injectability of the ELP-CPC composites, which transformed into nanocomposites at body temperature in vitro. We believe that our results are not limited to applications involving HAP but can be expanded to explore composites containing other metals and minerals. Our design and synthesis approaches may yield strong and tough materials for future load-bearing applications such as hard-tissue replacements.

Figure 8. Sequence specific elastin-based bionanocomposite: (A) A: Backbone-backbone Van der Waals interactions or hydrogen bonding; B: ELP-HAP binding; C: Backbone-backbone ionic interactions; D: Ion-mediated crosslinks. (B) Mechanical characterization of bone cements with and without ELP composites (Wang et al, Biomacromol. 2011).

Light responsive hydrogel elastin-graphene nanocomposite materials: We expanded our elastin-based composite material design theme to develop a bio-inspired light-responsive soft-robotic materials using protein engineering. Hydrogel actuators (HAs) are water swollen polymer networks that reversibly change their dimensions or shapes when external stimuli (e.g., solvent composition, temperature, and external fields) cause local or global changes in network swelling. HAs with tunable speed and motion are highly sought after to fulfill applications in various fields, including biology, medicine, microfluidics, and robotics. To fulfill the bio-related applications, HAs which utilize materials and stimuli that are compatible with biomolecules and cells are also desired.

Light-driven HAs are particularly attractive systems but have limitations that must be addressed to expand their utility. Light is applied remotely, so solution-wide changes and the introduction of potentially invasive wires or electrodes are avoided. In addition, light is easily controlled with higher spatial and temporal resolution than other stimuli. However, macroscale, photothermal HAs exhibit slow actuation kinetics (on the order of minutes), due to limited rates of water diffusion into and out of the networks. In addition, previous photothermal HAs had isotropic structures that changed size relatively evenly in each dimension, making non-linear motions difficult to achieve. In contrast, actuation into more complex shapes can be achieved by creating hydrogel networks with anisotropic composition, crosslinking, or porosity. With these limitations in mind and with an eye towards future bio-related applications, we created light-driven HAs that could rapidly undergo non-linear motions by combining reduced graphene oxide (rGO) nanosheets and elastin-like polypeptides (ELPs).

In our recent work (Wang et al, Nano Letters 2013) we synthesized a new chimeric ELP fused with the graphene binding (GB) peptide 'HNWYHWWPH'. This ELP-GB can physically bind to reduced graphene oxide (rGO) and graphene oxide (GO) via π-π stacking and keep the nanomaterials dispersed. The composite created as a result is light-responsive as rGO and GO act as photothermal heaters by absorbing light and locally generating heat while ELP absorbs the heat and responds mechanically by contracting. The ELP-GB chimera was further modified with the addition of an/the 'RGD' cell adhesive peptide (Wang et al, Langmuir 2014) to show the ease of improving cell behavior on these composites. We also synthesized hydrogels using the ELP-GB composites along with an additional ELP containing a lysine residue near the C-terminus; the primary amine groups were cross-linked together using an N-hydroxysuccinimide functional 4-armed PEG linker in organic solvents. A simple method of creating anisotropic gels was employed by allowing water vapor to freely diffuse into the water-miscible organic solvents as the gel cross-linked and the resulting gel contained distinct porous and solid layers. This gel is near infrared (nIR) light responsive as the rGO absorbs nIR and generates heat that causes ELP to transition and the gel to contract. Instead of an isotropic contraction, the porous layer of the gel contracts much faster than the solid side, resulting in bending of the gel. We demonstrated precise spatial and temporal control of the light-responsive actuation of these hydrogels (Fig. 9) raising the potential of these soft actuators for application as light-responsive drug delivery devices as well as remotely controllable soft actuators.

Figure 9. Light responsive nanocomposite. Lee research group synthesized near-infrared light-driven hydrogels by interfacing genetically engineered elastin-like polypeptides with reduced-graphene oxide sheets. The resulting nanocomposites exhibited rapid and tunable motions controlled by light position, intensity, and path, including finger-like flexing (Wang et al Nano Letter 2013).

E. PROTEIN-ELECTRIC INTERFACES

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A. Protein-Protein Interfaces
B. Protein-Cellular Interfaces
C. Protein-Organic Material Biointerfaces
D. Protein-Inorganic Interfaces
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The primary goal of this research is to interrogate protein structures and electric dipole relationships for the development of clean and green electric energy from the bio-inspiration of collagen-based piezoelectric properties in our body.

Biopiezoelectric energy generation: Piezoelectric materials are an attractive means of producing renewable and clean energy by utilizing ubiquitous vibrational and mechanical energy sources. The piezoelectric effect can be defined as the interconversion between mechanical and electrical energies induced by charge redistribution and separation upon the application of a mechanical or electrical stimulus to materials that lack inversion symmetry. Various inorganic piezoelectric materials exhibit strong piezoelectric properties, and have been used to generate electrical energy through commonplace activities, such as walking. Recently, several nanomaterials have been used to efficiently produce electrical energy by scavenging vibrational energy enough to operate small electronic devices. In addition, aligned, electrospun organic nanofibers have exhibited piezoelectric energy conversion efficiencies an order of magnitude higher than those of bulk films. Although previous piezoelectric materials have provided useful schemes for energy generation, they are often made using environmentally harmful chemicals and/or energy intensive conditions, e.g., high temperatures, high electric fields, and extreme-pressures. In contrast, nature produces diverse, hierarchically organized functional materials at near-ambient temperatures using benign and abundantly available resources. These materials, which span in size from the nano to macroscale, include viral particles, abalone shells, teeth and bones. Interestingly, the piezoelectricity of biomaterials has been associated with critical physiological activities such as bone growth and remodeling. Such biopiezoelectricity occurs because biological building blocks (nucleotides and amino acids), biopolymers (DNA and polypeptides), and their higher-order assemblies lack inversion symmetry. Although biopiezoelectric materials pervade natural systems and present advantages over synthetic materials, they have not been utilized for engineered systems. This is mainly due to a lack of cost effective fabrication methods and little understanding of how to control biopiezoelectric properties at the molecular level.

Figure 10. Schematic of piezoelectric M13 phage structure. a. The M13 phage is 880 nm in length and 6.6 nm in diameter, is covered by 2,700 pVIII coat proteins and has five copies each of pIII (grey lines) and pIX (black lines) proteins at either end. b. Side view of the electrostatic potential of M13 phage. The dipole moments generated by ten α-helical major coat proteins are directed from the N-terminus (blue) to the C-terminus (red). Yellow arrows indicate dipole direction. (Lee et al, Nature Nano. 2012)

In our recent work (Lee et al, Nature Nanotechnology 2012), we fabricated novel bioinspired piezoelectric materials by exploiting the naturally aligned dipole structure of a bacterial virus, M13 phage. Structurally, M13 phage possess piezoelectric material features. The M13 phage has a long rod-like shape, 880 nm in length and 6.6 nm in diameter (Fig. 10a). Each phage is covered by 2,700 copies of a major coat protein (pVIII). The pVIII proteins have an a-helical structure with a dipole moment directed from the amino- to the carboxy-terminal direction and cover the body of the phage with five-fold rotational and two-fold screw symmetry (Fig. 10b). Because the M13 aligned protein coat structure lacks inversion symmetry, we hypothesized that the phage will possess intrinsic piezoelectric properties. Therefore, we first characterized the piezoelectric property of the M13 phage and developed a virus-based electric energy generator. Using piezoresponse force microscopy techniques through the collaboration with Dr. Ramesh, we characterized the structure-dependent piezoelectric properties of the phage at the molecular level. We discovered that the M13 phage possess axial and lateral piezoelectric properties. Their intrinsic piezoelectric response is 0.30 pm/V. Through the genetic engineering of the M13 phage, we enhanced the piezoelectric properties of the phage upto 0.70 pm/V. We also discovered that through the multi-layered films, we could enhance the piezoelectric properties up to 7pm/V. In 2012, we successfully demonstrated that a phage-based piezoelectric device could produce up to 6 nA and 400 mV which is enough to operate an LCD display panel (Fig. 11). More recently, we further improved the phage dipole strength and its self-assembly process in order to enhance the efficiency of the energy conversion of the phage-piezoelectric materials upto 100 nA and 1 V level (Manuscript is currently under preparation). Our phage-based piezoelectric material presents an adaptable and cost-effective means of harvesting energy from the environment and is an important step toward accessing the largely untapped potential of piezoelectric biomaterials.

Figure 11. The first virus-based piezoelectric device that can operate a LCD display panel to show "1" by tapping the virus thin film device (Lee et al, Nature Nanotech. 2012).

The newly developed biopiezoelectric technology has attracted a lot of attention. Our virus electric energy generation was chosen as one of the 17 Biggest Scientific Breakthroughs of 2012 by iOS9. Scientific American chose this energy generating technology as one of top five future nanomanufacturing technologies, and it was also awarded R&D 100 award in 2013 as well.

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