June 18th 2015, ASRC
Enzyme-Instructed Self-Assembly: A Multi-Step Process for Potential Cancer Therapy
Bing Xu Brandeis University
The central dogma of the action of current anticancer drugs is that the drug tightly binds to its molecular target for inhibition. The reliance on tight ligand-receptor binding, however, is also the major root of drug resistance in cancer therapy. In this talk, we highlight enzyme-instructed self-assembly (EISA) - the integration of enzymatic transformation and molecular self-assembly - as a multi-step process for the development of cancer therapy. After the introduction of enzyme-instructed self-assembly of small molecules in the context of supramolecular hydrogelation, we describe several key studies to underscore the promises of enzyme-instructed self-assembly for developing cancer therapy. Particularly, we will highlight that enzyme-instructed self-assembly allows one to develop approaches to target "undruggable" targets or "untargetable" features of cancer cells and provides the opportunity for simultaneously interacting with multiple targets. We envision that enzyme-instructed self-assembly, used separately or in combination with current anticancer therapeutics, will ultimately lead to a paradigm shift for developing anticancer medicine that inhibit multiple hallmark capabilities of cancer.
Pathological Crystals: From Spirals to Therapies for Stone Disease
Michael Ward New York University
The crystal growth of conventional materials like silicon has been refined for decades and has led to textbook crystal growth models. Confidence in these models quickly evaporates when considering complex inorganic solids and molecular crystals, however, despite the importance of these materials to technology, biology, and human health. In particular, many crystalline materials are associated with diseases, from malaria to kidney stones. This presentation will illustrate the beauty and complexity of crystal growth, through mechanisms often hidden and deceptive, in pathological molecular crystals, including kidney stones. Armed with an understanding of some crystal physics and crystal surface structure at the nanoscale level, we can design crystal growth inhibitors that bind to specific crystal sites and prevent the formation of certain kidney stones, suggesting a pathway to therapies for crystal-based diseases in general.
A Modular Method to Synthesize Multimodal High-density Lipoprotein-derived Nanoparticle Contrast Agents Using Microfluidics
Francois Fay Icahn School of Medicine at Mount Sinai
High Density Lipoprotein (HDL) is a natural nanoparticle involved in the transport of cholesterol throughout the body. HDL has been shown to exhibit atheroprotective properties as it promotes cholesterol efflux from atherosclerotic plaque macrophages in the arterial wall. Various laboratories have focused on the reconstitution of HDL (rHDL) for a variety of reasons, ranging from a better understanding of the structural biology of apolipoproteins to the use of rHDL as an injectable therapeutic. A recent effort centers around the use of rHDL as a natural nanoparticle platform for the delivery of contrast agents such as gadolinium chelates, iron oxide or gold nanoparticles, and employing them as molecular imaging contrast agents. To date, multistep production protocols pose a limit on the synthesis of batch quantities and are sensitive to inter-batch variations. In order to scale up the production process and to judiciously control rHDLs composition we have developed a modular single-step approach based on recently introduced microfluidics technology that enables the standardized mass production of such lipoprotein-based nanoparticles.
Organic solutions containing phospholipids and imaging agents (QD, FeO-NP, Au-NP, DiO) were injected into a microfluidic chip alongside an aqueous solution containing ApoA1. Within the chip the controlled flow streams generate microvortices where fast mixing of the solutions leads to the instantaneous formation of HDL-like nanoparticles. HDL particles produced by this microfluidics method, which we refer to as µHDL, had similar physicochemical properties (size, morphology) to particles produced by conventional methods and natural HDL. Moreover cell based assays demonstrated that µHDL nanoparticles displayed a similar bioactivity profile to natural HDL. µHDL that encapsulated hydrophobic dies (DiO) or nanocrystals such as quantum dots (QD), gold (Au) or iron oxide (FeO) nanoparticles were characterized and evaluated in model murine macrophage cell line. We observed all the different versions to have excellent diagnostic properties, to be specifically taken up by macrophages and rendered these cells visible for either magnetic resonance imaging (FeO-µHDL), computed tomography (Au-µHDL), or fluorescence imaging (QD-µHDL).
We have developed a microfluidics-based method to produce bioactive multifunctional HDL-like nanoparticles that can be used for molecular imaging. This single step production process will facilitate the optimization of existing HDL nanoparticle platforms and could accelerate the development of new formulations and applications.
Directed Self-Assembly and Crystallization of Colloids
Marcus Weck New York University
The self-assembly of colloidal particles into well-defined 3D structures is of great interest for potential applications in biomaterials, catalyst supports, and photonics. The presentation will introduce a general method to functionalize patchy colloidal particles with well-defined symmetries with either sticky-ended DNA or metal-coordination recognition sites creating colloids with directional interactions; the "valence" is determined by the number of patches. Patch-patch attractions are realized by specific DNA hybridization or by a single metal-coordination step and the binding directionality is predetermined by the patch geometry. This strategy allows for the possibility of building new low-coordinated open structures, both amorphous and crystalline.
DNA-Programmable Nanoparticle Assembly
Oleg Gang Brookhaven Laboratory
In the last decades nanoscale inorganic objects emerged as a novel type of matter with unique functional properties and a plethora of prospective applications. Although a broad range of nano-synthesis methods has been developed, our abilities to organize these nano-components into designed architectures and control their transformations are still limited. In this regard, an incorporation of bio-molecules into a nano-object structure allows establishing highly selective interactions between the components of nano-systems. Such bio-encoding may permit programming of complex and dynamically tunable systems via self-assembly: biomolecules act as site-specific scaffolds, smart assembly guides and reconfigurable structural elements.
I will discuss our advances in addressing the challenge of programmable assembly using the DNA platform, in which a high degree of addressability of nucleic acids is used to direct the formation of structures from nanoscale inorganic components. Our work explores the major leading parameters determining a structure formation and methods for creating targeted architectures. The principles and practical approaches developed by our group allow for assembly of well-defined three-dimensional superlattices, two-dimensional membranes and finite-sized clusters from the multiple types of the components. I will also discuss how interplay of polymeric and colloidal effects can result in the novel interactions effects in these systems. Our recent progress on the assembly by-design, including super-lattices with pre-defined crystallographic symmetries and particle clusters with pre-determined architectures will be demonstrated. Finally, I will present several approaches for the dynamical control of assemblies, which allow for the post-assembly structural manipulation and selective triggering of system transformations.
Research is supported by the U.S. DOE Office of Science and Office of Basic Energy Sciences under contract No. DE-AC-02-98CH10886.
Hydrophobic Collagen Peptide Nanodiscs Act as Substrates and Scaffolds for the Noncovalent Organization and Higher Order Assembly of Natural Proteins
Kenneth McGuiness Rutgers University
Multi-component functional structures are prevalent in the cellular environment and difficult to design. Chromosomes, the tropomyosin-actin complex, and collagen fibers are examples of functional multi-component structures nature has evolved to store genetic information, build muscle, and provide bones flexibility and support. Our goal is to understand and create design principles for developing functional self-assembling multi-component biomaterials. We do this through the use of a scaffold-substrate technique involving noncovalent interactions between synthetic peptides and natural proteins. We target exposed hydrophobic groups along the fibrous proteins collagen, tropomyosin and alpha-synuclein to act as nascent scaffolding sites for the organized binding of hydrophobic collagen peptide nanodisc substrates. Binding of the nanodiscs further promotes higher-order assembly of collagen and tropomyosin. Additionally, the nanodiscs are used as a scaffold for the organization of the light harvesting complex I membrane protein. The use of hydrophobic interactions to drive multi-component assembly provides a means for dynamic processes to occur such as cargo delivery. Engineering peptides to bind to non-evolutionary designed interactions sites on proteins is a promising technique for developing novel functional biomaterials.
Soft Supramolecular Nanotubes for Robust Light Harvesting?
Dorthe Eisele City College of New York
The most remarkable materials that demonstrate the ability to capture solar energy are natural photosynthetic systems such as found in rather primitive marine algae and bacteria. Their light-harvesting (LH) antennae are crucial components, as they absorb the light and direct the resulting excitation energy efficiently to a reaction center, which then converts these excitations (excitons) into charge-separated states. Nature's highly efficient light-harvesting antennae, like those found in Green Sulfur Bacteria, consist of supra-molecular building blocks that self-assemble into a hierarchy of close-packed structures.
In an effort to mimic the fundamental processes that govern nature's efficient systems, it is important to elucidate the role of each level of hierarchy: from molecule, to supra-molecular building block, to close-packed building blocks. Here, I will discuss the impact of hierarchical structure. I will present a model system that mirrors nature's complexity: cylinders self-assembled from cyanine-dye molecules. I will show that even though close-packing may alter the cylinders' soft mesoscopic structure, robust delocalized excitons are retained: internal order and strong excitation-transfer interactions - prerequisites for efficient energy transport - are both maintained. These results suggest that the cylindrical geometry strongly favors robust excitons; it presents a rational design that is potentially key to Nature's high efficiency, allowing construction of efficient light-harvesting devices even from soft supra-molecular materials.
Computational Design of Hierarchic Peptide Self-Assembly
Vikas Nanda Rutgers Univeristy
Advances in protein structure modeling has allowed the design synthetic proteins with enhanced properties and new functions. However, the engineering of protein fibers such as collagen has proved challenging, given our limited understanding of how these proteins fold and the molecular forces that hold them together. Collagen assembles in a hierarchic fashion from nanometer scale molecules to micron and millimeter scale fibers and gels. We explore the properties of synthetic collagen-like assemblies whose properties are providing insights into the mechanisms by which natural collagens fold and assemble.
Visualizing Biomolecular Structure, Hybridization, and Enzymatic Accessibility on a Carbon Nanotube Surface
Jena Prakit Memorial Sloan Kettering Cancer Center
Single walled carbon nanotubes have unique physico-chemical properties that have promoted their development for imaging and sensing applications in vitro and in vivo. Nanotubes non-covalently functionalized with single strand DNA maintain their intrinsic near-infrared fluorescence. We present a fluorescence-based single molecule imaging platform to determine whether DNA on the nanotube retains its native recognition ability for nucleic acids and proteins i.e. is it biologically active? We quantify the ability of ssDNA on a nanotube to form duplexes as a function of sequence, and the accessibility of DNA to restriction endonucleases. Exploiting the existence of multiple emitting fluorophores in a diffraction limited spot, we demonstrate super resolution imaging to localize DNA duplexes on a carbon nanotube surface with sub-20 nm resolution.
Medical Nanotechnology: Generation of Nanoparticles in Complex Shapes and Label-free Cancer Cell Detection/enrichment
Hiroshi Matsui Hunter College/Cornell Medical School
1) It has been difficult to fabricate nanoparticles less than 10 nm with complex shapes that display a number of atomic edges known to catalyze chemical reactions. We developed a new method to fabricate such inorganic nanoparticles by etching specific crystalline faces in micelles and/or peptide assemblies. In this compartment, atoms are desorbed from specific crystalline faces of nanoparticle seeds, dependent on the coverage of capping peptides, and we could control this balance to evolve shape and structure of inorganic nanoparticles into targeted ones. This approach is universal to any inorganic nanoparticles. Pd, Au, and iron oxide nanocages were demonstrated to be synthesized for catalytic and medical applications. These nanoparticles are also coated by a new ligand, dextran-functionalized catechol. By this capping approach, each nanocage has an extremely stable and biocompatible surfactant. Dextran is also porous so that this capping does not compromise drug release when drug molecules are loaded in the cavity of nanocage. And the dextran-functionalized catechol displays carboxylic acid groups so that various drugs, antibodies, and biomarkers can be conjugated inside and outside of inorganic nanocage.
2) A new cancer detection platform incorporating electric cancer cell sensors on silicon chips will be discussed. This sensing platform was designed to distinguish cells in different sizes and shapes by measuring their characteristic impedance signals on polysilicon microelectrodes. Due to the softness of cancer cells as compared to normal cells, cancer cells were observed to swell three times more than normal cells under hyposmotic pressure. By using this sensor chip and protocol, cancer cells can be distinguished from other cells electronically without biomarkers; as strong hyposmotic stress is applied to cells, only cancer cells increase impedance signals due to the distinguished mechanical property. For example, we have examined six different cancer cell lines from prostate, kidney, ovarian, and breast, and all of these cancer cells were observed to expand their size about 35-50 % under osmotic pressure and their swellings could be detected sensitively and selectively by the robust impedance measurements of the sensor chip on the order of 10 cells/mL in less than 30 minutes even in contaminated samples. The aggressive breast, prostate, and bladder cancer cells could also be distinguished from less aggressive ones by measuring impedance values of the samples, opening the possibility that this technique could help grade various cancers. After the detection, these cancer cells can be separated selectively by applying specific AC frequency via negative dielectrphoresis and they are enriched through a microfluidic device. Nanoscale force measurements with AFM reveal that depolymerization of cytoskeleton leads to soften cells, indicating that actin polymers paly important roles in the cell rigidity. The development of non-invasive screening device for cancers with high specificity and selectivity enables more frequent monitoring of the early stage disease development, progress, recovery, and recurrence of cancers.
Functional Electrospun Polymeric Tubes as Biodegradable Micro-rockets
Amit Sitt Columbia University
Autonomous motion of micrometer scale objects through a fluid environment has long been a challenge. One method for propelling microsystems is by bubble ejection, where movement is obtained through expelling gas bubbles formed by decomposition of fuels such as hydrogen peroxide inside tube-shaped particles. Such systems are usually referred to as "micro-rockets" because of the resemblance of their propulsion mechanism to that of macroscale rockets.
In this work we present a novel type of biodegradable micro-rockets which are based on microscale polymer tubes fabricated using electro-spinning. Throughout the fabrication process, the chemistry of both the interior and exterior of the tubes can be defined and controlled, allowing the transformation of the tubes into micro-rockets by selective deposition of enzymes or inorganic nanoparticles in the interior. Furthermore, under external stimulus these particles can shape-shift into crescent-like form, which allows entrapment of materials inside them. The combination of simple fabrication, high control over the dimensions, biocompatibility and chemical modification make these micro-rockets attractive for a large range of biological and industrial applications.
Engineering of the Small Laccase (SLAC) from Streptomyces coelicolor for Incorporation into Bioelectrochemical Systems
Scott Banta Columbia Univeristy
The small laccase (SLAC) from Streptomyces coelicolor has many beneficial features for use in bioelectrochemical systems including recombinant expression in E. coli and high activity at neutral pH. We are using protein engineering strategies to improve the enzyme for bioelectrocatalysis applications.
First we have engineered the SLAC enzyme to self-assemble into amorphous proteinaceous hydrogels and we have included peptide-bound osmium complexes to improve electrical communication with an electrode. We have also focused on the integration of SLAC and carbon nanomaterials to improve biocathode performance. We have genetically appended the SLAC with a designed helical peptide, which binds to single-walled carbon nanotubes (CNTs) with high affinity. We have also exploited the interactions between zinc finger (Znf) protein domains and DNA. SLAC was genetically fused to Znf-268 and this fusion protein retained laccase activity while gaining the ability to bind DNA. The DNA has been attached to CNTs which provides a proof-of-concept for the nano-scale templating and arrangement of individual proteins during immobilization.
For the final approach, computationally designed biomolecular interactions were identified to engineer SLAC to self-assemble into crystalline-like assemblies. This was performed using the Rosetta methodology and led to the addition of strategically placed disulfide bonds at protein/protein interfaces. The new protein assemblies spontaneously form in an oxidative environment after purification and they are robust. Performance was further enhanced by entrapping CNTs in the crystals. The CNT containing crystals enable improved electrode kinetics and activity at high temperatures.
All of the new SLAC mutant proteins have been expressed, purified, and kinetically characterized. Different techniques have been explored to evaluate the assembly of the SLAC-SWNTs complexes and to characterize their activities. All these approaches have been successful in improving protein/nanomaterial interactions, which can lead to increased functional protein loading and improvements in overall activity in real electrochemical systems.
Self-Delivering Supramolecular Nanomedicine
Honggang Cui Johns Hopkins University
The creation of vehicles for the effective delivery of hydrophobic anticancer drugs to tumor sites has garnered major attention in cancer chemotherapies for several decades. A successful strategy promises immense benefits to cancer sufferers through both the reduction of side effects and a greater treatment efficacy. Current approaches focus on the use of nanosized carriers, whereby the drugs pharmacokinetic properties and biodistribution profiles are manipulated by encapsulation within, or by conjugation to the carrier. While these methods can be effective, there are concerns regarding the short-term and long-term toxicities arising from the synthetic nanomaterials other than the drug being delivered. Furthermore, there are inherent difficulties in achieving a quantitative and high drug loading per carrier (typically less than 10%). Our strategy presented here is to devise approaches that enable anticancer drugs to directly assemble into discrete well-defined nanostructures with the potential for self-delivery. In this presentation, I will cover two aspects of our recent research effort in the design and creation of self-delivering supramolecular assemblies for both cancer therapeutics and diagnosis. In the first part, I will detail our rational design of monodisperse, amphiphilic anticancer drugs - which we term drug amphiphiles (DAs) - that can spontaneously associate into discrete, stable supramolecular nanostructures with a high and fixed drug loading. Depending on the number and type of the drug in the molecular design, the resulting nanostructures could assume various morphologies, such as nanofibers, nanotubes or toroids. Our results suggest that formation of nanostructures provides protection for both the drug and the biodegradable linker from the external environment and thus offers a mechanism for controlled release. The second part of the presentation is focused on the design of supramolecular nanoprobes for cancer diagnosis. This strategy employs a molecular beacon approach to take advantage of the specific cleavage of certain peptide sequences by enzymes that are closely associated with cancer progression. Precisely probing the activities and expression level of these enzymes provides opportunities for cancer staging and possible early stage cancer diagnosis. Our results have shown that the designed supramolecular nanoprobe can be used as effective sensors for visualization and quantification of cathepsin B.
Short Peptides Self-assemble in the Presence of Metals to Produce Catalytic Amyloids
Ivan V. Korendovych Syracuse University
Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement that they be stably folded is a likely factor that contributes to their relatively large size (> 10,000 Dalton). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of 7-residue peptides that act as Zn2+-dependent esterases. Zn2+ helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. The fibril activity is on par with the most active to date zinc-protein complex. Such remarkable efficiency is due to the small size of the active unit (likely a dimer of 7-residue peptides), while the protein is at least 15-fold larger in molecular weight. The observed catalytic activity is by no means limited to ester hydrolysis. We have also designed copper binding peptides that are capable oxygen activation with remarkable efficiency.
These results open new opportunities for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions.