Symposium Organizers
Yizhou Dong, The Ohio State University
Christopher Alabi, Cornell University
Daniel Anderson, Massachusetts Institute of Technology
Bozhi Tian, University of Chicago
Symposium Support
Alnylam Pharmaceuticals, Inc.
Nanobio Delivery Pharmaceutical Co., Ltd.
Precision NanoSystems Inc.
BM07.01: Biomaterials for Therapeutic Applications I
Session Chairs
Christopher Alabi
Yizhou Dong
Monday PM, November 27, 2017
Sheraton, 2nd Floor, Grand Ballroom
8:00 AM - *BM07.01.01
Nano/Meso-Scale Principles and Applications with Flexibility—From Delivery and Self-Recognition to Differentiation
Dennis Discher 1
1 , University of Pennsylvania, Philadelphia, Pennsylvania, United States
Show AbstractFrom viruses to tissue matrices, biology is filled with remarkable polymeric structures that motivate mimicry with goals of both clarifying and exploiting biological principles. Filamentous viruses inspired our development and computations of worm-like polymer micelles – ‘filomicelles’ – that persist in the circulation and deliver even better than spheres [1,2]. However, particles of any type interact with innate immune phagocytes while nearby ‘Self’ cells are spared due to a polypeptide that limits phagocytic clearance [3]. The phagocyte’s cytoskeleton forcibly drives the decision downstream of adhesion, leading to a materials-inspired cell therapy [4] with pathways related to how matrix elasticity directs stem cell fate [5,6].
Key Words: block copolymer, self-assembly, shape, immunocompatability, hydrogel, mechanobiology, differentiation
References
[1] Y. Geng, P. Dalhaimer, S. Cai, R. Tsai, M. Tewari, T. Minko, and D.E. Discher. Shape effects of filaments versus spherical particles in flow and drug delivery. Nature Nanotechnology (2007) 2: 249-255.
[2] P.R. Nair, K. S. Anbazhagan, K.R. Spinler, M.R. Vakili A. Lavasanifar, and D.E. Discher. Filomicelles from aromatic di-block copolymers increase paclitaxel-induced tumor cell death and aneuploidy compared to aliphatic copolymer. Nanomedicine 11(12):1551-69 (2016).
[3] P.L. Rodriguez, T. Harada, D.A. Christian, D.A. Pantano, R.K. Tsai, and D.E. Discher. Minimal 'Self' peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science (2013) 339: 971-975.
[4] C.M. Alvey, K.R. Spinler, J. Irianto, C.R. Pfeifer, B. Hayes, Y. Xia, S. Cho, P.C.P.D. Dingal, J. Hsu, L. Smith, M. Tewari, and D.E. Discher. SIRPA-inhibited, marrow-derived macrophages engorge, accumulate, and differentiate in antibody-targeted regression of solid tumors. Current Biology 27:### (2017).
[5] A. Engler, S. Sen, H.L. Sweeey, and D.E. Discher. Matrix elasticity directs stem cell lineage specification. Cell (2006) 126: 677-689.
[6] J. Swift, I.L. Ivanovska, … and D.E. Discher. Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-directed Differentiation. Science (2013) 341: 1240104-1 to 15.
8:30 AM - BM07.01.02
Supramolecular Biomaterials Enabling Innovations in Drug Formulation and Delivery
Eric Appel 1
1 , Stanford University, Stanford, California, United States
Show AbstractSupramolecular biomaterials exploit rationally-designed non-covalent interactions to enable innovative approaches to drug formulation and delivery. For example, supramolecular interactions can be used to dynamically cross-linking polymer networks, yielding shear-thinning and self-healing hydrogels that allow for minimally invasive implantation in vivo though direct injection or catheter delivery to tissues. Alternatively, rationally designed high-affinity interactions can be used to non-covalently modify therapeutic proteins, endowing them with prosthetic function such as enhanced stability in formulation or extended activity in vivo. Herein, we discuss the preparation and application of shear-thinning, injectable hydrogels driven by non-covalent interactions between modified biopolymers (BPs) and biodegradable nanoparticles (NPs) comprised of poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA). Owing to the non-covalent interactions between PEG-b-PLA NPs and BPs, the hydrogels flow under applied stress and their mechanical properties recover completely within seconds when the stress is relaxed, demonstrating the shear-thinning and injectable nature of the material. The hierarchical construction of these biphasic hydrogels allows for multiple therapeutic compounds to be entrapped simultaneously and delivered with differential release profiles in vitro and in vivo. Moreover, delivery of loaded therapeutics can be tuned over several months, enabling novel long-term treatment strategies for a variety of chronic diseases. Further, we will discuss the use of supramolecular interactions to append functionality to therapeutic proteins for applications in formulation and therapy. This non-covalent approach to modification of authentic proteins is highly modular and allows for formulation of historically incompatible proteins. Overall, this presentation will demonstrate the utility of a supramolecular approach to the design of biomaterials affording unique opportunities in the formulation and controlled release of therapeutics.
8:45 AM - BM07.01.03
Heat-Generating Nanocomposite for Eradicating Bacterial Infections Associated with Indwelling Medical Implants
Nicole Levi-Polyachenko 1
1 , Wake Forest Health Sciences, Winston Salem, North Carolina, United States
Show AbstractStaphylococcus aureus is a common pathogenic bacterium prevalent in a wide variety of diseases, including progressive blood and tissue infections such as chronic ulcers, sepsis and osteomyelitis. Additionally, there exist many S. aureus subtypes which have developed resistance to broad-spectrum antibiotics. Part of the acquired antibiotic resistance may be attributed to the capacity for S. aureus to thrive in colonies within a protective matrix of polymers, polysaccharides, extracellular DNA and water (biofilm). Biofilm residing bacteria are known to be many times more resistant to select antibiotics than their planktonic counterparts.
One mechanism proposed for mediating biofilm-associated infections is to disrupt the polymer structure of the biofilm, leading to the release of planktonic bacteria which are more susceptible to antibiotic therapy. Chemical agents have been evaluated for accomplishing this; however, physical means, such as heat or ultrasound can serve two purposes by first disrupting the biofilm structure while also altering individual bacterial cell walls. Physical disruption may be especially advantageous for eradicating biofilms associated with implanted medical devices, alleviating the need for their removal and maintaining device function.
Silicone is a biocompatible elastomer that can either compose an entire medical device (catheters) or be used as a pliable surface coating on metal or ceramic implants. For the generation of heat, we have included photothermal donor–acceptor conjugated polymer nanoparticles based on poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b’]dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe). The composite materials were evaluated for the killing of planktonic and biofilm-residing S. aureus, in either the presence or absence of the antibiotic gentamicin. In the absence of near-infrared laser stimulation of the composite, addition of the nanoparticles exhibited an unexpected bactericidal effect. Stimulation of silicone alone with 612 J/cm2 of 800nm light had no effect on planktonic or biofilm S. aureus, whereas ablative temperatures in the nanoparticle doped composites resulted in a 95-100% reduction in viable bacteria. Sub-ablative temperatures resulted in a 75% reduction in planktonic bacteria treated with gentamicin and a 90% reduction in biofilm-residing S. aureus, compared to controls treated with gentamicin alone and no infrared stimulation. Our results demonstrate the potential for heat-generating biocompatible nano-composites to augment antibiotics used to treat S. aureus biofilms. These results offer a new avenue for the use of photothermal nanocomposites to eliminate challenging medical infections associated with in-dwelling medical devices.
9:00 AM - *BM07.01.04
Engineering Targeted Therapeutics for Breast Cancer
Debra Auguste 1
1 , Northeastern University, Boston, Massachusetts, United States
Show AbstractCells sense changes in their environment and respond by altering their gene expression. I investigate how cells manipulate membrane proteins, which has profound effects on disease progression. Cells orchestrate the density of proteins and lipids to govern adhesion and migration. From this knowledge, one can engineer drug delivery vehicles that complement the molecular patterns observed on cells to achieve strong, cooperative binding. I have employed these strategies in a model system of endothelial inflammation and in breast cancer metastasis. My lab has identified a new target and biomarker for triple negative breast cancer, examined the role of ligand/receptor cell adhesion by atomic force microscopy, and synthesized targeted drug delivery vehicles that demonstrate that nanoparticle ligand surface density alters gene expression. The basis for the targeted drug delivery platform lies at the intersections of biology, engineering, and medicine. We have used these vehicles to identify new strategies in the delivery of molecules and nucleic acids.
10:00 AM - BM07.01.05
Bioinspired Antioxidative Nanomaterials for Controlling Ischemic Brain Injury
Jinjun Shi 1
1 , Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts, United States
Show AbstractAntioxidative nanomaterials are emerging as a novel strategy for treating a myriad of important diseases through scavenging excessive reactive oxygen and nitrogen species (RONS), a mechanism critical in disease development and progression. However, similar to antioxidative enzymes, currently studied nano-antioxidants have only demonstrated scavenging activity to specific RONS, and sufficient antioxidative effects against multiple RONS generated in diseases remain elusive. Herein, we develop bioinspired melanin nanoparticles (MeNPs) for more potent and safer antioxidative therapy. We provide exhaustive characterization of the activities of MeNPs against multiple RONS including O2●−, H2O2, ●NO, ●OH, and ONOO–, the main toxic RONS generated in diseases. The potential of MeNPs for antioxidative therapy has also been evaluated in vitro and in a rat model of ischemic stroke. In addition to the broad defense against these RONS, MeNPs can also attenuate the RONS-triggered inflammatory responses through suppressing the expression of inflammatory mediators and cytokines. In vivo results further demonstrate that these unique multi-antioxidative, anti-inflammatory and biocompatible features of MeNPs contribute to their effective protection of ischemic brains with negligible side effects.
10:15 AM - BM07.01.06
Clickable Microgel Scaffolds as Platforms for 3D Cell Encapsulation
Alexander Caldwell 1 2 , Gavin Campbell 1 2 , Kelly Shekiro 1 2 , Kristi Anseth 1 2
1 Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, United States, 2 The Biofrontiers Institute, University of Colorado Boulder, Boulder, Colorado, United States
Show AbstractPorous hydrogels have proven to be valuable cell culture scaffolds both in vitro and in vivo, as they permit increased cell motility, proliferation, and matrix formation. While top-down approaches to pore formation are commonly used, bottom-up assembly methods afford the opportunity to create complex and unique microenvironments. We investigated the use of microgel building blocks for the bottom-up fabrication of porous cell-laden scaffolds. 8-arm, 20kDa and 4-arm, 10kDa poly(ethylene glycol) (PEG) macromers were functionalized with dibenzocyclooctyne (DBCO) and azide groups, respectively. An inverse suspension type polymerization was used, with polymer networks forming through a strain promoted alkyne-azide cylcoaddition (SPAAC) reaction. Polymerizations were performed off-stoichiometry to yield microgels with either excess surface DBCO or azide moieties. While the initial formulation led to relatively stiff microgels (compressive moduli between 10-15kPa), variation of PEG macromer size and functionality, as well as polymer weight percent during polymerization, could be used to alter microgel mechanical properties. Furthermore, variation of applied shear stress during polymerization allowed for control over particle size, yielding disperse microgel populations with mean diameters of approximately 10 and 100 μm. Microgels with complementary reactive groups were then assembled into interconnected networks, with covalent interactions forming between complementary microgels through the same SPAAC reaction. These networks were softer, with compressive moduli of 3kPa and 2kPa for scaffolds consisting of 10 and 100 μm microgels, respectively. There was a distribution of pore sizes, with average major axes lengths on the order of particle diameters (10 and 100 μm), while overall porosity was 10% and 30% for scaffolds comprised of 10 and 100 μm microgels, respectively. The rapid, cytocompatible nature of the reaction allows for human mesenchymal stem cells to be incorporated throughout the scaffolds with high viability. Furthermore, the used SPAAC chemistry provides the opportunity for facile incorporation of bioactive moieties. We demonstrate this concept with the incorporation of an azide functionalized adhesive peptide, GRGDS, resulting in cell spreading throughout the matrix. The distinct microenvironments created by the varying pore sizes exhibited control over cell morphology and cytoskeletal formation. Cells in large pores were highly spread with visible actin filament formation, while those confined to smaller pores were more circular with disorganized cytoskeletons. These “building block” systems offer the opportunity to direct many aspects of cellular growth independently, as well as the ability to recreate complex biological environments.
10:30 AM - BM07.01.07
Peptide Hydrogels for Three-Dimensional Cell Culture and High Throughput Drug Screening
Peter Worthington 1 2 , Zhiqin Li 2 , Katherine Drake 2 , Andrew Napper 3 , Sigrid Langhans 2 , Darrin Pochan 1
1 , University of Delaware, Newark, Delaware, United States, 2 , AI duPont Nemours Hospital for Children, Wilmington, Delaware, United States, 3 , Fxbio, San Francisco, California, United States
Show AbstractHigh throughput screening (HTS) is a method of drug discovery that utilizes automation to rapidly test hundreds of thousands of drug compounds against a designed disease state model to quickly hone in on promising drug candidates. The power behind HTS is its ability to screen such a high amount of compounds, but this requires the disease state model to be fairly simple in concept and amenable to standard liquid handling practices. The result is most HTS cell culture assays are cellular monolayers on treated plastic or a solution of suspension cells; using this type of model results in hits that are therapeutically effective in an environment much different from the in vivo environment that the disease model is attempting to reproduce. The goal of this research is to create a three-dimensional (3D) cell culture model that will result in more clinically relevant HTS hits and at the same time not impede standard HTS practices. Beta-hairpin peptide hydrogels can serve as the matrix because of their tunable physical properties, cytocompatibility, and shear thinning/rehealing properties. The hydrogel is formed by addition of peptides to cell culture medium with desired cells suspended and eventually encapsulated due to peptide nanofibrillar network assembly. A unique advantage of the hydrogel is its ability to shear thin and flow during shear common during syringe injection. When the shear is stopped the network immediately reheals back into a hydrogel solid. MAX8 (VKVKVKVKDPPTVKVEVKVKV) is the parent peptide used for the HTS assay development; it forms a hydrogel at physiological conditions, ideal for cell culture. Different hydrogel concentrations were tested and cell attachment ligands (RGDS, IKVAV YIGSR) were added to the hydrogel backbone to investigate the hydrogels effect on encapsulated cell proliferation. The 3D HTS assay development, first pilot screen results, and initial hit investigation will be presented.
10:45 AM - *BM07.01.08
Engineering Hearts and Other Body Parts
Tal Dvir 1
1 , Tel Aviv University, Tel Aviv Israel
Show AbstractThe heart is a non-regenerating organ. Consequently, the loss of cardiac cells and formation of scar tissue after extensive myocardial infarction frequently leads to congestive heart failure. Given the scarcity of cardiac donors, a potential approach to treat the infarcted heart is to repopulate the ‘dead zone’ with cells capable of spontaneous contraction. Cellular therapy evolved to introduce cells into diseased areas and regain function. However, two main drawbacks of this approach are the lack of control of cell accumulation site after injection, and cell death before forming cell-cell or cell-matrix interactions. These shortfalls motivated the development of the tissue engineering concept, where 3-dimensional (3D) biomaterials serve as extracellular matrix-like scaffolds to the cells, enabling the cells to assemble into effective tissue substitutes, that may restore tissue or organ function. After transplantation the scaffolds either degrade or metabolize, eventually leaving a vital tissue instead of the defected tissue. In this talk I will discuss the recent advancements in the field of cardiac tissue engineering. I will describe cutting-edge technologies for engineering functional cardiac tissues, focusing on the design of new biomaterials mimicking the natural microenvironment of the heart, or releasing biofactors to promote stem cell recruitment and cardioprotection. In addition, I will discuss the development of patient-specific materials and 3D-printing of personalized vascularized cardiac patches. Finally, I will show a new direction in tissue engineering, where, micro and nanoelectronics are integrated within engineered tissues to form cyborg tissues. In this new concept the built-in electronic network is used to on-line record cellular electrical activity and when needed to provide electrical stimulation for synchronizing cell contraction. Furthermore, electroactive polymers containing biological factors can be deposited on designated electrodes to release drugs in the cellular microenvironment on demand, affecting the engineered tissue or the host.
11:15 AM - BM07.01.09
Sequential Tethering of Proteins to Hydrogels through a Reversible thiol-ene Reaction
Joseph Grim 1 , Brian Aguado 1 , Kristi Anseth 1
1 , University of Colorado-Boulder, Boulder, Colorado, United States
Show AbstractPolyethylene glycol (PEG) hydrogels have emerged as promising in vitro cell culture platforms because their mechanical and biochemical properties can be controlled to mimic native extracellular matrix (ECM). To impart biological activity into PEG hydrogels, biomolecules are covalently tethered to the hydrogel network to elicit a cellular response. While this approach is commonplace for peptides, signaling proteins are less amenable due to their low stability in culture media. As such, researchers generally add proteins solubly to the culture media, where they can be replenished as needed. Increasing evidence suggests, however, that the context in which proteins are presented to cells (e.g, as soluble verses tethered siganls) can influence their bioactivity. Indeed, many proteins are bound to the ECM in vivo suggesting cells frequently interact with tethered proteins via the ECM. By covalently tethering proteins to hydrogels, one can recapitulate protein-ECM interactions in vitro. For this approach to be practical, it is necessary that the tethering method be reversible, so proteins can be refreshed or exchanged with different proteins as needed. We were particularly interested in the thiol-ene reaction as a method to achieve protein tethering because it is bioorthogonal, so it can be performed in situ. The reaction is also light-mediated, so it can be performed with precise spatial control, greatly expanding the in vitro experimental space. Unfortunately, the thiol-ene reaction is irreversible, so it is not possible to release the tethered biomolecule or exchange it with a new biomolecule once immobilization has occurred. To circumvent this issue, we drew inspiration from chain-transfer agents employed in RAFT polymerization to identify an allyl sulfide functionality which should enable a reversible thiol-ene reaction (addition of a biomolecule regenerates the 'ene' functionality and releases the previously tethered biomolecule). Importantly, this process should be repeatable multiple times to sequentially tether and release proteins with spatial control. We generated polyethylene glycol hydrogels that contain pendant allyl sulfide functionality. Thiolated human transferrin was tethered to the hydrogels using photolithography and visualized via immunostaining. Complete release of transferrin was achieved through a subsequent thiol-ene reaction with PEG1K-SH. The efficiency of protein tethering was quantified by ELISA, and we observed that protein tethering and release scaled linearly from pg/mL to μg/mL transferrin concentrations. Studies are ongoing to demonstrate that tethering and release of the signaling protein TGF-β can be performed in the presence of cells, and that cells respond to TGF-β in regions where it is tethered. However, our initial data demonstrates that the allyl sulfide handle is a valuable chemical tool to achieve sequential protein tethering to hydrogels through reversible thiol-ene reactions.
BM07.02: Synthetic Mimics of Biological Structures and Scaffolds
Session Chairs
Monday PM, November 27, 2017
Sheraton, 2nd Floor, Grand Ballroom
1:30 PM - *BM07.02.01
Bioinspired Adhesives
David Mooney 1 2
1 , Harvard University, Cambridge, Massachusetts, United States, 2 , Wyss Institute, Boston, Massachusetts, United States
Show AbstractAdhesives that can bond strongly to biological tissues would have broad applications, including placement of biomedical devices. However, existing tissue adhesives suffer from incompatibility with wet environments, inability to accommodate dynamic tissue movements, and weak adhesion to tissues. We have developed an approach, inspired by a mucus secreted by slugs, for adhesives consisting of both an adhesive surface and a energy dissipating matrix. These materials strongly adhere to wet surfaces, and adhesion is maintained with dynamic, repeated movement. We have demonstrated multiple potential applications, including tissue adhesion of devices, and serving as a sealant and a hemostatic agent.
2:00 PM - BM07.02.02
Developing a Tissue Glue by Engineering Adhesion and Bioactivity of Metal Oxide Nanoparticles
Martin Matter 1 2 , Fabian Starsich 2 , Sergio Bertazzo 3 , Sotiris Pratsinis 2 , Inge Herrmann 1
1 , Swiss Federal Laboratories for Materials Science and Technology, St. Gallen Switzerland, 2 , ETH Zürich, Zurich Switzerland, 3 , University College London, London United Kingdom
Show AbstractToday’s surgeons are challenged by a variety of poorly healing wounds due to an aging population and the rise in the incidence of diabetes worldwide. Current wound management solutions do not satisfy the patients’ needs. In this work, we show how bioactive nanoparticles made in a flame-spray reactor can be engineered to tackle problematic wounds by tailoring their bioactivity.
In a first step, silica, iron oxide, ceria, borate glass, and bioglass nanoparticles were produced by liquid-feed flame spray pyrolysis and several biological assays were carried out to assess their hemostatic properties, and adhesion to soft tissue. In a second step, the bioactivity of the nanoparticles was tailored by creating hybrid and doped versions by the same synthesis method. More clinically relevant properties including antimicrobial, angiogenic, and antioxidant activities, were assessed.
The experiments show exciting results for bioglass-based particles. The material exhibits exceptionally strong adhesive properties and promotes a manifold increase in blood clotting speed. Adding low levels of silver as a dopant to the particle increases antimicrobial activity, while adding strontium stimulates angiogenesis. Bioglass-ceria hybrid nanoparticles produced in a two-spray setup show high biocompatibility and express antioxidant activities. Not only have bioglass-ceria hybrid nanoparticles showed exciting properties connected to wound healing, but both materials have well-grounded clinical benefits that have been excessively stated in literature. The angiogenic and adhesive features of bioglass combined with the anti-inflammatory and antioxidant properties of ceria make for a promising and comprehensive wound management solution which can be further tuned by a wide spectrum of bioactive metal ion dopants.
2:15 PM - BM07.02.03
Culturing Hydrogels with Mucosa Mimicking Properties
Anna Duraj-Thatte 1 2 , Noemie-Manuelle Dorval Courchesne 1 , Jarod Rutledge 1 , Pichet Praveschotinunt 1 2 , Neel Joshi 1 2
1 Wyss Institute for Biologically Inspired Engineering, Harvard University , Boston, Massachusetts, United States, 2 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractProtein-based hydrogels have a variety of biomedical and nanotechnology applications including drug delivery, tissue engineering and development of biosensors and nanowires. Proteins as polymeric hydrogel building blocks are uniquely adept at simultaneously playing structural and bioactive roles. Alternative approaches to modifying hydrogels to facilitate their interaction with biological systems requires time consuming and expensive synthesis and purification steps. Here, we demonstrate a novel method for the rapid biosynthesis of tailored bioactive hydrogels in a single step, directly from bacterial culture, using a protocol that requires no protein purification. The hydrogel scaffold is based on the engineered extracellular matrix protein CsgA, which self-assembles into a fibrous mesh-like network. By concentrating curli fibers on the filter and subsequent addition of the ionic surfactant sodium dodecyl sulfate (SDS), gelling agent we achieved rapid gelation.
To engineer hydrogel with specific properties, we have rationally genetically modified a curli system of E. coli, which is a functional amyloid that is produced by cell during biofilm formation. To create mucoadhesive hydrogel, we selected the trefoil factors (TFFs), the family of human cytokines that exhibit specific binding affinities for various mucins, to be a genetically fused to the C-terminus of the gene product of the curli operon, CsgA, which is the main structural component of curli fibers. We demonstrated that the genetically engineered hydrogel is able to adhere to mucosal epithelium surface of the goat colon. The shear-thinning behavior of this hydrogel created an opportunity to deliver it directly to the gut by injecting or spraying by an endoscopic device. In vivo studies have shown that we are able not only to deliver successfully hydrogel into mice gut but also the engineered hydrogel demonstrated muccoadhesive properties. The fluorescent labelled hydrogel has been detected in the gut up to 48h after administration.
Here, we present the unique method to provide quick and cost effective generation of amyloid-based hydrogels that possess high stability and mechanical strength. Moreover, by fusing functional proteins to the amyloid fibers we can bio-fabricate a variety of customized hydrogels for several applications.
2:30 PM - *BM07.02.04
3D Silicon Microscale Networks for Studying and Manipulating Cell Growth and Proliferation
John Rogers 1 , Ralph Nuzzo 2
1 , Northwestern University, Evanston, Illinois, United States, 2 Chemistry, University of Illinois, Urbana, Illinois, United States
Show AbstractComplex three-dimensional (3D) organizations of materials represent ubiquitous structural motifs, the most notable of which are in life-sustaining hierarchical structures found in biology but where simpler examples also exist as dense multilayered constructs in high performance electronics. Each class of system evinces specific enabling forms of assembly to establish their functional organizations. Here we describe materials and means of assembly that extend and join these disparate systems—schemes for the functional integration of soft and biological materials with 3D microscale, open frameworks that can leverage the most sophisticated advanced forms of multilayer electronic technologies. The cell migration behaviors, temporal dependencies of growth, and contact guidance cues provided by the non-planarity of these frameworks illustrate design criteria useful for their functional integration with living matter (e.g. NIH/3T3 fibroblast and primary dorsal root ganglion cultures).
3:30 PM - BM07.02.00
Molecular Design and Activity of Sequence-Defined Macromolecules
Christopher Alabi 1
1 Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States
Show AbstractControl over primary sequence and structure is critical to the development of new functional materials such as catalysts, synthetic affinity ligands and therapeutics, sequence responsive scaffolds, programmable biomaterials and much more. Motivated by these opportunities and the need for sequence-control and structural diversity in polymer research, we present a versatile methodology for the assembly of a new class of sequence-defined macromolecules called oligoTEAs. With sequence-control in hand, we are currently working to establish sensitive solution-phase structural characterization methods to determine their conformational dynamics and to formulate sequence-structure relationships for biological applications. We focus on applications that leverage the advantages of these novel macromolecules such as increased serum stability, precise control of backbone and pendant group sequence, and a large scope of chemically diverse monomers. Current applications under exploration in our lab include the design of cleavable linkers to quantitate intracellular cleavage kinetics, development of novel sequences and conjugates for intracellular drug delivery, and the design of membrane selective antibacterial compounds. In this talk, I will discuss the antibacterial properties of oligoTEAs in detail by examining the kinetic phenomenon behind their mechanism of action and investigations into the effect of primary sequence, composition and structure on antibacterial properties.
3:45 PM - BM07.02.05
Development of Nanomaterials for mRNA Therapeutics and Genome Editing
Yizhou Dong 1
1 Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States
Show AbstractMessenger RNA (mRNA) therapeutics have shown great promise for purpose of expressing functional proteins. However, the efficient and safe delivery of mRNA remains a key challenge for the clinical application of mRNA based therapeutics. Lipid and lipid-like nanoparticles possess the potential for mRNA delivery. Based on our previous experiences, we have designed and synthesized N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT). We applied an orthogonal experimental design to investigate the impacts of formulation components on delivery efficiency. TT3 LLNs, a lead material fully recovered the level of human factor IX (hFIX) to normal physiological values in FIX-knockout mice. In addition, we demonstrated that TT3 LLNs were capable of effectively delivering Cas9 mRNA and guide RNA to the mouse liver for genome editing.
4:00 PM - BM07.02.06
From Bacteria Compartmentalization within Inorganic Foams to the Assembly of Microbial Consortia
Armand Roucher 1 2 , Mickael Morvan 1 , Pekin Deniz 1 , Martin Depardieu 3 , Jean-Luc Blin 2 , Veronique Schmitt 1 , Manfred Konrad 4 , Jean-Christophe Baret 1 , Renal Backov 1
1 , Centre de Recherche Paul Pascal, Pessac France, 2 , Structure et Réactivité des Systèmes Moléculaires Complexes, NANCY France, 3 , Max Planck Institute of Colloids and Interfaces, Postdam Germany, 4 , Max Planck Institute for Biophysical Chemistry, Goettingen Germany
Show AbstractMicro-organisms play an essential role in the transformation of raw materials. These transformations have a fundamental function for the micro-organism to maintain its out-of-equilibrium state [1]. Yet making use of their capabilities for transformations requiring synergistic interactions between different organisms is challenging as the competition for resources might reduce the diversity and ultimately disrupt the synergies. Here, we propose a new method usable to construct microbial consortia for the integration of complex transformations. We used a sol-gel process as one of the most promising paths to compartmentalize bacterial proliferation as it allows their confinement while maintaining both viability and metabolic activity [2]. One of the main drawback of using hybridized gels is the lack of free space which does not allow bacterial division and proliferation. Our systems are based on Silica High Internal Phase Emulsion (Si(HIPE)) in order to obtain highly porous foams. It was already demonstrated that this materials allow bacterial proliferation inside the porous structure without motion restriction [3] and further display programmable enzymatic functions. These latters are assembled in complex enzymatic cascades. We manipulate and assemble them to perform cycles or pre-programmed sequences of reactions [4]. Our solid-supported versatile biocatalysts should find applications in a wide range of practical and industrial systems where complex tasks have to be performed by controlled consortia of micro-organisms.
References :
[1] E. Schroedinger, What is life ? The physical aspect of the living cell, , 1944, , pg.
[2] N. Nassif et al., Living bacteria in silica gels, Nature Materials, 1, pp. 42-44 (2002).
[3] M. Depardieu et al., A multiscale study of bacterial proliferation modes within novel E. coli@Si(HIPE) hybrid macrocellular living foams, J. Mater. Chem. B, 4, pp. 2290-2303 (2016).
[4] A. Roucher et al., From Bacteria Compartmentalization within Inorganic Foams to the Assembly of Microbial Consortia, submitted
4:15 PM - BM07.02.07
Towards the Development of a More Realistic In Vitro 3D Tumor Model
Mahboobeh Rezaeeyazdi 1 , Thibault Colombani 1 , Sidi Bencherif 1 2 3
1 Department of Chemical Engineering, Northeastern University, Boston, Massachusetts, United States, 2 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 3 , University of Technology of Compiègne, Compiègne France
Show AbstractTwo-dimensional (2D) cell cultures growing on plastic do not recapitulate the three-dimensional (3D) architecture and complexity of human tumors. Recently, Multicellular Tumor Spheroids (MCTS) aimed to reproduce the 3D architecture of solid tumors and fill the gap between monolayer cultured cells and animal models. These 3D MCTS cultures were set up either as free-floating, microencapsulated into inert non-porous hydrogels, or embedded in mechanically-instable ECM. These methodologies lack either a physiological context and/or reproducible format for assessing tumor cells in vitro. To overcome these limitations, our team has developed a new 3D culture tumor model taking into consideration both the need to have a mechanical support around MCST, a biomimetic matrix enabling local cell remodeling, and the presence of interconnected macropores. This unique macrostructure allows cells invasion and trafficking into the surrounding matrix but also enables physiological gradients for nutrients, oxygen, pH, and catabolites. Based on a combination of MCTS, generated with a new technique allowing a better control over spheroid dimensions and uniformity, and their integration into macroporous 3D biomimetic cryogel-based scaffolds, we created a more realistic in vitro 3D tumor model. This approach offers an opportunity to evaluate processes and modulators of cancer cell dissemination from the tumor to the matrix, possibly modeling tumor cells invasion and early metastatic events. Additionally, the impact of pharmacological compounds and genetic/pathway manipulation can be evaluated to determine the functional impact on these processes.
Acknowledgement: This research was financially supported by Northeastern University (Tier 1 Provost grant).
4:30 PM - *BM07.02.08
An Inflammation Responsive Hydrogel Platform for Controlled Drug Delivery
Nitin Joshi 1 2 , Jeffrey Karp 1 2
1 , Brigham and Women's Hospital, Cambridge, Massachusetts, United States, 2 , Harvard Medical School, Boston, Massachusetts, United States
Show AbstractWe have developed a self-assembled hydrogel based drug delivery platform from small molecule amphiphilic gelators, which we have identified through our screening of the FDA’s generally recognized as safe (GRAS) list of compounds. This platform can titrate drug release to the level of inflammation, ensuring that the drug is released only when needed at a therapeutically relevant concentration. Another interesting characteristic of this platform is it’s selective adhesion to inflamed tissue, which results in high local drug concentrations at the disease site, maximizing the therapeutic effect. We have demonstrated the utility of this platform for drug delivery in multiple conditions, including rheumatoid arthritis, osteoarthritis, vascularized composite allo-transplantation and inflammatory bowel disease. This talk will discuss our previously published and currently ongoing work to advance the self-assembled hydrogel platform.
BM07.03: Poster Session I
Session Chairs
Tuesday AM, November 28, 2017
Hynes, Level 1, Hall B
8:00 PM - BM07.03.01
In Vitro Prodrug Activation over Platinum Nanoparticles for Side Effect Reduction in Lung Cancer Treatment
Antoine Herzog 1 , Elia Schneider 1 , Wendelin Stark 1
1 , ETH Zürich, Zurich Switzerland
Show AbstractDespite the extensive research efforts in the last decades, lung cancer remains a staggering cause of human deaths and accounts for around 25% of cancer deaths thereby making it the deadliest form of cancer. The main treatment options, especially for small cell lung cancer, are based on untargeted chemotherapies.1,2 Such chemotherapeutics block the molecular mechanisms of mitosis, eventually leading to apoptosis. Because chemotherapeutics target molecular mechanisms involving a high degree of similarity between healthy and cancer cells, they display cytotocity towards both cancer and healthy cells. The destruction of healthy cells (notably rapidly dividing cells) by untargeted chemotherapies ultimately leads to the so-called side effects.
In order to reduce side effects, we designed a general system composed of three elements.3 Namely, a prodrug, a catalyst and a gas mixture containing hydrogen. The cytotoxic drug is chemically protected thus resulting in an inactive prodrug. Its activity can be restored at the desired locus (e.g. where the lung cancer resides) by removing the protecting group with hydrogen in the presence of platinum nanoparticles.
Using the A549 cell line (human lung carcinoma) and gemcitabine, a FDA-approved lung cancer chemotherapeutic, we performed an in vitro proof of the system. The cytotoxity of the drug was significantly reduced when protected and its cytotoxicity was fully recovered after activation over platinum nanoparticles with hydrogen gas.
In the perspective of in vivo use, we employed hydrogen-containing air mixtures that have applications as deep diving breathing gases (e.g. hydrox)4. The nanoparticles can be administered by inhalation and reside in the airways thus conferring a spatial control over the drug distribution. A temporal control is achieved by the tunable air mixture administration. Indeed, by changing the concentration of hydrogen and the pace of administration one can control the rate of deprotection. Additionally, the cytotoxicity of the material and of hydrogen in the here relevant concentrations were investigated in vitro.
(1) Bryant, J. L.; Meredith, S. L.; Williams, K. J.; White, A. Lung Cancer 2014, 86, 126–132.
(2) Powell, H. A.; Tata, L. J.; Baldwin, D. R.; Potter, V. A.; Stanley, R. A.; Khakwani, A.; Hubbard, R. B. Br. J. Cancer 2014, 110, 908–915.
(3) Herzog, A. F.; Stark, W. J. Combination Medicament Comprising a Prodrug and Inhalable Catalyst. EP17176199, 2017.
(4) Fife, W. P. The Use of Non-Explosive Mixtures of Hydrogen and Oxygen for Diving; 1979.
8:00 PM - BM07.03.02
Low-Cost and Cleanroom-Free Fabrication of Microneedles for Transdermal Drug Delivery
Hojatollah Rezaei Nejad 1 , Aydin Sadeqi 1 , Gita Kiaee 1 , Sameer Sonkusale 1
1 , Tufts University, Medford, Massachusetts, United States
Show AbstractWe present a facile, low-cost and cleanroom-free technique for the fabrication of microneedles using molds created by laser ablation. Microneedle mold with high aspect ratios is achieved on acrylic sheet by engraving a specific pattern of cross over lines (COL) using CO2 laser cutter. The fabrication starts by engraving lines that overlap only at their center cross point. The engraving depth at the cross point is higher since this point is traversed multiple × the depth is then proportional to the number of times that the laser beam passes through the point. As the laser beam passes the cross point, a sharp cone starts to shape in the middle, which eventually leads to the formation of microneedle mold at the cross point. The depth of sharp cone can simply be adjusted by changing the number of lines used in the initial 2D CAD drawing. In the next step, the mold’s replicate was created by casting PDMS on the acrylic sheet. The PDMS-casted sheet was degassed and subsequently cured in the oven at 80°C for two hours. The PDMS microneedles were detached from the acrylic mold and were treated with oxygen plasma. The microneedles were then silanized with Trichloro silane under vacuum in a desiccator overnight. PDMS was casted on the silanized microneedles followed by degassing and curing. The silane layer creates a barrier between PDMS microneedles and PDMS mold avoiding them from bonding to each other and facilitates their detachment. The final PDMS mold was used to create microneedles from different polymers.
Microneedles of different shape, size and angle were fabricated by altering three parameters of laser scanning speed, number of lines used to fabricate each needle and length of the lines. Height and tip angle are two major characteristics of the microneedles. These two factors can be readily controlled by either changing the number of lines or engraving speed.
Biodegradable microneedles were fabricated using PVA polymer. We used gelatin hydrogel as a model tissue for drug release studies which is optically transparent and has similarity to the skin tissue. We placed gelatin 10 % in the refrigerator for solidification. Then, we placed the polymer membrane (Parafilm® M) over on top of the hydrogel surface. A microneedle patch of 1×1 cm2 containing phenol dye was then pressed against the membrane surface to insert the needles into the gelatin hydrogel. The polymer membrane acted as a diffusion barrier and prevented the abrupt release of drug from microneedle’s bulk PVA substrate into the underlying gelatin hydrogel. The phenol red was used as the model drug, and the standard curve was drawn to quantify the drug release. Almost half of the encapsulated phenol red inside microneedle was released in 20 minutes and the remaining was released in the next five hours.
8:00 PM - BM07.03.03
Structural Motifs on the µm and nm Level Entail Failure Resistance to Pike Bone Fish
Katrein Sauer 1 , Paul Zaslansky 1 2 , Ron Shahar 4 , Alexander Rack 3 , Ivo Zizak 5
1 Julius Wolff Institute, Charite, Universitaetsmedizin Berlin, Berlin Germany, 2 Charite - Universitaetsmedizin Berlin, Department for Restorative and Preventive Dentistry Centrum für Zahn-, Mund- und Kieferheilkunde, Berlin Germany, 4 Laboratory of Bone Biomechanics, Koret School of Veterinary Medicine, Rehovot Israel, 3 , European Synchrotron Radiation Facility - ESRF, Grenoble France, 5 , Helmholtz-Zentrum Berlin, Berlin Germany
Show AbstractUnosteocytic fish bones such as the cleithra of pikes (Esox lucius) consist of the same building blocks as mammalian bones: carbonated apatite nanoparticles, collagen fibrils and water. They are however different from mammalian bone since they lack osteocytes. The flat, broadly curved “wing-shaped” cleithra undergo significant loading, originating from motion of the pectoral fins which are attached directly to the bone surface.
In order to explore the structure and mechanical functional relation within this material, we combined several methods, including high-resolution synchrotron radiation micro-computed tomography (µCT), second harmonic generation laser scanning confocal microscopy (SHG) and XRD using micron sized beam.
High-resolution µCT in both absorption and phase contrast enhanced modes revealed layers slightly varying mineral densities. Phase contrast enhancement, highly sensitive to very small density differences, revealed that most layers contain fields of low mineral density 'spots' appearing connected in adjacent layers. SHG revealed that the pores are filled with isolated, quasi-parallel, unmineralized collagen bundles, extending at right angles to the outer bone surfaces. This matches previous reports in tilapia fish bones, observed by Raman spectroscopy and FIB-SEM. A woven-like cross-hatched collagen mesh is observed in plane, matching the mineral layers, suggesting that mats of mineralized collagen fibrils with markedly different collagen orientations dominate the structure, in a plywood manner.
XRD of bone cross-sections demonstrated that mineral crystal orientation is highly aligned with the mineral dense layers, i.e. the longitudinal bone layers. This provides additional support to the notion that unmineralized collagen bundles serve to pin neighboring bone layers together, presumably improving resistance to bending of the whole structure. Three point bending tests demonstrate that fracture across the cleithra is hindered with cracks repeatedly stopped successively layer by layer.
We hypothesize that this 3D arrangement on several levels of organization is the key to the impressive macroscopic failure resistance of these bones and learning from this evolutionarily advanced bone structure that is markedly different from mammalian bone could be useful to mimic the structural complexity of biogenic crystals in artificial composites.
8:00 PM - BM07.03.04
Magnetic Microtubes Decorated with Nanowires and Cell for Bio-Micro Robot Applications
Jose Souza 1 , Cesar Pomar 1 , Tamiye Goia 1 , Herculano Martinho 1 , Andrea Rodas 1 , Sydney Santos 1
1 , Federal University of ABC, Santo Andre-SP Brazil
Show AbstractDevelopment of micro-bio-magnetic systems based components have attracted great attention from scientific community. On the other hand, magnetic micro/nanotubes have involved great consideration not only in material science, but also in technological applications due to their magnetic property counterpart. Magnetically driven systems include magnetic-bio micro-robots, water treatment, drug delivery, localized therapy via wireless intervention due to external magnetic field actuation. All these applications involve transport phenomena at micro/nanoscale which can be implemented by using biocompatible and magnetic actuated agents. As far as these points are concerned, in this work, antiferromagnetic hematite and ferromagnetic magnetite microtubes decorated with nanowires and cells have been obtained. The synthesis route of these hollow structured materials including heat treatment and electrical current along with growth mechanism will be presented. Most important is that one is able to tune in the magnetic moment magnitude going from ferrimagnetic (high magnetic moment) to nonmagnetic-like hollow microstructures. We have also calculated both torque and driving magnetic force of hollow bio-microtubes as a function of external magnetic field gradient which were found to be robust opening the possibility for magnetic micro-robot device fabrication and possible application in biotechnology. The field gradient also exerts a force producing a linear movement along the z-axis. Interesting, it is possible, for example, to equilibrate the weight of nano/microtube while keeping it levitating inside a solenoid. We have observed that our nano/microtube hematite, which is the outer layer of our microtubes, has biocompatibility and affinity with cells. Indeed, experiments involving cell culture on them reveal biocompatibility. In this case, fibroblast cell adhesion was observed on the surface of the microtubes corroborating their non-cytotoxicity behavior. Cell cytotoxicity reveals the ability of certain compounds to destroy living cells before adherence and migration. Fibroblast cells adhered homogeneously on surface of the microtubes without any preferred area. The highly rough surface of samples due to the presence of nanowires allowed cells to interact and proliferate on the nano engineered topography.
8:00 PM - BM07.03.05
Utilization of Biocompatible Photosensitizing Nanocomplex for Photodynamic Therapy via Intraocular Injection on Retinoblastoma
Dong Hyun Jo 1 , Keunsoo Jeong 2 , Jin Hyoung Kim 1 , Sehoon Kim 2 , Jeong Hun Kim 1 3 4
1 Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul Korea (the Republic of), 2 Center for Theragnosis, Korea Institute of Science and Technology, Seoul Korea (the Republic of), 3 Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul Korea (the Republic of), 4 Department of Ophthalmology, Seoul National University College of Medicine, Seoul Korea (the Republic of)
Show AbstractIn the treatment of retinoblastoma, the most common intraocular malignant tumor in children, vitreous seeds in the vitreous body of the eye remain as a clinical conundrum, which cannot be effectively controlled by currently available treatment options, thermotherapy by laser application or chemotherapy. In this study, we investigated the therapeutic potential of the biocompatible photosensitizing nanocomplex for the treatment of retinoblastoma by intraocular injection. The photosensitizing nanocomplex was formed with self-assembly of a photosensitizer and a biocompatible surfactant. In contrast to free methylene blue, which poorly penetrates to tumor cells, the nanocomplexed methylene blue demonstrated effective penetration to retinoblastoma cells. In addition, further laser irradiation effectively induced apoptosis of retinoblastoma cells after treatment with the nanocomplexed methylene blue. In vivo experiments using an orthotopic transplantation mouse model showed that this photosensitizing nanocomplex penetrated well-formed in vivo tumors after intraocular injection. As in vitro experiments, laser treatment after intraocular injection of the nanocomplexed methylene blue decreased the degree of tumor formation in the vitreous of mice. Laser irradiation and photodynamic therapy are widely utilized treatment options in the field of ophthalmology. We expect that this biocomplatible photosensitizing nanocomplex based on clinically approved materials can expand the use of photosensitizing materials and photodynamic therapy in the treatment of retinoblastoma and other intraocular cancers.
8:00 PM - BM07.03.06
Peptide-Enriched Nanocarriers for the Growth Inhibition of Antibiotic-Resistant Bacteria
Nicole Bassous 1 , Thomas Webster 1 2
1 , Northeastern University, Boston, Massachusetts, United States, 2 , Wenzhou Institute of Biomaterials and Engineering, Wenzhou China
Show AbstractPrevalent research underscores efforts to engineer highly sophisticated nano-vesicles that are functionalized to combat antibiotic-resistant bacterial infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA), and that aid with wound healing or immunomodulation. This is especially relevant for patients who are susceptible to S. aureus infections post-operatively. Technical challenges associated with this aim require a thorough assessment of the chemical properties of synthesis materials and methods. Here, formulations were incorporated into polymeric, biocompatible vesicles called polymersomes that self-assemble via hydrophobicity interactions of admixed aqueous and organic substances. Nano-polymersomes were synthesized using a high molecular weight amphiphilic block copolymer, and were conjugated to include antimicrobial peptides (AMPs) along the peripheral hydrophilic region and silver (Ag) nanoparticles inside their hydrophobic corona. In vitro testing on bacterial and human cell lines indicated that finely tuned treatment concentrations of AMP and Ag nanoparticles in polymersomes synergistically inhibited the growth of MRSA without posing significant side effects, as compared with other potent treatment strategies. In particular, a ratio of silver-to-AMP of about 1:1.9, corresponding to approximately 0.1 mg/ml of silver nanoparticles and 40 μM of the peptide, yielded complete MRSA inhibition over a 23-hour time frame. Conceivably, the silver nanoparticles generate bacterial cell wall indentations that prompt the influx of external entities and the outflow of vital proteins or nutrients. This weakening of lipopolysaccharide permeability barriers promotes the unimpeded cellular translocation of contiguous AMP molecules, which can then disrupt internal processes including DNA or protein synthesis. This bacteriostatic activity, coupled with nominal cytotoxicity towards native human dermal fibroblast cells, extends the potential for AMP/Ag nanoparticle polymersome therapies to replace antibiotics in the clinical setting. This study will facilitate contemporary research that is feasible, important, and highly relevant, and possesses the potential to advance knowledge in several fields, including drug delivery, nanotechnology, and artificial immunity.
8:00 PM - BM07.03.07
Innovative Nano Assembly by Link Chemistry
Markus Schütz 1 , Khan Lê 1 , Shaista Ilyas 1 , Ania Jurewicz 1 , Thomas Fischer 1 , Sanjay Mathur 1
1 Institute of Inorganic Chemistry, University of Cologne, Cologne, North Rhine-Westphalia, Germany
Show AbstractMetal oxides and metal nanostructures play a key role in many emerging applications, for example in photodynamic therapy, renewable energy, magnetic resonance imaging (MRI), therapeutic agents for hyperthermia based cancer treatments or other medical applications. Many confined surface properties are only suitable for their well-defined applications. In this study we have assembled different nanomaterials to get different surface properties to use them in various applications.
We demonstrate for the first time an approach that allows attachment of single nanoparticles on the surface of other nanoparticles via link chemistry. Here, we show the use of the copper-catalyzed azide-alkyne cycloaddition reaction, the carbodiimide coupling reaction between an amino group and a carboxyl group by using N,N′-dicyclohexylcarbodiimide and the reversible Diels-Alder reaction between a conjugated diene and a substituted alkene. All reactions are very stereospecific, give a very high chemical yield and have simple reaction conditions. In this work we have combined nanostructures of different sizes, morphologies and compositions. Different silica (SiO2) nanostructures as a model system, iron oxide (Fe2O3, Fe3O4) nanostructures due of their different magnetic properties and gold (Au) nanoparticles because of their specific optical properties. The combination of these selected properties creates completely new materials to be applicable in various fields. The crystalline nature of the particles, before and after their assembly was evaluated by X-ray diffraction analysis. The quantitative and qualitative confirmation of various functionalities was determined by Fourier-transform infrared spectroscopy, ultraviolet–visible spectroscopy and thermogravimetric analysis. With scanning and transmission electron microscopy we were able to show the differences and specific features of various as synthesized assemblies.
After the formation of assembly, the available active groups can be used for conjugation of various targeting ligands. The functionalized nanostructures can be used for the cancer diagnosing imaging and the therapy.
8:00 PM - BM07.03.08
Silk-Proteoglycan Biointerfaces for Blood Contacting Applications
Kieran Lau 1 , Fengying Tang 1 , John Whitelock 1 , Megan Lord 1 , Marcela Bilek 2 , Jelena Rnjak-Kovacina 1
1 , University of New South Wales, Sydney, New South Wales, Australia, 2 , The University of Sydney, Sydney, New South Wales, Australia
Show AbstractBiomaterial platforms that present superior physical and mechanical features frequently require functionalization with bioactive molecules to achieve appropriate biological function. The complex and dynamic interactions at the biomaterial surface can have great impact on the biomaterial performance, especially in blood contacting applications. Silk biomaterials have been modified using a range of techniques to achieve improved biological function, including passive adsorption, covalent cross-linking and bulk loading with bioactive molecules. Passive adsorption can result in a random distribution of molecular orientations and thus compromised biological performance. Covalent coupling has the advantage of being a more stable modification compared to adsorption and bulk loading, and the resultant interface is often more uniform, but silk has a very limited number of modifiable amino acid side chain groups, making physical surface modifications attractive. The goal of this work was to elucidate the governing principles driving molecular presentation on silk biomaterials under different immobilization conditions.
Silk biomaterials were functionalized with recombinantly expressed domain V of human perlecan using passive adsorption, chemical (EDC-NHS) and physical cross-linking (plasma ion immersion implantation, PIII). Domain V is the C-terminal region of human perlecan, a large, conserved extracellular matrix proteoglycan that plays a major role in the vascular niche, including modulation of platelet, endothelial and smooth muscle cell interactions. Additionally, it makes an attractive model molecule due to the multiple features that allow probing of the molecular orientation and function on the biomaterial surface, including a protein core presenting an α2β1 cell binding site and different glycosaminoglycan (GAG) chains.
The findings confirmed successful functionalization of silk biomaterials with domain V, resulting in enhanced endothelial cell interactions and reduced platelet and smooth muscle cell binding, making this an attractive blood contacting material. Domain V binding was significantly higher on PIII activated silk biomaterials, compared to functionalization using passive and chemical immobilization techniques as demonstrated via surface plasmon resonance and ELISA analyses. Further, the presentation of Domain V on the silk biomaterial surface was dependent on the immobilization technique, reaction pH and the presence of GAG chains on domain V, which in turn affected its biological function. Taken together, this data demonstrate a promising blood contacting biomaterial and a highly tunable model to study molecular interactions on the biomaterial surface.
8:00 PM - BM07.03.09
Bioluminescence-Activated Photodynamic Therapy of Cancer
Seonghoon (Sean) Kim 1 2 , Sun-Joo Jang 1 2 , Seok-Hyun Yun 1 2
1 Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States, 2 Harvard Medical School, Harvard University, Boston, Massachusetts, United States
Show AbstractConventional photodynamic therapy (PDT) is emerging therapeutic modality, which generates reactive oxygen species (ROS) from the combination of photosensitizers and light. However, delivery of the light has been limited to the superficial layer of the tissue since intrinsic absorption and scattering causes exponential decay of photons being delivered to targets. Here, we demonstrate bioluminescence resonance energy transfer (BRET)-based photochemical activation which can overcome the limitation of optical penetration depth. Bioluminescence molecules were conjugated with photosensitizers to allow intramolecular energy transfer. Light energy generated by enzymatic interaction of luciferase with substrate was non-radiatively transferred to photosensitizers and generated reactive oxygen species (ROS). BRET-based ROS generation occurred in cancer cells showed similar cytotoxicity compared with conventional PDT at the same intracellular concentration of photosensitizers. Our results show that BRET-based intramolecular ROS generation may be a novel technique to overcome the penetration depth limitations of conventional PDT for deep-tissue tumor therapy
8:00 PM - BM07.03.10
Photo-Patterned Oxygen Sensing Films for Controlling Cell Growth and Studying Metabolism
Fei Zeng 1 , Zengju Fan 1 , Siying Wu 1 , Shanshan Wu 1 , Xing Cheng 1 , Yanqing Tian 1
1 , Southern University of Science and Technology, Shenzhen China
Show AbstractSurface properties play a significant role in establishing the responses of cells and tissues to substrates [1]. It is popular to use chemical and topographic cues to fabricate the micro-patterned surfaces in various fields like tissue engineering, cell-based sensor and implant for modulating cell viability, adhesion preference, orientation, migration, proliferation, and cell-to-cell communications [2,3].
In this study, we have developed micro-patterned oxygen sensing hydrogel films via photolithography. Biocompatible poly(2-hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAM), and their composites were chosen as the matrices. A polymerizable oxygen probe of OS2 derived from the highly stable and efficient oxygen probe of Platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6- pentafluorophenyl)-porphyrin (PtTFPP) was used for oxygen sensing. A series of grating patterns with 1um height but different sizes were designed, and the topographical properties for these micro-patterned hydrogel films were examined by SEM and AFM. The oxygen sensing films showed excellent oxygen sensing ability characterized through an oxygen-nitrogen titration. It was found that 5um-sized granting showed the highest sensitivity to oxygen. Cell attachments on non-patterned and patterned sensor films were investigated using HeLa and 3T3-L1 as two representative cell lines and observed by a phase contrast fluorescent inverted microscope. It was observed that most of cells seeded attached on the surfaces of the biocompatible sensing materials; however, size affected cell alignment and elongation. HeLa cells oriented randomly on the non-patterned films; most of the cells aligned on the 50 μm-grating patterned regions without elongation; however, cells elongated and aligned well along the 5 μm gratings. Herein, we defined the length ratios of the long axis to the short axis of cells as “A” as well as angles between long axis of cells and axis of grating as “θ”, we noticed that “A” decreased by increasing gratings’ sizes while “θ” increased. For 3T3-L1 cells, we found that they formed long “cell-strips” along the 50 μm grating, and big “cell-sheets” upon the 5 μm grating surface. Comparing with epithelial cells, fibroblasts have lower ability of attachment to the surfaces. Therefore, they prefer attaching to each other to form cell-strips or -sheets with the guidance of the patterned hydrogel films. Thus, we have found sizes of the micro-pattered structures affected significantly cell elongations and alignments. Further research will be carried out for the monitoring of cellular oxygen respiration and metabolisms using the micro-structured sensors.
Reference:
1. Brunette D M, Chehroudi B. J Biomech Eng., 1999, 121: 49-57.
2. Charest J L, Eliason M T, García A J, et al. Biomaterials, 2006, 27(11): 2487-2494.
3. Lim J Y, Donahue H J. Tissue Eng., 2007, 13(8): 1879-1891.
8:00 PM - BM07.03.11
Virus-Assisted Immobilization of Glucose Oxidase for High Power Enzymatic Biofuel Cells
Sangwook Chu 1 , Pradeep Ramiah Rajasekaran 1 , Adam Brown 1 , James Culver 1 , Reza Ghodssi 1
1 , University of Maryland, College Park, College Park, Maryland, United States
Show AbstractEnzymatic biofuel cells (EBCs) receive significant attention as potential energy sources for autonomous operation of wearable or implanted medical electronics. EBCs harvest electrical energy from catalytic reactions induced by oxidoreductases (e.g. glucose oxidase, bilirubin oxidase, laccase, etc.) with their target substrates/biofuels (e.g. glucose, oxygen, etc.) which are abundant and essential in human physiological fluid. While their reliable features, including biocompatibility, high-specificity, and renewability, offer an attractive prospect for advanced biomedical devices, one of the major challenges towards their practical implementation is their low current/power density. This is mainly limited by the charge transfer kinetics from the enzymatic reactions to the electrode, inefficient oxidation of the biofuels, and low enzyme immobilization density.
In this work, we report Tobacco mosaic virus (TMV)-templated glucose oxidase (GOx) electrodes for the development of advanced enzymatic biofuel cells (EBCs). TMV is a plant virus featuring a nanoscale pipe structure (300nm length, 4nm inner diameter, 18nm outer diameter) formed through helical assembly of the ~2130 coat proteins encapsulating its genome. The thousands of functional receptors expressed at the nanoscale provide an excellent template for high-density immobilization of biological receptors onto electrodes. Particularly, a genetically-engineered TMV expressing high density thiol residues on its surface (TMV1cys) allows both 1) robust surface immobilization of TMV particles onto Au electrodes, and 2) a simple chemical conjugation strategy for loading GOx onto the pre-immobilized virions.
All chemical conjugations were performed on an Au surface (0.42cm2) with sequential loading and rinsing of 10μl sample droplets. All processes were performed at pH7.2 (1x PBS) in a humid chamber. First, TMV1cys particles (0.1mg/ml in 1x PBS) were self-assembled onto the Au surface via thiol-gold binding. This was followed by chemical conjugation steps for bridging streptavidin-conjugated GOx with TMV1cys via heterobifunctional cross-linkers (EZ-Link Maleimide-PEG11-Biotin). The enhanced electrode-enzymatic activity has been characterized using cyclic voltammetry within a -0.8 to 0.2V potential range (vs. Ag/AgCl and Pt as reference and counter electrodes, respectively). Comparing the change in redox current levels of two different electrodes (Au/TMV/GOx vs. Au/GOx), a significant 2-fold increase in the redox current level is observed (at -0.3V vs. Ag/AgCl) with Au/TMV/GOx compared to Au/GOx electrodes (-32.7uA/cm2 vs. -16.5uA/cm2), indicating successful immobilization of GOx onto the surface-functionalized TMV1cys. We believe that the results are sufficiently significant to propose TMV as an effective carrier for high-density enzyme immobilization towards the development of high performance EBCs.
8:00 PM - BM07.03.13
A Biocompatible T1 MRI Contrast Agent Based on Iron Oxide Nanoparticles Made by Scalable Flame Aerosol Technology
Fabian Starsich 1 , Christian Eberhardt 2 , Andreas Boss 2 , Ann Hirt 1 , Sotiris Pratsinis 1
1 , ETH Zurich, Zurich Switzerland, 2 , Universitätsspital Zürich, Zürich Switzerland
Show AbstractContrast agents for magnetic resonance imaging (MRI) are essential to clinicians for crisp visualization. MRI can be classified into T1 and T2 weighted protocols that require paramagnetic and superparamagnetic materials, respectively, as contrast agents. T1 weighted imaging is frequently preferred over T2, as it induces a bright contrast, that allows a sharper image analysis. Commonly used and FDA-approved T1 contrast agents , however, were shown to cause nephrogenic systematic fibrosis due to released Gd ions from the injected complexes [1]. Iron oxide, which is at larger sizes a clinically applied superparamagnetic T2 contrast agent, shows paramagnetic properties for crystal sizes below 5 nm. Here, such ultra-small iron oxide nanocrystals are produced via highly scalable and dry flame synthesis [2] and investigated as promising T1 MRI contrast agents, focusing on structure and biocompatibility. Most importantly, via the addition of SiO2 as a spacing material [3], morphology could be altered and the contrast enhancement efficiency finely tuned. The produced nanocrystals are a promising biocompatible alternative as they attain relaxivity values comparable to commercially used Gd-complexes.
[1] Penfield, J. G.; Reilly, R. F. What Nephrologists Need to Know about Gadolinium. Nat. Clin. Pract. Nephrol. 3, 654–668 (2007).
[2] Starsich, F. H. L.; Sotiriou, G. A.; Wurnig, M. C.; Eberhardt, C.; Hirt, A. M.; Boss, A.; Pratsinis, S. E. Silica-Coated Nonstoichiometric Nano Zn-Ferrites for Magnetic Resonance Imaging and Hyperthermia Treatment. Adv. Healthc. Mater. 5, 2698–2706 (2016).
[3] Sotiriou, G. A. ; Starsich, F.; Dasargyri, A.; Wurnig, M. C.; Krumeich, F.; Boss, A.; Leroux, J. C.; Pratsinis, S. E. Photothermal Killing of Cancer Cells by the Controlled Plasmonic Coupling of Silica-Coated Au/Fe2O3 Nanoaggregates. Adv. Funct. Mater. 24, 2818–2827 (2014).
8:00 PM - BM07.03.14
Ultrasensitive, Highly Durable Nanoscale Crack Based Strain-Gauge Sensors Inspired by Spider Sensory Receptors
Byeonghak Park 1 2 , Daeshik Kang 3 , Tae-il Kim 1 2
1 , Sungkyunkwan University, Suwon Korea (the Republic of), 2 Institute for Basic Science, Center for Neuroscience Imaging Research, Suwon Korea (the Republic of), 3 , Ajou University, Suwon Korea (the Republic of)
Show AbstractArachinds are among the most senstiive creatures on the Earth. Especially, their system for mechanosensory embedded in the crack-shpaed slit organ made of stiff exoskeleton over a cuticular pad near leg joints is known to sense a tiny variation of mechanical stress, thereby, serve as a ultra-sensitive vibration sensor. In this talk, we introduce mechanosensors inspired by spiders having an ulltrasensitivity, durability. It also serve as a multifunctional sensor for a vibration and pressure sensing. The device fabricated on a sheet of plastic is reproducible, mechanically flexible and shape-deformable so that they can be easily mounted on human skin as skin electronic with multi-pixel arrays. We also show that the sensory system is applicable for highly selective speech pattern recognition even in noisy environment (~82dB).
For ultrasensitivity and durability, we considered the geometrical effects in cracks and self-healable polymers. By controlling crack depth by simple propagating process, the sensitivity of our sensor shows ~15,000 in 2% strain, which is the world best sensitivity value. Due to the high sensitivity, the signal-to-noise-ratio is 6 times higher than before, up to ~35 so that it can be used in sensing human voice clearly. Also, self-healable polymer helps to recover the crack gaps after 25,000 cycles. We introduce the possilibility of semi-permanent uses over 1,000,000 cycles in our sensors. The spider inspired sensory system with high sensitivity and durability would provide versatile novel applications such as E-skins, devices for medical applications, and IoT applications etc.
8:00 PM - BM07.03.15
Modular “Petri Dishes” for the Study of Organismal Interactions
Oskar Siemianowski 1 , Kara Lind 1 , Xinchun Tian 1 , Ludovico Cademartiri 1
1 , Iowa State University, Ames, Iowa, United States
Show AbstractThe study of chemical signaling between organisms in a laboratory is fundamentally hampered by the slow pace of diffusion, which requires organisms (e.g., plants and microbes) to be separated by small distances (<1mm). Therefore, organisms are either mixed together in simple cm-scale habitats (which prevents the independent control and monitoring of the individual organisms and their chemical signaling) or are fluidically connected in microfluidic devices (which do not scale well to plants and are expensive and complex).
We here present a scalable platform that allows for the creation of communities of organisms in which the effective signaling distance between organisms can be controlled independently of the physical distance, therefore enabling the quantitative study of chemical signaling between cm-scale organisms.
8:00 PM - BM07.03.16
microRNA Functionalized Magnetic Nanobeads for the Selective Capturing and Identification of Liver Cancer Associated Proteins
Isabel Gessner 2 , Xiaojie Yu 1 , Thomas Fischer 2 , Markus Schütz 2 , Margarete Odenthal 1 , Sanjay Mathur 2
2 Institute of Inorganic Chemistry, University of Cologne, Cologne Germany, 1 Institute of Pathology, University Hospital Cologne, Cologne Germany
Show AbstractLiver cancer is one of the most frequent but also deadliest cancer types worldwide. The early diagnosis of liver cancer is thus of major importance for the efficient treatment of patients. MicroRNAs (miR) which are small endogenous non-coding RNAs have been found to play an important role in posttranscriptional regulation. In this regard, miR-198 has been identified as tumor suppressor in liver cancer cells as it inhibits cell growth and proliferation. However, it has been previously revealed that miR-198 is actively transported out of liver cancer calls via exocytosis [1]. In this work, we focused on the identification of proteins responsible for the exosomal release of these oligonucleotides. Therefore, magnetic beads were surface functionalized with miR-198 antisense to selectively capture miR-198 and attached proteins and magnetically isolate them out of liver cancer cells. In this context, miR-198 antisense was customized with fluorophore at 3’ end and with amino and alkyne C6 at 5’end, respectively. Iron oxide nanoparticles of 27 nm diameter were prepared through thermal decomposition with oleic acid as solvent and surface active ligand. A reverse microemulsion method was used to coat the particles with an ultrathin silica shell and render them hydrophilic. As-prepared particles were further functionalized with functional azide and carboxylic acid groups, respectively and mir-198 antisense was subsequently covalently bound to the particle surface via click and carbodiimide chemistry. No free oligonucleotides could be detected in the supernatant upon magnetic separation. After adding miR-198 antisense conjugated nanoparticles in cell growth medium followed by incubation with transgenic miR-198 overexpressing liver cancer cells, cells were broken by use of a sonicator and nanoparticles were collected by magnetic stones. After RNA isolation from nanoparticles using the TRIZOL method, real time PCR was performed and captured miR-198 by the nanoparticles was quantified. 5-fold higher amounts of miR-198 were captured with miR-198 antisense conjugated nanoparticles in comparison to the scramble oligonucleotide conjugated nanoparticles, serving as a control. Moreover, red fluorescence in liver cancer cells was detected, proving their successful cellular uptake. Furthermore, proteins eluted from the miRNA conjugated nanoparticles were studied by mass spectrometry. Proteomic analysis identified a wide panel of proteins, which are putatively involved in miR-198 sorting and secretion. For the next step, functional analysis of the eluted proteins will be further investigated. The results indicate that customized miR-198 antisense conjugated nanoparticles can capture intracellular miR-198 as well as miR-198 binding proteins.
[1] X. Yu, H. Eischeid, R. Büttner, M. Odenthal, Z Gastroenterol, 54 (2016) 1343-1404.
8:00 PM - BM07.03.17
Optimization of Highly Aligned Fibers for Drug Delivery Application
Milad Khorrami 1 , Ning Yi 2 , Mohammad Reza Abidian 1
1 , University of Houston, Houston, Texas, United States, 2 , The Pennsylvania State University, State College, Pennsylvania, United States
Show AbstractThe ability to create materials with well-controlled structure in micrometer scale has been extensively practiced for drug delivery systems in the brain. While anti-inflammatory drugs have been widely used to reduce reactive response of implanted electrodes at electrode-neural tissue interface, the drug encapsulation and sustain release have been challenging due to inefficient method of drug encapsulation.
In this research, we optimized the electrospinning technique to create highly aligned and monodisperse microfibers and enhance sustained drug release capability from the fibers. The fabrication process includes electrospinning of poly(lactic-co-glycolic acid) (PLGA) (75:25) loaded with 7% (w/w) organic soluble dexamethasone (DEX) on 5mm×10mm gold-coated Si wafer substrates. The substrates were attached to a rotating wheel. The fibers were characterized using scanning electron microscopy. The experimental results revealed that only inner diameter of syringe tip and speed of rotating wheel have significant influence on fiber diameters. The diameter of fibers reduced from 2.06µm to 1.27µm (~38%) when the tip diameter decreased from 0.69mm to 0.34mm and from 1.61µm to 1.23µm (~24%) when the speed of rotating wheel increased from 100RPM to 600RPM. The alignment of fibers was predominantly affected by inner dimeter of syringe tip, electrical field, and speed of rotating wheel. The alignment was enhanced about 1.6%, 1.2%, and 3.6% when diameter of tip decreased from 0.69mm to 0.34mm, electrical field increased from 42.9kV/m to 85.7kV/m and the speed of rotating wheel increased from 100RPM to 1000RPM, respectively. The surface chemistry of DEX-loaded PLGA fibers was studied by Fourier transform infrared spectroscopy (FTIR). In-vitro release profile of DEX from aligned and random PLGA fibers was characterized using UV-Vis spectrophotometry. Results showed an initial burst release ~33% in 4days from the surface of fibers, followed by slower rate of release ~27% in next 20days due to bulk degradation and drug diffusion mechanism. However, random fibers showed slower rate of release than aligned fibers (~37% in 24days), presumably due to the smaller surface area to volume ratio of random PLGA fibers. Future studies will focus on coating of PLGA fibers with conducting polymers and study on-demand release of drug/biomolecules using electrical actuation of conducting polymers.
Symposium Organizers
Yizhou Dong, The Ohio State University
Christopher Alabi, Cornell University
Daniel Anderson, Massachusetts Institute of Technology
Bozhi Tian, University of Chicago
Symposium Support
Alnylam Pharmaceuticals, Inc.
Nanobio Delivery Pharmaceutical Co., Ltd.
Precision NanoSystems Inc.
BM07.04/BM08.03/BM09.03: Joint Session I: Flexible and Stretchable Electronics for Neural Interfaces
Session Chairs
Christelle Prinz
Bozhi Tian
Tuesday AM, November 28, 2017
Sheraton, 2nd Floor, Grand Ballroom
8:30 AM - *BM07.04.01/BM08.03.01/BM09.03.01
Soft and Stretchable Epidermal Electronics and Biosensors for Personalized Medicine
Roozbeh Ghaffari 1
1 Center for Bio-Integrated Electronics, Simpson Querrey Institute for BioNanotechnology, Northwestern University, Evanston, Illinois, United States
Show AbstractSoft bioelectronics systems enabled by recent advances in materials science are approaching the softness and curvilinear format of human skin. These systems are referred to as 'epidermal electronics' by virtue of their stretchable form factors and 'skin-like' mechanics compared to conventional packaged electronics and sensors. Here we present recent results for an emerging class of fully-integrated epidermal electronics. These devices incorporate arrays of sensors, microprocessors, memory and wireless connection (via Bluetooth low energy) configured in ultrathin, stretchable formats for continuous monitoring of neuromuscular and biomechanics signals. Quantitative analyses of strain distributions and circuit performances under mechanical stress highlight the utility of these systems in clinical operating rooms or in the home. We conclude with pilot clinical studies showing the utility of these epidermal systems in neurophysiological monitoring compared to clinical standards of care in operating rooms.
9:00 AM - BM07.04.02/BM08.03.02/BM09.03.02
Syringe-Injectable Mesh Electronics Integrate Seamlessly with Minimal Chronic Immune Response in Central Nervous System
Tao Zhou 1 , Guosong Hong 1 , Tian-Ming Fu 1 , Xiao Yang 1 , Robert D. Viveros 1 , Charles M. Lieber 1
1 , Harvard University, Cambridge, Massachusetts, United States
Show AbstractSeamless integration of minimally-invasive electrical probes into animal tissues is of central importance to both neuroscience research and biomedical applications. Previously we designed ultra-flexible mesh electronics that can be injected into animal brains through syringes. Here we report systematic histology studies of the interface between ultra-flexible mesh electronics and central nervous system. We also conducted histology studies of conventional electrical probes implanted in mice brain for comparison. Compared with conventional electrical probes, mesh electronics introduces little or no inflammation to brain tissues after chronic implantation. Unlike conventional rigid probes, which introduce depletion regions in brain tissues, neurons and axons surrounding the mesh electronics exist at endogenous tissue levels. More intriguingly, axons and neuron somata even penetrate into the interior of mesh electronics, allowing for the formation of seamless interfaces between brain tissues and mesh electronics. Seamless incorporation of minimum invasive ultra-flexible mesh electronics with tissues allows for a wide range of applications, including recordings, stimulation and repairing of the brain and other tissues such as spinal cord, opening a new window for brain-machine interfaces and cyborg animals.
9:15 AM - BM07.04.03/BM08.0.03/BM09.03.03
Dynamic Devices for Neural Interfacing
Christopher Proctor 1 , Vincenzo Curto 1 , Jolien Pas 1 , Adam Williamson 2 , George Malliaras 1
1 , Ecole des Mines St Etienne, Santa Barbara, California, United States, 2 , Aix Marseille University, Marseille France
Show AbstractSignificant advances have been made in the last two decades in interfacing electronic devices with the nervous system. Organic electronic materials in particular have emerged as ideal materials for interfacing with the brain due to their flexibility, biocompatibility and moreover their electronic and ionic conductivity. To that end, significant research efforts are being pursued to develop minimally invasive, implantable organic electronic devices integrating recording, stimulating, and drug delivery features. Here we report recent developments towards such dynamic devices for neural interfacing that take full advantage of the favorable properties offered by conducting polymers and polymer substrates. It is shown that thin, flexible devices can incorporate microfluidic channels to enable new sensing and therapeutic functionalities. Furthermore we show such features also open the door to novel implantation strategies that can reduce inflammatory tissue response as well as the surgical footprint required for implantation. We anticipate this work will accelerate the development of a new generation of devices for neural interfacing.
9:30 AM - BM07.04/BM08.03/BM09
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10:00 AM - BM07.04.04/BM08.03.04/BM09.03.04
Microfluidic Actuation of Flexible Microelectrodes for Neural Recording
Daniel Vercosa 2 3 , Flavia Vitale 1 , Alex Rodriguez 3 , Sushma Sri Pamulapati 1 , Frederik Seibt 4 , Eric Lewis 3 , Stephen Yan 5 , Krishna Badhiwala 5 , Mohammed Adnan 1 , Micheal Beierlein 4 , Caleb Kemere 3 5 6 , Matteo Pasquali 1 7 , Jacob Robinson 3 2 5
2 Applied Physics Program, Rice University, Houston, Texas, United States, 3 Department of Electrical and Computer Engineering, Rice University, Houston, Texas, United States, 1 Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, United States, 4 Department of Neurobiology and Anatomy, McGovern Medical School at UTHealth, Houston, Texas, United States, 5 Department of Bioengineering, Rice University, Houston, Texas, United States, 6 Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States, 7 Department of Chemistry, The Smalley-Curl Institute, Rice University, Houston, Texas, United States
Show AbstractNew tools for the recording and stimulation of neurons are key to advancing basic neuroscience research and developing new treatments for neural dysfunction. Despite tremendous advances, chronic electrodes for high-resolution electrical recording and modulation of neural activity at the cellular level still rely on rigid metal or silicon materials, which poorly match soft brain tissue and cause extensive acute and chronic injury, eventually leading to electrode encapsulation and the loss of signal over the scale of weeks or months.
Flexible electrodes and ultra-small microwires have been shown to significantly reduce brain damage during chronic implantation and increase the quality and longevity of neural recordings compared to rigid electrodes. However, unsupported flexible electrodes easily buckle on contact with the brain and require temporary stiffening agents to overcome the force of implantation. These agents increase the device footprint and may cause additional damage to the brain during implantation.
Here, we present the microfluidic drive, a novel solution to precisely actuate and implant flexible electrodes without changing the profile of the implanted electrode. After constructing a multi-layer polydimethylsiloxane (PDMS) microfluidic device to constrain electrodes, we utilize viscous fluid flow to push electrodes into tissue. The viscous fluid distributes force along the length of the electrode, allowing it to enter the brain without buckling. Computational analysis on electrodes made from flexible carbon nanotube fibers (CNTfs) suggests that implantation using the microfluidic drive increases the critical buckling force of CNTf microelectrodes by three-fold compared to standard methods.
The device’s hydraulic design with embedded valves enables precise control of the electrode position with minimum fluid output. In vitro experiments in brain phantoms show that microfluidic actuated CNTf electrodes can be implanted up to a 4-mm depth with 30 µm precision, while keeping the total volume of fluid ejected with the electrode below 0.5 µL.
10:15 AM - BM07.04.05/BM08.03.05/BM09.03.05
Hybrid Integration of Stiff Active Electronic Components on Stretchable Carrier Substrate
Florian Fallegger 1 , Aaron Gerratt 1 , Stephanie Lacour 1
1 , Bertarelli Foundation Chair in Neuroprosthetic Technology, Laboratory for Soft Bioelectronic Interfaces, Centre for Neuroprosthetics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne Switzerland
Show AbstractMost implantable neuroprostheses consist of electrodes reading neural signals and/or stimulating the neural tissues. These sites are usually treated as passive components in the system with raw signals treatment and logic processing being traditionally performed by external electronic circuits. To increase electrode density and signal bandwidth, closer integration of electronic hardware is being explored.
Here, we explore how to integrate (CMOS) active electronic components with soft surface electrodes embedded in thin silicone membranes. This hybrid integration combines conventional microfabrication techniques with innovative polymer processing techniques. The system consists of three main parts to enable the transition from the hard components to the stretchable substrate: the chip integrated with a soft via material, a stiff platform to isolate the component and contacts area from strain, and stretchable interconnects.
The hard components are picked and placed manually or semi-automatically and then embedded in a PDMS matrix. The stiff platform consists of 150μm thick SU8 disk integrated under the chips. The components are electrically contacted with a “soft via” consisting of a composite of platinum microparticles and PDMS, self-aligned to the contacts by screen-printing. Interconnects consist of stretchable gold thin films patterned by shadow masking then assembled in a multilayer structure in order to achieve simple circuits. The different layers (i.e. CMOS Integrated chip, soft vias and multiple layers of interconnects) are aligned and bonded using a custom-made alignment tool.
The hybrid circuit is characterized mechanically up to global uni-axial strain of 30% showing that the stiff components do not experience local strain greater than 0.2%. Furthermore the strain profile at the surface of the circuit, running from the mechanically-isolated rigid chips to the fully stretchable carrier is smooth suppressing any peak strain at the rigid-elastic interface. The developed method allows contacting thin (< 250μm) but rigid chips with contact sites with sizes in the range of 100μm to 100s μm. This system enables functions such as multiplexing or addressing individual electrode sites with a switch matrix scheme, which will allow for an increase in the number of electrode sites.
10:30 AM - BM07.04.06/BM08.03.06/BM09.03.06
Three-Dimensional Silicon Mesostructures for Biointerfaces
Yuanwen Jiang 1 , Bozhi Tian 1
1 , University of Chicago, Chicago, Illinois, United States
Show AbstractSilicon-based materials exhibit biocompatibility, biodegradability as well as a spectrum of important electrical, optical, thermal and mechanical properties, leading to their potential applications in biophysical or biomedical research. However, existing forms of silicon (Si) materials have been primarily focused on one-dimensional (1D) nanowires and two-dimensional (2D) membranes. Si with three-dimensional (3D) mesoscale features has been an emerging class of materials with potentially unique physical properties. Here, we incorporated new design elements in traditional synthetic methods to prepare various forms of 3D Si mesostructures and studied their functional biointerfaces with cellular components. In the first example, an anisotropic Si mesostructure, fabricated from atomic gold-enabled 3D lithography, displayed enhanced mesoscale interfacial interactions with extracellular matrix network. This topographically-enabled adhesive biointerface could be exploited for building tight junctions between bioelectronics devices and biological tissues. Another Si mesostructure with multi-scale structural and chemical heterogeneities, was adopted to establish a remotely-controlled lipid-supported bioelectric interface. We further adapted the bioelectric interface into the non-genetic optical modulation of single dorsal root ganglia neuron electrophysiology dynamics. Our results suggest that the dimensional extension of existing forms of Si could open up new opportunities in the research of biomaterials manufacturing and application.
10:45 AM - BM07.04.07/BM08.03.07/BM09.03.07
Wireless Photometers for In Vivo Behavioral Studies in the Deep Brain
Luyao Lu 1 , Philipp Gutruf 1 , Li Xia 2 , Dionnet Bhatti 2 , Michael Bruchas 2 , John Rogers 1
1 , Northwestern University, Evanstan, Illinois, United States, 2 , Washington University in St. Louis, St Louis, Missouri, United States
Show AbstractMonitoring the neural dynamics at the cellular level in behaving animals is a central goal of modern neuroscience. This is critical to understand neural computations and communications that create diverse brain functions. Current Ca imaging techniques such as fiber photometry provides some capabilities for recording neuron activities in animals during behaviors. However, the rigid optical fibers are not mechanically compliant with soft brain tissues, and the wired set up will restrict movements of animals, therefore impeding studies of natural behaviors. Here we present an integrated wireless photometry device capable of recording calcium transient activity in awake, freely behaving animals. The wireless photometry platform consists of a microscale inorganic light-emitting diode (μ-ILED) and a microscale inorganic photodetector (μ-IPD) for stimulating and recording Ca fluorescence, a detachable transponder, a control unit, a miniature power supply and an external receiver system. These μ-ILED and μ-IPD mount on ultrathin, flexible kapton substrate with overall dimensions (~350 μm wide and ~150 μm thick) significantly smaller than fiber optic cables. The wireless data transmission fully eliminates physical tethers and reduces motion artifacts. Detailed in vivo studies demonstrate that the wireless photometry platform allows high fidelity recording of calcium fluorescence in the deep brain, with results that are comparable or better than those obtained from fiber photometry system.
11:00 AM - *BM07.04.08/BM08.03.08/BM09.03.08
Wireless, Implantable Optoelectronics for Stimulating, Inhibiting and Monitoring Neuronal Dynamics in the Deep Brain
John Rogers 1
1 , Northwestern University, Evanston, Illinois, United States
Show AbstractThe recent development of materials and design designs for flexible, filamentary optoelectronic probes opens up opportunities for wireless stimulation, inhibition and monitoring of neuronal dynamics in the deep brain regions of freely-moving, untethered animals. This talk summarizes some of the latest results in this field of research, with a focus on fluorescence photometers that integrate sub-mm scale light sources and photodetectors on narrow, needle-shaped polymer supports, suitable for delivery into the brain at sites of interest. The ultrathin geometry and compliant mechanics of these probes allow minimally invasive implantation and stable chronic operation. In vivo studies in freely moving animals demonstrate high fidelity recording of calcium fluorescence in the deep brain, with measurement characteristics that match or exceed those associated with the most advanced, tethered fiber photometry systems. The capabilities in optical recordings of neural dynamics in untethered, freely moving animals have potential for widespread applications in neuroscience research.
BM07.05/BM08.04/BM09.04: Joint Session II: Conductive Polymers for Biointerfaces
Session Chairs
Polina Anikeeva
Anastasia Elias
Tuesday PM, November 28, 2017
Sheraton, 2nd Floor, Grand Ballroom
1:30 PM - *BM07.05.01/BM08.04.01/BM09.04.01
Skin-Inspired Electronic Materials and Devices
Zhenan Bao 1
1 , Stanford University, Stanford, California, United States
Show Abstract
Flexible organic electronics have attracted considerable attention over the past decade. Stretchable electronics represent another type of optoelectronic devices that are intrinsically elastic, that is they are foldable, twistable, and stretchable while maintaining performance, integrity and durability. Incorporated into devices, properly designed stretchable materials may result in more robust devices under bending and strain compared to flexible but not stretchable materials. For intrinsically stretchable electronics, it is desirable to have intrinsically stretchable materials, ranging from stretchable conductors, stretchable dielectric to stretchable semiconductors. In this talk, I will present various molecular design concepts for realizing stretchable electronic polymers without compromising electronic properties. Some applications of such materials will also be presented.
2:00 PM - BM07.05.02/BM08.04.02/BM09.04.02
Measuring Evoked Electrocorticography on Cortical Surface of Optogenetics Rat Using Transparent Organic Electro Chemical Transistors
Wonryung Lee 1 , Dongmin Kim 1 , Naoji Matsuhisa 1 , Masaki Sekino 1 , Tomoyuki Yokota 1 , George Malliaras 2 , Takao Someya 1
1 , The University of Tokyo, Tokyo Japan, 2 Department of Bioelectronics, Ecole Nationale Supérieure des Mines, Gardanne France
Show AbstractOptogenetics tools have been developed to control spatial and temporal neuronal function for making it possible to investigate complex neural circuitry. In general, it is hard to measure evoked response while strong light stimulation directly applying on the device due to light artifact, and nontransparent metallic wires.
In this work, we developed world first transparent organic amplifier and measured evoked electrocorticography (ECoG) signals from rat which has light sensitive neuron by using 3-μm-thick flexible transparent organic electro-chemical transistors (OECTs) with small light artifact. The trans-conductance (gm) of OECTs showed 1.1 mS with 70 μm/20 μm channel dimension. The transparent OECTs was fabricated on 1.2-μm-thick parylene (diX-SR) substrate. The 70-nm-thick Au grid for source/drain of OECTs deposited on the substrate, while it showing 60% transparency. The mechanical stability of Au grid was tested by applying compression. The sheet resistance of Au grid film changed 3 Ω/sq to 7 Ω/sq after 50% compression, while sheet resistance of ITO (70 nm) changed 80 Ω/sq to 400 Ω/sq at same condition. The SU-8 for passivation layer was patterned. The PEDOT:PSS for active material of OECTs was patterned by etching process [1].
The applicability of transparent OECTs was demonstrated by measuring light evoked signal on optogenetic rat [2]. The cortical surface was stimulated by laser at a wavelength of 473 nm through the transparent and non-transparent OECT. The transparent OECT could record evoked ECoG (ΔIds/gm = 700 µV) which has double amplitude of bio response from non-transparent OECT (ΔIds/gm = 350 µV) at the same light intensity (40 mW). Finally, non-light artifact was confirmed by control experiment using non optogenetic rat. The light artifact was less than peak to peak noise level (100 nA). The non-light artifact can be obtained because of wide bandgap and high capacitance of PEDOT:PSS. We concluded that measuring evoked ECoG on optogenetic rat showed that possibility of transparent OECTs for investigation on more complicate neural circuit.
[1] M Braendlein et al, Advanced Materials 29, 13 (2017).
[2] E Boyden, et al Nature Neuroscience 8, 1263 (2005).
2:15 PM - BM07.05.03/BM08.04.03/BM09.04.03
Biodegradable and Biocompatible Force Sensor Based on a New Piezoelectric Polymer to Monitor Important Bio-Physiological Pressures
Thanh Nguyen 1
1 , University of Connecticut, Storrs, Connecticut, United States
Show AbstractMeasuring vital bio-physiological pressures such as trans-pulmonary pressure, intra-articular pressure, intra-abdominal pressure, intra-ocular pressure, intra-cranial pressure etc. is important for monitoring health status, preventing dangerous internal force build up in impaired organs, and enabling novel approaches of using mechanical stimulation for tissue regeneration. Pressure sensors are often required to be implanted and directly integrated with native soft tissues and organs, therefore they should be flexible and at the same time, biodegradable to avoid any invasive removal surgery, which could damage the interfaced tissues. There has been recent achievements of biodegradable force sensors which are based on either Silicon piezo-resistive probe or capacitive biopolymer. Although exhibiting excellent sensing performance, these devices rely on (1) passive materials which need to be electrically powered, (2) exotic electronic materials (e.g. Silicon), which have not been confirmed to be completely degradable and safe for use inside human body, and (3) complex clean room micro-fabrication processes. Recently, triboelectric sensors fabricated with biodegradable polymers have been reported. Yet, friction-induced triboelectric charges, while ideal for energy harvesting, are often susceptible to variation of force-response due to the delay of charge dissipation in the sensor. Here, for the first time, we present the study and processing of a novel piezoelectric biopolymer of poly-l-lactid acid (PLLA) and create a biodegradable PLLA force-sensor which only relies on medical materials, used commonly in FDA-approved implants, to monitor tiny biological forces. The sensor is able to sensitively detect a wide range of pressure from 1 – 18 kPa, relevant to many biological pressures such as intracranial pressure (0 – 2.7 kPa), intraocular pressure (0 - 5.3 kPa), and intrabladder pressure (0 - 3.92 kPa). With 150 µm thick poly-lactide (PLA) encapsulators, we show the sensor can sustain its performance inside a buffer solution for 2-3 days. As a proof of concept, we implanted this sensor into a mouse thorax to measure trans-pulmonary/trans-diaphragmatic pressure of the animal for detection of respiratory disorder from obstructive pulmonary diseases. This novel biodegradable force-sensor, based solely on common medical biomaterials, offers an extremely useful tool to monitor important biological pressures. The sensor could be also integrated with native/engineered tissues and organs, forming a bionic self-sensing systems which could enable many applications in regenerative medicine, drug delivery, and medical devices.
2:30 PM - BM07.05/BM08.04/BM09
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3:00 PM - BM07.05.04/BM08.04.04/BM09.04.04
Organic Bioelectronic Materials and New Opportunities for Neural Interfacing
Jonathan Rivnay 1
1 , Northwestern University, Evanston, Illinois, United States
Show AbstractDirect measurement and stimulation of electrophysiological activity is a staple of neural interfacing for mapping of circuits, diagnosis and/or therapy. Such bi-directional interfacing can be enhanced by the low impedance imparted by organic electronic materials that show mixed conduction properties (both electronic and ionic transport). Many high performance bioelectronic devices are based on conducting polymers such as poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS. However, new structure-property and device based design rules have led to a new class of formulations/materials. The incorporation of glycol side chains into carefully selected backbone motifs, for example, has enabled a new class of high performance bioelectronic materials that feature high volumetric capacitance, transconductance >10mS (device dimensions ca. 5μm), and steep subthreshold switching characteristics. We explore the implications of these new materials for neural interfacing, including the effect of device operation regimes, and their effect on recording sensitivity and power consumption.
3:15 PM - BM07.05.05/BM08.04.05/BM09.04.05
New Approach for High Performance PDMS Based Electrodes for Neuronal Recording and Stimulation
Aline Renz 1 , Klas Tybrandt 1 2 , Flurin Stauffer 1 , Greta Thompson-Steckel 1 , Janos Voros 1
1 , ETH Zürich, Zürich Switzerland, 2 , University Linköping, Linköping Sweden
Show AbstractNew approaches for the fabrication of stretchable electronic implants in healthcare applications have attracted increased attention in the past years. Enhancement of the implant-tissue interface to both reduce the foreign body response as well as achieve improved electrode properties has been the main focus of many new devices. Stretchable implants have shown promise in their ability to reduce the foreign body response, however, there are still many limitations to the successful implementation of these devices. Specifically, achieving the combination of reliable electrical recording, as well as stimulation have yet to be established to gain long-term stable implants.
Here we present a new microelectrode array fabrication method, in which electrodes with controllable diameters ranging from 30 µm to 1 mm and tunable height can be generated on PDMS. These porous nanomaterial-based electrodes exhibit stable stimulation characteristics for several thousand pulsing repetitions, and demonstrate excellent impedance values of approximately 4 kΩ at 1 kHz for a 30 µm electrode. Additionally, this method can be used for a broad range of electrode designs. Overall, these electrodes can be utilized for the recording and stimulation of electrically excitable cells and tissues for both in vitro as well as in vivo applications.
3:30 PM - BM07.05.06/BM08.04.06/BM09.04.06
Soft and Intrinsically Stretchable Inkjet-Printed Transistor Arrays with Sub-Volt Operation for Skin-Like Bioelectronics
Francisco Molina-Lopez 1 , Theo Gao 1 , Ulrike Kraft 1 , Yeongin Kim 1 , Yuxin Liu 1 , Zhenan Bao 1
1 , Stanford University, Stanford, California, United States
Show AbstractSoft and stretchable electronic materials are receiving increasing attention in the fields of biology and biomedicine. Part of the reason for this interest resides in their mechanical properties, which match those of human body and other living organisms, allowing intimately integration with them in a minimally invasive manner. On the other hand, printing electronics presents the possibility of additive low-cost deposition and patterning of a wide range of solution-processed functional materials at ambient conditions and over large areas. These characteristics suit the requirements for integration of stacked organic electronic materials in the fabrication of skin-like electronics. Among the different printing methods, inkjet has special interest as it is a digital fabrication method with the capability of depositing materials on-demand and without physical contact, facilitating prototyping and patterning on different surface topologies.
In this work, we present a soft and stretchable array of transistors with sub-volt operation for application in skin-like bioelectronics. The transistors are composed of stacks of intrinsically stretchable functional materials, namely networks of semiconducting carbon nanotubes (CNTs), ionic dielectric and conducting and stretchable PEDOT:PSS. Since these materials can be only processed from solution and do not withstand high temperatures, inkjet printing has been used to facilitate their integration at ambient conditions (below 60°C) on an elastomeric substrate. Each material of the system was first formulated as an inkjet-printable ink using orthogonal solvents, and subsequently deposited and patterned using a commercial lab-scale tabletop inkjet printer for electronics. Good resolution of few tens of micrometers over large-areas of several cm2 was achieved for every printed material. The double-layer capacitor effect of the utilized ionic gate dielectric permitted over 1 µm-thick irregular printed films to operate below 1 volt and without risk of gate current leakage. Sub-volt operation is paramount in bioelectronics to avoid water splitting and to emulate neuron synapsis behavior. Furthermore, Inkjet printing-patterning of the gate dielectric suppressed cross talk between neighboring transistors. High mobility and large on/off current ratio were achieved for the printed transistors by fine-tuning the CNT network density through controlling the number of printed passes. The excellent electrical properties of the fabricated transistors along with their mechanical softness and the versatility offered by the non-contact and maskless nature of inkjet printing, makes this system a promising general platform easily customizable for different applications in bioelectronics. Indeed, the potential of the fabricated transistors array to work as a soft wearable system for neuron interfacing will be tested, advancing the new generation of brain-machine interfacing devices and prosthetics with sensing capabilities.
3:45 PM - BM07.05.07/BM08.04.07/BM09.04.07
Organic Electronics for High-Resolution Electrocorticography of the Human Brain
Dion Khodagholy 2 , Jennifer Gelinas 1 , Gyorgy Buzsaki 3
2 Electrical Engineering, Columbia University, New York, New York, United States, 1 , Columbia University, New York, New York, United States, 3 Neuroscience Institute, NYU Langone Medical Center, New YorK, New York, United States
Show AbstractLocalizing neuronal patterns that generate pathological brain signals may assist with tissue resection and intervention strategies in patients with neurological diseases. Precise localization requires high spatiotemporal recording from populations of neurons while minimizing invasiveness and adverse events. We describe a large-scale, high-density, organic material–based, conformable neural interface device (“NeuroGrid”) capable of simultaneously recording local field potentials (LFPs) and action potentials from the cortical surface. We demonstrate the feasibility and safety of intraoperative recording with NeuroGrids inanesthetized and awake subjects. Highly localized and propagating physiological and pathological LFP patterns were recorded, and correlated neural firing provided evidence about their local generation. Application of NeuroGrids to brain disorders, such as epilepsy, may improve diagnostic precision and therapeutic outcomes while reducing complications associated with invasive electrodes conventionally used to acquire high-resolution and spiking data.
4:00 PM - BM07.05.08/BM08.04.08/BM09.04.08
Real Time Monitoring of 3D Cell Cultures In Vitro Using Conducting Polymer Scaffolds
Charalampos Pitsalidis 1 , Magali Ferro 1 , Donata Iandolo 1 , Isabel del Agua 1 , Sahika Inal 2 , Roisin Owens 1
1 , Ecole des Mines de Saint-Etienne, Gardanne France, 2 , King Abdullah University of Science and Technology, Saudia Arabia (KAUST), KAUST Saudi Arabia
Show AbstractThree-dimensional (3D) cell cultures are sought to improve the physiological relevance of cell-based assays and provide a better alternative to animal testing compared to the conventional cell-monolayer based cultures. We report herein an in vitro toxicology screening platform based on 3D conducting polymer scaffolds consisting of poly(3,4-ethylene dioxythiophene (PEDOT). The conducting scaffolds are used concurrently as a biocompatible host to support 3D cell cultures as well as an electrode to electrically probe cell behavior. Dynamic electrochemical impedance spectroscopy of the 3D conducting scaffolds reveal in real time the different features of the cell culture including adhesion, growth and proliferation. By tuning the composition parameters and the microstructural properties of the fabricated scaffolds we were able to provide a suitable 3D environment for the cells without affecting the electrical sensing capability of the device. The proposed platform tested with various cell types including fibroblasts and epithelial cells represents a nondestructive and label-free in-situ cell-based toxicity screening platform, paving the way towards next generation in vitro toxicology assays toward the reduction of animal tests.
4:15 PM - BM07.05.09/BM08.04.09/BM09.04.09
Elastic Microelectrodes for Bioelectronic Recording from Peripheral Nerves
Tobias Cramer 1 , Francesco Decataldo 1 , Davide Martelli 2 3 , Marta Tessarolo 1 , Mauro Murgia 4 , Beatrice Fraboni 1
1 Department of Physics and Astronomy, University of Bologna, Bologna Italy, 2 Department of Biomedical and Neuromotor Sciences-Physiology, University of Bologna, Bologna Italy, 3 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia, 4 Istituto per lo Studio dei Materiali Nanostrutturati (CNR-ISMN), Consiglio Nazionale delle Ricerche, Bologna Italy
Show AbstractMonitoring of bioelectric signals in peripheral nerves is crucial to gain understanding of how the autonomic nerve system controls specific body functions related to disease states such as inflammatory response.1,2 In order to achieve long-term, chronic recordings, that do not interfere with nerve function or animal behaviour, a low-invasive wireless electrode technology has to be developed.
In this work, we present our efforts to achieve a wireless peripheral nerve interphase based on stretchable electrodes to record from the splanchnic and renal nerve in rats. Polydimethylsiloxane (PDMS) is used as elastic substrate and encapsulation material for electrodes and interconnects made of thermally evaporated Ti/Au. A kinked electrode shape has been introduced to facilitate the surgical procedure to position and fix the electrodes at the nerve. An electropolymerized layer of the doped organic semiconductor Pedot:Pss is deposited on the electrodes to reduce impedance and improve signal quality. The impact of strain on electronic and morphologic properties of the electrode are investigated. In in-vivo recordings, bioelectronic signals are amplified and digitized by a subdermal battery operated transmitter. We show that our electrode is able to record neural activity of peripheral nerves during chronic experiments in free moving animals.
1. D. Martelli, S. T. Yao, M. J. McKinley and R. M. Mc Allen, Reflex Control of Inflammation by Sympathetic Nerves, Not the Vagus, J Physiol 592.7, 1677-1686 (2014).
2. D. Martelli, D. G. Farmer, and S. T. Yao, The Splanchnic Anti-Inflammatory Pathway: Could It Be the Efferent Arm of the Inflammatory Reflex?, Exp Physiol 101.10, 1245-1252 (2016).
BM07.06: Poster Session II
Session Chairs
Wednesday AM, November 29, 2017
Hynes, Level 1, Hall B
8:00 PM - BM07.06.01
Tumor Cell Invasion Using FPGA Based Directional Control of Magnetospirillum Magneticum
Sushila Silwal 1 , Marvin Xavierselvan 1 , Prabir Patra 1 , Isaac Macwan 1
1 , University of Bridgeport, Bridgeport, Connecticut, United States
Show AbstractMagnetospirillum magneticum (AMB-1) are a species of magnetotactic bacteria that are capable of orienting along the earth’s magnetic field lines through their organelles called magnetosomes. Even though MTB’s have been shown to have applications in the fields such as MEMS, micro total analysis systems (μTAS) and lab-on-chip, there is very little information available on a directional control mechanism that can harness the true potential of these microorganisms in dealing with individual tumor cells at the microscale. Typically techniques such as magnetic hyperthermia and magnetic drug delivery using nanoparticles are used to target the tumor cells. However, these techniques pose another problem in terms of nanoparticle accumulation over a period of time leading to a potentially toxic conditions for the surrounding healthy cells. Preliminary studies have shown that AMB-1 can infect the U87 MG tumor cells and it was found that all the tumor cells were killed in 24 hours. Thus, guiding AMB-1 along a predefined path by controlling the intensity of a local magnetic field can pave a way for not only selectively doping the tumor cells but also isolating the tumor cells from a group of healthy cells through the invasive assays. Here we come up with a novel platform of standing solenoid coils to generate a local magnetic field that is used for the directional control of AMB-1. This is achieved by a controlled switching of the current through the coils, which in turn guides the AMB-1 along the predetermined path over a glass slide containing the cells to be invaded. Each coil is ~200 micrometers in diameter and has five turns that make up a tiny mesh to create a path for the bacteria to move. The arrangement of the coils is such that it creates an intersection consisting of three standing coils on the X axis and three on the Y axis thereby producing the magnetic field along the Z axis. Additionally, Cyclone II FPGA is used to control the direction of 0.5A of current through a driver IC, which in turn controlled the magnetic field of 15.7 gauss. It is found that the motility of AMB-1 is controlled in a particular direction along the X-Y axes. The experimental demonstration involved the AMB-1 invading the HCF tumor cells. Statistical analysis is performed to know the number of AMB-1 and HCF cells before and after the invasion to confirm their viability. The viable HCF cells were then mixed with the non-AMB-1 containing HCF cells. On the reverse switching of the coils, the AMB-1 containing HCF cells were directed back from the mixture, tracing the same path in order to understand the efficacy of AMB-1 in isolating selective HCF cells. The bacterial invasion also indicated how the AMB-1 and the HCF cells interacted clarifying the survival rate of the AMB-1 after the invasion.
8:00 PM - BM07.06.02
PEGylated Artificial Antibodies—Plasmonic Biosensors with Improved Selectivity
Jingyi Luan 1 , Srikanth Singamaneni 1
1 , Washington University in St. Louis, St Louis, Missouri, United States
Show Abstract
Molecular imprinting, which involves the formation of artificial recognition elements or cavities with complementary shape and chemical functionality to the target species, is a powerful method to overcome a number of limitations associated with natural antibodies. An important but often overlooked consideration in the design of artificial biorecognition elements based on molecular imprinting is the non-specific binding of potentially interfering species to non-cavity regions of the imprinted polymer. Here, we demonstrate a universal method, namely, PEGylation of the non-cavity regions of the imprinted polymer, to minimize the non-specific binding and significantly enhance the selectivity of the molecular imprinted polymer for the target biomolecules. The non-specific binding, as quantified by the localized surface plasmon resonance shift of imprinted plasmonic nanorattles upon exposure to common interfering proteins, was found to be more than 10 times lower compared to the non-PEGylated counterparts. The method demonstrated here can be broadly applied to a wide variety of functional monomers employed for molecular imprinting. The significantly higher selectivity of PEGylated molecular imprints takes biosensors based on these artificial biorecognition elements closer to real-world applications.
8:00 PM - BM07.06.03
Wafer Scale Fabrications of Polydimethylsiloxane Fluidics for Fluorescence Imaging of DNA Molecules
Sung-Wook Nam 1
1 Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu Korea (the Republic of)
Show AbstractPolydimethylsiloxane (PDMS) fluidics are important for biomedical applications including diagnostic and therapeutic devices. Nanofabrication methods for PDMS fluidics are essential parts to pursue low-cost and large-scale productions of the biomedical devices. Here, we report a method to create PDMS nano-microfluidics for 200 mm wafer-scale productions. We employed conventional photolithography to print the design of fluidic channels on photoresist (PR) spun on silicon (Si) wafers. The PR patterns were transferred to the silicon wafer by reactive ion etching (RIE) consisting of a repeated CF4 and SF6-based plasma procedure which is known as Bosch process. The photolithography followed by the RIE processes allowed us to prepare high-quality Si micropatterns with vertical sidewall structure which is well suited for PDMS fluidic channel fabrications. For a given mold structure on the Si wafer, we poured PDMS and cured the samples to produce microfluidic channels with 2 to 5 micrometer size. The PDMS fluidic devices were uniformly created over the Si wafers. Each PDMS device was peeled off from the Si wafer and attached with slide-glass. We examined channel-wetting properties by both optical microscope imaging and electrical characterization of the conductance of KCl electrolyte across the microfluidic channels. We confirmed the wetting of the microfluidic channels and inserted the fluorescence dye-tagged lambda-DNA molecules. Fluorescence imaging of the DNA single molecules and the dynamics of DNA translocations will be discussed.
8:00 PM - BM07.06.04
Triggered Release of Hydrophobic Drugs with Magnetic Nanoparticles and Alternating Magnetic Fields
Irene Andreu 1 2 , Ian Esdale 1 , Sarah Poteryko 1 , Courtney van Ballegooie 2 , Donald Yapp 2 , Byron Gates 1
1 , Simon Fraser University, Burnaby, British Columbia, Canada, 2 , BC Cancer Agency, Vancouver, British Columbia, Canada
Show AbstractWe propose the use of polymeric nanospheres as vehicles to contain, deliver and controllably release low-solubility anticancer compounds. These nanoparticles can release drug molecules on demand. The low solubility in physiological conditions of certain compounds with anticancer potential is a hurdle that inhibits their clinical applications. It is estimated that more than 80% of compounds in pharmaceutical research get discarded at the early stages of research merely based on their poor solubility in aqueous media.1 Although several methods have been developed to increase the solubility of hydrophobic compounds for their use as drugs, these methods usually involve the use of other substances, such as vehicles or surfactants, which might compromise the efficacy of the drug or produce unwanted side effects.2 In addition, control of the drug concentration in tumours is highly desirable in oncological treatments, as it would reduce side effects to the patient. Spatial and temporal control of drug concentrations can be achieved using nanoparticles with enhanced tumour accumulation and through the triggered release of the therapeutic agents.
Our approach combines the use of poly(lactic-co-glycolic acid) (PLGA) nanoparticles and iron oxide magnetic nanoparticles (IONPs), each approved by the FDA for their use in therapeutic devices. The hydrophobicity of PLGA permits a high loading of hydrophobic compounds, while the IONPs are able to generate heat by using alternating magnetic fields; this heat subsequently triggers the release of drugs on demand from the PLGA to cells in the vicinity of these particles. Furthermore, the magnetism of the IONPs can be used as MRI contrast agents, to locate the nanoparticles and serve as a guide to trigger the drug release only after observing nanoparticle accumulation in the tumour. In this work, we present the synthesis and characterization of these nanoscale drug delivery systems, and demonstrate the loading and triggered release of hydrophobic molecules using alternating magnetic fields that are safe for use in clinical patients.
1. Babu et al. Solubility Advantage of Amorphous Drugs and Pharmaceutical Cocrystals; Crys Growth & Design (2011) 11, 2662-2679
2. Savjani et al. Drug Solubility: Importance and Enhancement Techniques; ISRN Pharm (2012), 195727
8:00 PM - BM07.06.05
Immunostimulatory Microgels for Immune Cells Recruitment and Activation
Mahboobeh Rezaeeyazdi 1 , Thibault Colombani 1 , Sidi Bencherif 1 2 3
1 , Northeastern University, Boston, Massachusetts, United States, 2 , Harvard University, Cambridge, Massachusetts, United States, 3 , University of Techn ology of Compiègne, Compiègne France
Show AbstractDespite monumental advances in cancer therapy, cancer still is the second cause of death worldwide representing a major public health challenge. Although traditional therapies such as surgery, radiation and chemotherapy have improved cancer survival rates, detrimental side effects combined with limited therapeutic benefits against aggressive types of metastatic cancer, the development of new therapeutic strategies remains vital. Recently, cancer immunotherapy has been gaining momentum due to its ability to potentially stimulate the immune system and induce a specific, effective and long lasting immune response to eradicate tumors even in remote areas. However, the outcome of a number of current immunotherapeutic approaches (e.g. cell-based therapies) remain quite disappointing suggesting the need to develop new alternatives. Based on significant advances in biomaterials design and engineering of multifunctional and versatile polymeric vehicles for in vivo molecular delivery, our team developed innovative approaches to design the next generation of immunostimulatory biomaterials. To this end, we designed an injectable and ionically sensitive microgel system loaded with (i) GM-CSF, a growth factor responsible of dendritic cells accumulation; and (ii) CpG ODN 1826 (cytosine-phosphodiester-guanine oligodeoxynucleotide), an adjuvant specific to TLR9. We hypothesize that intra-tumoral injection of microgels pre-loaded with immunomodulator factors can lead to the effective recruitment, proliferation, and activation of dendritic cells. These immunostimulatory microgels have the potential to induce a potent immunogenic environment at the tumor site while evoking a strong immune response against cancer.
Acknowledgement: This research was financially supported by Northeastern University (Tier 1 Provost grant).
8:00 PM - BM07.06.06
Protein Adsorption Property of Silicon/Titanium Carbide and Their Thin Films Grown onto NiTi by Radiofrequency Magnetron Sputtering
Chisaki Hiwatari 1 , Tadashi Shiota 1 , Kazuo Shinozaki 1 , Toshiyuki Ikoma 1
1 , Tokyo Institute of Technology, Tokyo Japan
Show AbstractStent placement has been used as a therapy for vascular diseases due to its low invasiveness and short operation time. Self-expanding stents utilizing super elasticity of shape memory alloy (NiTi) attract attention; mechanical load on vascular wall is moderate in expanding. There are still problems such as restenosis and elution of metal ions. Thus, stents with a non-degradable polymer with/without drugs have been developed; however inflammation or vascular occlusion was sometimes caused by the polymer. On the other hand, silicon carbide (SiC) and titanium carbide (TiC) have been described to be non-toxic substances, but there are few descriptions for medical applications. The objectives of this study were to elucidate protein adsorption ability of SiC and TiC powders and to make thin films of SiC or TiC onto NiTi by radiofrequency magnetron sputtering.
A 20 mg of SiC, 30 mg of TiC or 40 mg of NiTi was mixed and rotated in 1 mL of phosphate buffer saline including bovine serum albumin (BSA, pI:4.6, 0 to 6.0 mg/mL) or chicken egg white lysozyme (LSZ, pI:11, 0 to 6.0 mg/mL) at 20°C for 6 hours. The liquid phase was separated from the samples by centrifugation, and was supplied for UV-Vis spectrometer at 280 nm to measure adsorption amounts of proteins. The SiC and TiC sintered bodies with φ 50 × 5 mm in size were made by spark plasma sintering at the pressure of 60 MPa and the temperature of 1900°C and 1600°C. The sintered bodies were used as sputtering targets. SiC and TiC thin films were grown on NiTi by radiofrequency magnetron sputtering. Ar gas was introduced into the chamber at 6.5 SCCM and total pressure was fixed at 35 mTorr. The films were analyzed by X-ray photoelectron spectroscopy (XPS), and peeling strength between films and substrates was measured according to JIS8504.
The adsorption isotherms of BSA or LSZ onto SiC and TiC powders and NiTi plate were approximated by Langmuir equation; the saturated adsorption amounts of BSA were calculated to be 0.45 μg/m2, 5.2 μg/m2 and 350 μg/m2, and of LSZ were calculated to be 1.7 μg/m2, 0.30 μg/m2 and 503 μg/m2, correspondingly. The components of Ti-O binding on TiC was 57.9%, and that of Si-O binding on SiC was 7.5% from XPS spectra. These results indicate that the different adsorption amounts of the proteins were clearly due to the existence of oxide layers. Furthermore, the isoelectric point of SiO2 and TiO2 were 1.5 ~ 3.7 and 6.0 ~ 6.7, which was also affected to the adsorption phenomena of proteins with different pI values. BSA and LSZ were adsorbed onto SiC and onto TiC at less than 0.5% and 2% compared with that onto calcium phosphates at 1200 μg/m2. The bulk density and average grain size of SiC target was 79% and 2.1 mm, and those of TiC target was 96% and 4.7 mm. Thin films after Ar sputtering (2 kV and 2 min) contained 0% of Si-O or 9.6% of Ti-O as impurities from XPS spectra. Both thin films of SiC and TiC were not removed by the peeling test.
8:00 PM - BM07.06.07
Biosensors Based on Nano-Gold/Zeolite-Modified Ion Selective Field-Effect Transistors for Creatinine Detection
Berna Ozansoy 1 , Sergei Dzyadevych 2 , Burcu Akata Kurc 1
1 , Middle East Technical University, Ankara Turkey, 2 , Laboratory of Biomolecular Electronics, Kyiv Ukraine
Show AbstractCreatinine (CD) is one of the most important analytes used for determination of kidney and muscular dysfunction and control of patients receiving hemodialysis. It is usually determined by spectrophotometric methods. These methods require expensive instruments, time-consuming sample pre-treatment and skilled persons to operate them. Biosensors for CD determination have been proposed to reduce cost, time and complexity of routine analysis allowing home testing. Creatinine biosensors developed are mostly based on either amperometric or potentiometric detection methods. The sensing performance of these biosensors is greatly affected by the immobilization of the bioselective element onto the transducer surface. These methods, however, may suffer from low reproducibility and poor spatially controlled deposition. Recently, inorganic have been investigated to solve these problems. Zeolites are inorganic solids with large surface areas and well-defined internal structures of uniform cages, cavities or channels. In the field of biosensors, zeolites are promising materials for enzyme immobilization since they have a large surface area, thermal/mechanical stabilities, ion exchange capacity and controllable hydrophilicity/hydrophobicity. Additionally, conjugation of gold nanoparticles and host materials is known to enhance biocatalytic activity. In our recent study, owing to the unique advantages such as easiness, fastness, inexpensiveness and ability to analyse the biological content as electrical signal of electrochemical biosensors with an additional advantage of miniaturized silicon-based semiconductor nature of field-effect transistor (FET)-based sensors, we have developed a novel approach for constructing an ion selective field-effect transistor (ISFET) device for creatinine monitoring using silicalite. The objective of this study was to combine the advantages of using zeolites with different properties (i.e. structure and particle size) with an additional benefit of the ability to use zeolites as hosts for gold nanoparticles to be used for the first time to improve the characteristic properties of ISFET-based creatinine biosensors. In this study, creatinine-based ISFET biosensors have been successfully produced by modifying the gate of ISFET with four different types of zeolites separately. All zeolite-based biosensors had higher sensitivities, lower detection limits, response and regeneration time than traditional biosensors with good reproducibility showing that zeolites can be good alternatives for creatinine biosensor production. Specific type of Beta type biosensor resulted in threefold increased sensitivity compared to traditionally used biosensor, which could be attributed to favourable microenvironment for CD to avoid denaturation as well as increased surface area produced by gold nanoparticles with good stability. This result showed that gold nanoparticles can be used with zeolite to improve the characteristics of ISFET-based biosensors.
8:00 PM - BM07.06.08
A Novel Amperometric Glutamate Biosensor Based on Glutamate Oxidase Adsorbed on Silicalite
Berna Ozansoy 2 , Sergei Dzyadevych 3 , Burcu Akata Kurc 1 2
2 Micro and Nanotechnology Department, Middle East Technical University, Ankara Turkey, 3 Taras Shevchenko National University of Kyiv, Institute of High Technologies, Kyiv Ukraine, 1 , Middle East Technical University, Ankara Turkey
Show Abstract
Glutamate (glutamic acid) plays an important role in vital activity of humans and other mammals, especially in the functioning of the central nervous system. In particular, glutamate is the major excitatory neurotransmitter in the central nervous system of mammals. It is a part of many pharmaceuticals due to its ability to sensitize the taste receptors and stimulate the brain activity. Therefore, glutamate is often used as a flavor enhancer. Thus it is rather problematic to completely eliminate glutamate from the diet. Determination of glutamate is of significance in clinical biochemistry when diagnosing the diseases associated with abrupt changes of glutamate level in the body, including diseases of liver and cardiovascular system. The methods of accurate and rapid detection of glutamate are required in neurophysiology and neuropathology, fundamental and clinical medicine, pharmaceutical and food industries, and in analytical biochemistry and biotechnology. The up-to-date standard methods for highly accurate determination of glutamate require qualified personnel and complex expensive equipment. Currently, a number of biosensors and biosensor systems have been developed for glutamate determination in various real samples—foods and pharmaceuticals, cell cultures, blood serum and urine, microdialyzates at neurophysiological studies, and for monitoring fermentation in the food industry. However, many of these biosensors are based on a complex and time-consuming method of immobilization, often with the use of toxic reagents. Neither of them has not been commercialized so far. Therefore, the elaboration of new methods of creating glutamate-sensitive biosensors with improved analytical characteristics is an actual challenge. This study is aimed at creation of the amperometric biosensor for glutamate determination, which allows faster and more accurate analysis and can be suitable for mass production in the future. The problem is supposed to be solved using a new method of enzyme immobilization, the glutamate oxidase adsorption on transducers covered with a silicalite layer. For this purpose, a new amperometric biosensor for glutamate detection using a typical method of glutamate oxidase (GlOx) immobilization via adsorption on silicalite particles was developed. The main parameters of modifying amperometric transducers with a silicalite layer were determined along with the procedure of GlOx adsorption on this layer. The biosensors based on GlOx adsorbed on silicalite demonstrated high sensitivity to glutamate. The linear range of detection was from 2.5 to 450 μM, and the limit of glutamate detection was 1 μM. It was shown that the proposed biosensors were characterized by good response reproducibility during hours of continuous work and operational stability for several days. The developed biosensors could be applied for determination of glutamate in real samples.
8:00 PM - BM07.06.09
Computational and Experimental Design of Imprinted Polymeric Nanoparticles—A Novel Theranostic Detection and Neutralization Mechanism for Lipopolysaccharides
Sriharshita Musunuri 1 2 , Christopher Lausted 2
1 , Henry M. Jackson High School, Mill Creek, Washington, United States, 2 , Institute for Systems Biology, Seattle, Washington, United States
Show AbstractLipopolysaccharides (LPS) are harmful biomolecules found on the surface of gram-negative bacteria that initiate fatal inflammatory responses. Past efforts to detect and extract LPS have been hampered by the presence of certain proteins, high synthesis costs, or incompatibility with body fluids.
In this research, the lock-and-key mechanism of molecularly imprinted polymers was utilized to design a novel fluorophore-conjugated nanoparticle capable of simultaneous LPS detection and neutralization through competitive inhibition of the LPS-LBP complex. Functional monomers were screened for LPS affinity using GROMACS (molecular dynamics) software. Analysis of system stability, reaction spontaneity, and non-covalent interactions indicated that itaconic acid and hydroxypropyl methacrylate were prime candidates for LPS imprinting.
These computational results were translated to experimental synthesis of MIP nanoparticles by precipitation polymerization, with varying concentration ratios of EGDMA cross-linker and functional monomer; polymerization was initiated with AIBN in DMF solvent and nanoparticles were centrifuged, run through 3 cycles of Soxhlet extraction with distilled water, and vacuum-dried. SEM imaging indicated particles in sizes ranging from 50 to 200 nm in diameter, and FTIR comparisons between LPS-imprinted and non-imprinted polymers showed a significant increase in absorption intensity at 2975 cm-1, which was indicative of LPS-specific hydrogen-bonding interactions. Surface plasmon resonance confirmed this selective polymer composition as LPS-MIP binding was observed under plasma-like conditions.
Fluorescence spectroscopy established a statistically significant relation between LPS concentration and fluorophore intensity, confirming diagnostic potential and offering a quantitative method of assessing gram-negative sepsis for the first time. Based on comparisons to the standard LAL assay, these polymers are able to bind endotoxins for a fraction of the cost and be applied to decrease the effects of gram-negative sepsis and LPS contamination.
8:00 PM - BM07.06.10
Native Silk Enables High-Resolution Micro Patterning
Anastasia Brif 1 , Chris Holland 1 , Frederik Claeyssens 1
1 , University of Sheffield, Sheffield United Kingdom
Show AbstractHistorically silk’s applications have been limited to the area of textiles, where as a fibre it boasts good mechanical strength, lustre and dye uptake. However recently silk’s other attributes have garnered attention, such as its biocompatibility, stability in water and slow biodegradation rate. Furthermore, we now appreciate silk is much more than just a fibre and can be reprocessed into a variety of forms. Silk films, for example, are produced from a protein solution, and can be made to be soft, flexible and transparent, making them suitable for bio-electronic and bio-optical devices. However current approaches towards controlling the surface patterning of these devices is limited due to challenges surrounding the resolution of the structures generated, which is the focus of this study.
Here we show mask-less projection based lithography is well suited towards structuring silks. Our approach has several advantages over traditional mask-based lithography as it enables rapid and controllable fabrication of complex multi-layer patterns. In this technique, a digital micro-mirror device (DMD) is used to project the desired structure onto a silk solution containing a photocrosslinker, which serves to stabilise the silk structures after laser illumination. Traditionally, silk-based devices are made from a reconstituted silk solution, which is produced via the resolubilisation of spun fibres in a process that is known to decrease silk’s molecular weight. However in this work, we extract the native silk protein solution directly from the animal’s silk gland, without additional purification, rehydration or subsequent reduction in molecular weight. Through the application of image correlation analysis we find that native silk solutions offer a clear advantage over reconstituted solutions in the achieved complexity of surface patterns, allowing us to fabricate a high-resolution, detailed structures and images.
To understand the mechanism behind this process, the position of amide I band was compared before and after light exposure. This position depends on the secondary structure of the protein and indicates the formation of β-sheet structure. No shift in the position of band was recorded, indicating no protein denaturation following the light illumination. This result suggesting patterning via crosslinking but without conversion of the silk to the denatured state. By applying methanol solution with various concentrations, a control over the crystallisation level of the silk was achieved. The chemical crosslinking, which was found to be the main mechanism in the patterning process resulted from di-tyrosine bond formation.
We believe this work unveils a promising new area of research where the combination of mask-less, light based micro patterning and native photo-curable silk solutions, offer improved resolution and image complexity paving the way for novel silk-based applications.
8:00 PM - BM07.06.11
Enzyme Immobilization on Magnetic Particles via SpyTag/SpyCatcher Partners
Weizheng Shen 1 2 , Carol Ladner-Keay 1 2 , Valentyna Semenchenko 3 , Trent Bjorndahl 1 2 , Carlo Montemagno 1 2
1 , Ingenuity Lab, Edmonton, Alberta, Canada, 2 Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada, 3 , Nanotechnology Research Centre, Edmonton, Alberta, Canada
Show AbstractEnzymes help facilitate chemical reactions in cells and play a very important role in metabolic pathways. In order to utilize enzymes in continuous bioassay systems or microbioreactor configuration, enzymes need to be immobilized on surface and localized in a certain sequence. Exploiting magnetic particles for enzyme immobilization provides several advantages, such as the convenience of reaction allocation, easy separation from reaction mixtures, and the feasibility of repeat or continuous use, not to mention the enhanced stability. However, most of reported methods employed either direct covalent coupling or generic binding proteins, such as protein A and streptavidin. Direct covalent coupling may cause enzyme lose its conformation and activity, while generic binding proteins may not be stable in certain conditions.
Inspired by recent works on SpyTag/SpyCatcher, we believe that using peptide tags to form “unbreakable” covalent bonds in between the magnetic particles and target enzymes is a better option. SpyTag (13 residue) and SpyCatcher (116 residue) are a pair of complementary peptide/protein that spontaneously reconstitute to form an isopeptide bond under a broad range: temperatures (4-37oC), pH values (5-8), and buffer systems (no specific anion or cation required). Magnetic beads that functionalized with either SpyTag or SpyCatcher chemically bond to their complementary pair that is fused with the target enzyme irreversibly and specifically. With an external magnetic field, the bound enzymes can be easily isolated from lysate and used directly in downstream applications.
Using an amino acid with a propargyl side chain, we were able to synthesize a SpyTag peptide with an alkyne-functionalized group in solid phase synthesis, and to crosslink it to azide-modified magnetic particles surface via click chemistry. To prove the concept, we chose GAPDH as the model enzyme. GAPDH is an enzyme that catalyzes the sixth step of glycolysis and serves to break down glucose for energy as well as for carbon molecules. We are currently expressing the SpyCatcher-GAPDH fusion protein and immobilizing it onto magnetic particles through the SpyTag and SpyCatcher interaction. The feasibility of this approach will be evaluated by quantifying the GAPDH immobilization. The enzyme activity, thermal stability, reusability, and enzymatic reaction kinetics will be determined for both free and immobilized GAPDH. Eventually, we aim to place the immobilized GAPDH in a microbioreactor along with other immobilized enzymes for an efficient glycolysis.
8:00 PM - BM07.06.12
Design of De Novo Fibrin-Specific Targeting Peptide
Moon Young Yang 1 , Jeong Heon Yu 2 , Yoon Sung Nam 1 2
1 KAIST Institute for the NanoCentury, Korea Advanced Institute of Science and Technology, Daejeon Korea (the Republic of), 2 Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon Korea (the Republic of)
Show AbstractFibrin is a fibrous protein matrix involved in the clotting of blood and is formed in response to injury from its precursor protein, fibrinogen. In addition to the role in would healing, it has been reported the role of fibrin in a variety of pathologies including fibrosis, Alzheimer’s disease, cardiovascular disease, and cancer. Because of its prominent role in various diseases, the fibrin-specific targeting has a great potential for medical implications. However, it has been challenged due to the structural similarity with its precursor fibrinogen that shares 98% of its structure with fibrin and is present at the high concentration of 2–4 mg mL-1 in the blood.
Here, we present designing a heptameric peptide that selectively targets a γ-module of fibrin based on molecular dynamics and docking simulations. An insight from the structural change during the fibrinogen to fibrin conversion, a γ381-390 (P2 strand) region of the γ-module is targeted as a novel fibrin-specific epitope. From theoretical calculations, we show that the linear motif of the peptide is important for the binding, where hydrophobic amino acids are favorable. The capable of fibrin targeting of the designed peptide is validated by size exclusion chromatography and circular dichroism analyses for recombinant variants of the γ-module. In addition, the designed peptide is applied to selectively target a lung adenocarcinoma A549 cell line that has a high expression level of fibrinogen as a diagnostic model. In vitro and in vivo experiments show a high potential of the designed peptide for various diagnostic, imaging, and therapeutic implications of fibrin-related diseases.
8:00 PM - BM07.06.13
Metal–Organic Framework Based Dopamine Carrier for Parkinson Disease Treatment
Alessandra Pinna 1 , Luca Malfatti 2 , Rossana Migheli 2 , Plinio Innocenzi 2 , Julian Jones 1 , Paolo Falcaro 3
1 , Imperial College, London United Kingdom, 2 , University of Sassari, Sassari Italy, 3 , University of Graz, Graz Austria
Show AbstractParkinson’s disease is a neurodegenerative disorder of the central nervous system, due to loss of neurons in the substantia nigra pars compacta and to reduction of dopamine (DA) in the striatum. The conventional treatment for Parkinson’s disease consists of the administration of L-Dopa, the natural precursor of DA. L-Dopa can be metabolized in different regions of the body, producing side effects in patients. These can be overcome by controlled release of DA in the brain via nasal administration.
Metal-Organic Frameworks (MOFs) are an excellent competitor as carriers for DA. MOFs are a class of porous materials with extraordinary surface area, consisting of metal ion or cluster nodes connected by organic linkers. MIL-88 MOFs class is the most investigated for biomedical applications. The properties of MOFs can be improved by combining these porous crystals with iron oxide nanoparticles.
Herein, magnetic framework composites have been used as delivery systems for DA to treat Parkinson’s disease.
The magnetic framework composite (PMP@MIL-88A) has been prepared through repetitive growth cycles of MIL-88A (Fe) crystals on the surface of magnetite particles (PMPs). In a typical synthesis, iron (III) chloride hexahydrate (FeCl3 6H2O) and PMPs were mixed with the fumaric acid (C4H4O4) water solution. These 1st generation PMP@MIL-88A particles were washed and dispersed in water. The further growth cycles were obtained by mixing the 1st generation PMP@MIL-88A particles in a water solution of FeCl3 6H2O and C4H4O4 at 70°C for 30 min. The obtained n-generations PMP@MIL-88A particles were washed and treated at 150°C. DA was loaded into PMP@MIL-88A by mixing DA hydrochloride and PMP@MIL-88A particles of the desired generation in ethanol solution at 25°C for 72 h.
The physicochemical properties of the PMP@MIL-88A were characterized by SEM and TGA.
The results show that the repetitive growth cycles affect both the size of the PMP@MIL-88A (from 8 to 86 µm) and the crystal size (from 1.8 to 6.6 µm). The carrier was shown to be more efficient than other systems (e.g. silica particles) and is able to release 0.56 mg mL- 1 of DA in 6 h, preventing DA oxidation.
The amount of DA released by the carriers was measured in vitro, in PBS by HPLC, and in nerve pheochromocytoma cell line (PC12).
The data show that DA can be released in the intracellular compartment using the PMP@MIL-88A carrier, avoiding side effects due to its degradation in the extracellular environment.
The cytotoxicity of PMP@MIL-88A and DA-PMP@MIL-88A was evaluated by exposing PC12 cells to increasing concentrations of MOF particles (10, 20, 40 µg mL-1) for 24 h. The results show that 1st and 2nd growth of PMP@MIL-88A and DA-PMP@MIL-88A are not toxic. Conversely, the 3rd growth produces limited toxicity.
This work demonstrates that the effective DA release from PMP@MIL-88A reduces the pharmacological danger, enabling a targeted and long-term delivery, and improves the cost efficiency.
8:00 PM - BM07.06.14
N-Heterocyclic Carbene Based Nitric Oxide Systemic Delivery Triggered by High-Intensity Focused Ultrasound
Youngnam Kang 1 2 , Jeesu Kim 1 , Junbeom Park 1 2 , Kimoon Kim 1 2 , Chulhong Kim 1 , Eunsung Lee 1 2 , Won Jong Kim 1 2
1 , POSTECH, Pohang Korea (the Republic of), 2 , Institute for Basic Science, Seoul Korea (the Republic of)
Show AbstractNitric oxide (NO) has been widely regarded as an important biological molecule in numerous physiological processes. As NO also plays critical roles in therapeutic function, there have been a plethora of classes of NO donor development in past decade, such as organic nitrates/nitrites, nitrosamines, N-diazeniumdiolates (NONOates), and S-nitrosothiols (RSNOs). Of interests is to develop controllable NO releasing donors by various endogenous (pH, redox condition) or external stimuli (heat, light). Besides endogenous stimuli, several exogenous stimulating treatment methods have been tried to treat tumors. X-ray or proton radiation therapies are commonly applied to adjuvant therapy as well as cancer therapy by damaging the DNA of cancerous cells. High intensity focused ultrasound (HIFU) has recently been developed and used as a non-invasive and non-radiative method for treating tumors deep in the body. Compare to radiation therapies, HIFU is non-ionizing, cost-efficient, and safe. During HIFU treatment, focused ultrasound waves are delivered to the focal point of the transducer and generate a rapid temperature increment and a mechanical cavitation, which lead to tissue coagulation or ablation. Herein, we provide the first report of IMesNO as a HIFU-responsive NO donor and its systemic delivery in vivo using IMesNO loaded micelle (IMesNO@MCs). Recently, we reported synthesis and characterization of IMesNO. It is also found that the IMesNO is thermally decomposed to release nitrous oxide (N2O) and nitric oxide (NO) since the residue remained, changing to white powder from brown crystalline. However, the mechanism of thermolysis of IMesNO is demonstrated for the first time in this report as gives rise to the use of HIFU for NO releasing-stimuli. Owing to the therapeutic function of NO and HIFU treatment for cancer therapy in real clinical use, we designed the IMesNO and DOX-loaded polymer micelles (IMesNO/DOX@MCs), which preferentially accumulate in a targeted tumor and respond to HIFU irradiation for stimuli-responsive NO release, leading to site-specific and controllable NO release in vivo and the therapeutic effect. Therefore our findings may suggest a potential biomedical application for clinical translation. The details of use of IMesNO as a therapeutic agent with HIFU therapy is currently being investigated.
8:00 PM - BM07.06.15
Mechanically- and Chemically-Active Nanostructured Antibacterial Surfaces Fabricated by Glancing Angle Sputter Deposition
Nadine Ziegler 1 , Christina Sengstock 2 , Kristina Tschulik 3 , Manfred Köller 2 , Alfred Ludwig 1
1 Institute for Materials, Chair of MEMS Materials, Ruhr University Bochum, Bochum Germany, 2 Surgical Research, University Hospital Bergmannsheil, Bochum Germany, 3 Faculty of Chemistry and Biochemistry, Micro- & Nano-Electrochemistry and Center for Electrochemical Sciences (CES), Ruhr University Bochum, Bochum Germany
Show AbstractTo reduce implant associated infections, implant materials need to have antibacterial properties combined with good tissue compatibility. The nanostructure of Clanger cicada, was synthetically mimicked in this study by glancing angle deposition (GLAD) sputtering. The produced nano-columns are effective against gram-negative bacteria which can be related to their specific geometry [1]. Furthermore, antibacterial materials e.g. silver show the ability to kill gram-negative and gram-positive bacteria (chemical effect). To control the release of Ag from antibacterial surfaces, a sacrificial anode concept was realized [2]. Finally, the mechanically active nanocolumns will be combined with the antibacterial ion release to create novel antibacterial surfaces.
[1] C. Sengstock, M. Lopian, Y. Motemani, A. Borgmann, C. Khare, P. J. Buenconsejo, T. A. Schildhauer, A. Ludwig, and M. Koller, “Structure-related antibacterial activity of a titanium nanostructured surface fabricated by glancing angle sputter deposition,” Nanotechnology, vol. 25, no. 19, 2014.
[2] M. Köller, C. Sengstock, Y. Motemani, C. Khare, P. J. S. Buenconsejo, J. Geukes, T. A. Schildhauer, and A. Ludwig, “Antibacterial activity of microstructured Ag/Au sacrificial anode thin films,” Mater. Sci. Eng. C, vol. 46, pp. 276–280, 2015.
8:00 PM - BM07.06.16
Ferroelectric Polarization and Plasmonic Property Enhanced Photodynamic Bacteria Killing and Wound Healing
Yu Xin 1 , Linlin Li 3 , Hong Liu 2
1 , Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan, Shandong, China, 3 , Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, Beijing China, 2 , State Key Laboratory of Crystal Materials Shandong University, Jinan China
Show AbstractInfectious diseases caused by bacteria have threated the lives and health of millions of peoples each year. In the past several decades, unfortunately, the abuse of antibiotics in medicine and other fields have developed antibiotic-resistant bacteria, which induces serious problem to human health and eco-environment.[1] Recently, photodynamic therapy has been developed as a minimally invasive therapeutic modality to conquer multidrug resistance bacteria. Under the irradiation of light at a certain wavelength, photosensitizers can generate reactive oxygen species to destroy bacteria.[2] Titanium dioxide nanomaterials as the mostly used photoactive materials could absorb ultraviolet light to catalyze the ROS generation. Due to its large band gap, however, TiO2 can only absorb ultraviolet light to yield ROS.[3] In order to increase efficiency of ROS generation for PDT antibacterial effect, we developed a multilayered coaxial heterostructure with nanorod array structure as a coating for antibacterial effect and wound healing. Au NPs with localized surface plasmon resonance not only extend the absorbance of light into visible region, but also enhances the transfer of the photogenerated electons from TiO2 to ferroelectric. The ferroelectric polarization of BaTiO3 provides a driving force for the transport of photoinduced charge carrier, thus enhancing their separation when photoexcited by light.
8:00 PM - BM07.06.17
The Microfluidic Ion Pump—A New Approach to Problems in the Brain
Christopher Proctor 1 , Adam Williamson 2 , Ilke Uguz 1 , Vincenzo Curto 1 , Anna-Maria Pappa 1 , Isabel del Agua 1 , Christophe Bernard 2 , George Malliaras 1
1 , Ecole des Mines St Etienne, Santa Barbara, California, United States, 2 , Aix Marseille University, Marseille France
Show AbstractDespite tremendous research efforts, treatment options for many neurological disorders are inadequate. Systemic drug treatments suffer from side effects and long-term habituation; electrical stimulation is unspecific; and the fluidic injection of drugs often displaces the very cells that are being targeted due to the local pressure increase. Thus, there exists a pressing need to develop novel treatment strategies that overcome these limitations. One such technology is the recently introduced drug delivery platform known as the microfluidic ion pump (µFIP)1. The µFIP is an implantable device that electrophoretically pumps ions (eg. neurotransmitters, drugs, etc) to the target tissue. In addition to spatial and temporal control, a distinctive feature of the µFIP is that it delivers just the ion and not the solvent and thus does not increase pressure at the outlet. This “dry” delivery is of paramount importance for neural interfacing as it enables an intimate interface between the drug delivery outlet and the target cells. Here we report recent advances in incorporating µFIPs into implantable devices for treating neurological disorders including both depth probes and cortical arrays with recording capabilities. The efficacy of the µFIP platform is demonstrated by stopping epileptic seizures in vivo. This is the first in vivo demonstration of an ion pump for treating a neurological disorder and offers a glimpse of what can be achieved by tailored engineering of the µFIP platform. We anticipate this work to be the starting point for new stimulation, recording and drug delivery paradigms in chronic neural implantation.
Symposium Organizers
Yizhou Dong, The Ohio State University
Christopher Alabi, Cornell University
Daniel Anderson, Massachusetts Institute of Technology
Bozhi Tian, University of Chicago
Symposium Support
Alnylam Pharmaceuticals, Inc.
Nanobio Delivery Pharmaceutical Co., Ltd.
Precision NanoSystems Inc.
BM07.07: Engineering Macromolecular Therapeutics
Session Chairs
Christopher Alabi
Yizhou Dong
Wednesday AM, November 29, 2017
Sheraton, 2nd Floor, Grand Ballroom
8:00 AM - *BM07.07.01
Lipid-Like Materials for RNA Delivery—A How-to Guide for Hacking Gene Expression
Kathryn Whitehead 1
1 , Carnegie Mellon University, Pittsburgh, Pennsylvania, United States
Show AbstractDespite the promise of RNA therapeutics, progress towards the clinic has been slowed by the difficulty of delivering RNA drugs, such as short interfering RNA (siRNA) and messenger RNA (mRNA), into cellular targets within the body. RNA therapeutics are large (104 – 106 g/mol) and negatively charged; they do not have favorable biodistribution properties in vivo nor an ability to cross the cellular membrane of target cells. In response to these challenges, our lab has created and tested large libraries of biodegradable lipid-like materials called ‘lipidoids’ using high-throughput methodologies. Lipidoids are able to efficiently manipulate gene expression in a variety of biological systems, including hepatocytes, white blood cells and tumors. Furthermore, structure-function analysis has revealed material design criteria that reliably predict in vivo delivery efficacy without the need for any biological testing. Current efforts on therapeutic application of lipidoid nanoparticle technology will be discussed.
8:30 AM - BM07.07.02
Functional Nucleic Acid Nanotechnology for Gene Therapy
Hyukjin Lee 1 , Bora Jang 1
1 , Ewha Womans University, Seoul Korea (the Republic of)
Show AbstractNucleic acid nanotechnology has drawn tremendous attention, since it can provide a precise tool for building multi-dimensional structures with a defined size, shape, and surface property. This is particularly of interest to the field of gene delivery. For targeted delivery of nanoparticles, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition, and surface charge leading to suboptimal performance, lack of tissue specificity and potential toxicity. Here, we show that self-assembled functional nucleic nanostructures with a well-defined size can serve as a multi-functional platform to induce efficient RNA interference, mRNA expression. For the synthesis of functional RNA structures, we have utilized the rolling circle transcription of pre-designed DNA template to produce a large quantity of functional RNAs. For programmable and simultaneous gene silencing, we have design the dicer substrate RNA nanostructures to regulate three different fluorescent proteins (GFP, RFP, and BFP) in the target cells. Using three arm junction RNA nanostructures, we were able to show programmable regulation of fluorescent protein expression to generate 8 different fluorescent colored cells. For mRNA expression, in vitro transcription (IVT) of mRNA has been carried out to express three distinct fluorescent proteins (GFP, RFP, and BFP) in target cells. Long term expression can be obtained with the programmable assembly of mRNA into defined RNA structures.
8:45 AM - *BM07.07.03
Development of “CLAN” Nanomedicine for Nucleic Acid Therapeutics
Jun Wang 1
1 , South China University of Technology, Guangzhou China
Show AbstractClinical application of nucleic acid therapeutics is limited by their in vivo delivery. We will introduce the development of cationic lipid-assisted nanoparticles (CLANs) as an efficient platform for nucleic drug delivery. CLANs are constructed from cationic lipid-assisted assembly of clinically approved poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) or its analogs. CLANs show efficient encapsulation of various nucleic acids including plasmid DNA (pDNA), small interfering RNA (siRNA), messenger RNA (mRNA) etc., with encapsulation efficiency constantly higher than 90%. We have demonstrated that CLANs are robust in delivering therapeutic siRNAs into cancer cells to inhibit tumor growth in a variety of different tumor models. More interestingly, the properties of CLANs can be readily modulated by changing the types of lipids and the feed ratios of PEG-PLA to the lipids, which enable the facile establishment of a CLAN library. Taking advantage of this library, we can screen certain types of CLANs that show better efficacy in delivering the nucleic acid therapeutics into cancer stem cells, T cells, B cells, or macrophages etc. to further expand the applications of CLANs in the treatment of more diseases.
9:15 AM - BM07.07.04
Gold Nanoantenna Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer
Yu-Chuan Ou 1 , Joseph Webb 1 , Shannon Faley 2 , Daniel Shae 1 , Eric Talbert 1 , John Wilson 1 3 , Leon Bellan 2 , Rizia Bardhan 1
1 Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Tennessee, United States, 2 Mechanical Engineering, Vanderbilt University, Nashville, Tennessee, United States, 3 Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States
Show AbstractLow-temperature sensitive liposomes (LTSLs) are ideal drug delivery vehicles due to their low phase transition temperature ~41.7 °C. This enables drug release at mild hyperthermia, minimizing unnecessary heating of healthy tissues. For enhanced utility of LTSLs, improved hyperthermia strategies that are noninvasive are necessary. However, current clinical approaches to induce local hyperthermia can be invasive that requires heating probe directly in contact with solid tumors (e.g. radiofrequency ablation), or can give rise to heterogeneous tumor temperatures (e.g. ultrasonic energy) resulting in unpredictable drug release and toxicity to healthy tissues. In this work, we demonstrate controlled drug delivery from LTSLs mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple negative breast cancer model, MDA-MB-231. The unique geometry of MGNs enables the generation of mild hyperthermia by converting near-infrared light to heat and effectively deliver doxorubicin (DOX) from LTSLs in breast cancer cells. We confirmed cellular uptake of MGNs by both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs and compared them with free DOX, and DOX-loaded non-temperature sensitive liposomes (NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Due to the high phase transition temperature of NTSLs, no drug delivery was observed from DOX-loaded NTSLs. The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimum off-target toxicities.
10:00 AM - BM07.07.05
Facile Synthesis of DNA Flowers as Protein Vehicles for Cellular Delivery
Eunjung Kim 1 , Limor Zwi-Dantsis 1 , Natalie Reznikov 1 , Catherine Hansel 1 , Shweta Agarwal 1 , Molly Stevens 1
1 Department of Materials, Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, London United Kingdom
Show AbstractInspired by biological processes, biomimetic crystallization methods have been exploited to fabricate new functional materials with the same variety of specialized and complex properties that exist in nature. Such methods involve a homogenous or heterogeneous growth of organic and inorganic components in materials, providing highly ordered hierarchical structures with well-defined morphology and unique properties.1 An emerging class of such hybrid constructs is DNA-inorganic hybrid architectures, fabricated by the rolling circle technique. These are attracting increasing scientific interest due to their potential porosity and functionality. Although some rolling circle-based DNA constructs have been developed for drug/gene delivery and imaging,2 their fabrication methods are often complex and time consuming, involving several procedures. Here, we introduce a simple, generic, and effective way to formulate a protein-DNA hybrid composite for the intracellular delivery of cytotoxic proteins.3 By taking advantage of the well-established rolling circle amplification technique, we show that a wide range of bioactive proteins, including enzymes, can be simultaneously associated with the growing DNA strands and inorganic crystals during the process, leading to the direct entrapment of proteins into a three-dimensional DNA-inorganic hybrid constructs (termed DNA flowers or DNF, hereafter) while retaining the activity of protein payloads. Extensive characterization of the protein-containing DNF using a dual beam microscope (FIB-SEM) and structured illumination microscopy demonstrates how the protein entrapment influence their internal porosity and the proteins are localized within DNF. We further explore the potential of this method as a protein delivery system by demonstrating that the encapsulation of cytotoxic proteins, such as RNase A, in the DNF can induce significant toxic effects to the cells without a loss of structural integrity and its biological activity. Therefore, the simplicity of the manufacturing process and physiological reaction conditions combined with diverse selection pools of proteins for encapsulation and inherent functionality of DNA structures make our method highly preferential for biological applications.
References
1. a) S. Mann, Nature 1993, 365, 499; b) S. I. Stupp, P. V. Braun, Science 1997, 277, 1242.
2. a) W. Sun, T. Jiang, Y. Lu, M. Reiff, R. Mo, Z. Gu, J. Am. Chem. Soc. 2014, 14722; b) R. Hu, X. Zhang, Z. Zhao, G. Zhu, T. Chen, T. Fu, W. Tan, Angew. Chem.-Int. Edit. 2014, 53, 5821.
3. E. Kim, L. Zwi-Dantsis, N. Reznikov, C. S. Hansel, S. Agarwal, M. M. Stevens, Adv. Mater. 2017, 1701086.
10:15 AM - *BM07.07.06
Cell Penetrating Peptides to Improve Cytosolic Drug Delivery
Dehua Pei 1
1 , The Ohio State University, Columbus, Ohio, United States
Show AbstractThe greater majority (~80%) of disease-relevant human protein targets, including essentially all intracellular protein-protein interactions (PPIs) and defective/missing proteins caused by genetic mutations, are currently undruggable by conventional drug modalities (i.e., small molecules and biologics). These targets would become druggable with new drug modalities such as peptides, macrocycles, proteins, and nucleic acids, if the latter could be effectively delivered into the interior of mammalian cells. We have discovered a novel class of cyclic cell-penetrating peptides (CPPs) with unprecedented drug delivery efficiencies (e.g., up to 120% cytosolic delivery efficiency, compared to 2% for Tat). I will discuss their mechanism of action as well as their applications for delivering linear and cyclic peptides, proteins, and nucleic acids into the cytosol of mammalian cells in vitro and in vivo. Our efforts have led to potent, selective, and metabolically stable inhibitors against a variety of previously intractable intracellular proteins such as calcineurin and K-Ras.
10:45 AM - BM07.07.07
Biodegradable Periodic shRNA Systems for Enhanced Gene Silencing and Immunotherapy
Connie Wu 1 , Jiahe Li 1 , Paula Hammond 1
1 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractRNA interference (RNAi) provides a versatile therapeutic strategy via silencing of specific genes implicated in cancer and other diseases. However, clinical translation of RNAi for cancer therapeutics remains unrealized, due to challenges in delivery of small interfering RNA (siRNA) to tumors. The low valency and high rigidity of siRNA often requires high excesses of cationic delivery materials to condense into stable nanoparticles, leading to dose-limiting toxicities. To address this challenge, we adopt an RNAi platform based on periodic short hairpin RNAs (p-shRNAs). Consisting of siRNA sequences linked together, these polymeric RNAi molecules are generated by the repeated action of an RNA polymerase around a small circular DNA template. Through template design and selective enzymatic digestion, we can design p-shRNA structures that are efficiently processed inside cells into siRNAs and induce significant gene silencing. Furthermore, p-shRNA exhibits immunostimulatory properties that can be combined with RNAi to dramatically enhance therapeutic efficacy.
To develop an optimal delivery vehicle for op-shRNA, we used factorial design to synthesize a library of poly(beta-amino ester)s (PBAEs). Screening of this library showed that p-shRNA silencing efficiency increases with increasing alkyl side chain percentage and decreasing molecular weight. Our PBAEs are able to fully condense p-shRNA into sub-100 nm complexes with high silencing efficiency, at much lower polymer-to-RNA ratios than those typically required for PBAE gene delivery. We further modified the complexes with a poly(ethylene glycol) (PEG)-PBAE copolymer containing alkyl side chains, introducing an outer PEG layer that can enhance colloidal stability and decrease cytotoxicity. By directing op-shRNA against signal transducer and activator of transcription 3 (STAT3), which promotes an immunosuppressive tumor microenvironment, we can significantly reduce tumor progression and prolong survival in a B16F10 melanoma model. Thus, through nucleic acid engineering and rational carrier design, we have successfully developed a stable, potent RNAi delivery system that can trigger significant gene silencing at low doses, and enable higher therapeutic efficacy in vivo through RNA interference and immunomodulation.
11:00 AM - BM07.07.08
Click Nucleic Acids for Building PLGA Based Nanoparticles for Gene Silencing
Albert Harguindey 1 , Dylan Domaille 1 , Benjamin Fairbanks 1 , Justine Wagner 1 , Christopher Bowman 1 , Jennifer Cha 1
1 Chemical and Biological Engineering, University of Colorado at Boulder, Boulder, Colorado, United States
Show AbstractDrug resistance is a prominent issue in cancer treatment and the co-delivery of both therapeutic agents and siRNA has the potential to overcome these problems. However, due to the intrinsically different chemical nature between hydrophobic cancer drugs and hydrophilic nucleic acid, developing a single agent that can sequester and deliver both for in vivo targeted delivery is not trivial. In this study we modified the block copolymer poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG–PLGA) which have been extensively used for targeted drug delivery to contain a novel DNA analog, click nucleic acids (CNAs), which allowed us to fabricate PLGA based nanoparticles that can also hold the polymer significant amounts of DNA. For this, the block copolymer PEG-CNA-PLGA was first synthesized and then formulated into nanoparticles by using an oil-in-water emulsion. This CNA containing particles are highly capable of encapsulating DNA sequences that are complementary to the CNA, whereas minimal amounts of nucleic acid acids are encapsulated for non-complementary CNA sequences or when CNA is not present. The particles were further tested by encapsulating hydrophobic molecules as drug models and by successfully encapsulating larger and more complex DNA sequences as a demonstration of the potential this CNA containing particles have as co-delivery agents.
11:15 AM - BM07.07.09
Microfluidics-Based Manufacture of PEG-b-PLGA Block Copolymer Nanoparticles for the Delivery of Small Molecule Therapeutics
Andrea Armstead 1
1 , Precision NanoSystems Inc., Vancouver, British Columbia, Canada
Show AbstractS.M. Garg, M. Parmar, A. Thomas, E. Ouellet, M. DeLeonardis, P. Johnson, A. Armstead, S. Ip, T.J. Leaver, A.W. Wild, R.J. Taylor, E.C. Ramsay
Precision NanoSystems Inc., Vancouver, BC, V6P 6TZ, Canada.
Purpose: In recent years, numerous methods have been developed for the production of block copolymer nanoparticles as drug delivery vehicles. However, these methods pose numerous challenges in maintaining consistent nanoparticle quality, tuning size depending on the application, optimization for scale-up, and reproducibility. The NanoAssemblr™ platform is an automated microfluidics-based system that eliminates user variability and is capable of reproducible, and scalable manufacture of nanoparticles. Here, we describe the use of microfluidic mixing to manufacture PEG-b-PLGA nanoparticles using the NanoAssemblr™ Benchtop instrument. We further describe optimization strategies and investigate the physical encapsulation of a hydrophobic model drug coumarin-6.
Methods: PEG-b-PLGA nanoparticles were manufactured using the NanoAssemblr™ Benchtop instrument (Precision NanoSystems Inc., Vancouver, Canada). PEG5000-b-PLGAX of varying molecular weights of the hydrophobic block (X) was dissolved in suitable organic solvents (e.g. acetone, DMSO) at desired concentrations. PEG-b-PLGA nanoparticles were formed by nanoprecipitation achieved by the rapid and controlled mixing of two-inlet fluid streams containing PEG-b-PLGA in organic solvent, and aqueous solution, through proprietary staggered herringbone mixing (SHM) apparatus. Coumarin-6 was loaded into PEG-b-PLGA nanoparticles and compared against conventional manufacturing technique.
Results: Microfluidic mixing enabled the rapid and consistent manufacturing of PEG-b-PLGA nanoparticles having diameters below 100 nm. Instrument parameters such as aqueous:organic flow rate ratio and total flow rate had a significant impact on the size of the resulting nanoparticles. Increasing the molecular weight of the PLGA block from 10000 - 95000 resulted in an increase in the size of the nanoparticles from 25 - 60 nm. However, changes in the total flow rate of the instrument enabled all the nanoparticles to be tuned to a similar size of 60 nm which is difficult to control using conventional techniques. Coumarin-6 was successfully loaded into PEG-b-PLGA nanoparticles with an encapsulation efficiency of 52% w/w which was significantly higher than that obtained by co-solvent evaporation technique (34% w/w) (Table 1). The size of the nanoparticles prepared using the NanoAssemblr™ platform were smaller than that prepared using co-solvent evaporation.
Conclusions: Herein, we have successfully reported the development and manufacture of PEG-b-PLGA nanoparticles encapsulated with coumarin-6 using the NanoAssemblr™ microfluidics technology. We have further demonstrated the potential of this platform in the encapsulation and delivery of small molecule therapeutics.
BM07.08: Biomaterials for Therapeutic Applications II
Session Chairs
Christopher Alabi
Eric Appel
Wednesday PM, November 29, 2017
Sheraton, 2nd Floor, Grand Ballroom
1:30 PM - *BM07.08.01
Carrier-Free Drug Delivery and Drug-Free Therapeutics
Yoon Yeo 1
1 College of Pharmacy, Purdue University, Lafayette, Indiana, United States
Show AbstractOne of the primary requirements of a drug carrier is biocompatibility—the lack of tissue reactions against the material causing premature removal and/or unwanted immune responses. Various natural and synthetic polymers have been explored as biocompatible drug carriers, under an assumption that they have no other roles than delivering the payload to target tissues. However, many studies show that the carriers play more active roles in therapeutic effects of the product, although some of them may have been underestimated or inadvertently ignored. For example, we have reported a new chitosan derivative with unique anti-inflammatory activities, which attenuates the onset of endotoxin-induced sepsis upon intraperitoneal injection and protects animals from TNBS-induced colitis via prophylactic oral administration. We have also observed that a polycation-polysaccharide complex, initially developed for gene delivery, shows pro-apoptotic activity in a selected group of cancer cells and enhances anti-tumor activity of separately administered chemotherapy. Conversely, some of the carriers can interfere with pharmacological effects of drugs being delivered. These studies indicate that drug carriers can have biological effects to the host, which may be worthwhile to explore for new therapeutic applications, and the actual role of a carrier in drug delivery need to be revisited in some cases. Given the potential bioactivity of a drug carrier, we recently developed a new drug delivery approach involving a minimal amount of non-drug components. In this presentation, I will introduce examples of biomaterials with therapeutic potential and carrier-free drug delivery systems.
2:00 PM - BM07.08.02
Gene-Editing of Human Cells Using Modular CRISPR Ribonucleoprotein Nanoparticles
Amr Abdeen 1 , Jared Carlson-Stevermer 1 , Lucille Kohlenberg 1 , Madelyn Goedland 1 , Kaivalya Molugu 1 , Meng Lou 1 , Krishanu Saha 1
1 , University of Wisconsin-Madison, Madison, Wisconsin, United States
Show AbstractCRISPR-Cas9 gene editing is anticipated to generate revolutionary therapies for genetic conditions. However, efficacy using nonviral delivery methods is limited due to low efficiency, imprecise editing and lack of selection and monitoring methods. Using an RNA aptamer based strategy, we modified Cas9 ribonucleoproteins (RNPs) to complex with multiple biotinylated molecules. By complexing Cas9 RNP with quantum dots and fluorescent dyes, we monitored RNP complexes interacting with cells in vitro and, based on fluorescence intensity, selected for edited cells increasing editing efficiency ~2-3 times over conventional methods. Furthermore, by employing different fluorescent molecules we are able to enrich and select for multiplexed edited cells, which would otherwise require extensive screening and testing using conventional methods. Furthermore, by complexing Cas9 with single stranded donor DNA (ssODNs) and bringing the donor template in proximity to the cutting site, we saw an increase the ratio of precise editing to imprecise editing by up to 18-fold higher than standard gene editing methods. This method uses versatile, commercially available reagents and could make the gene editing process more accurate, effective and less costly for both in vitro and ex vivo applications of CRISPR-Cas9.
2:15 PM - BM07.08.03
Patch-Type Swellable Microneedle Adhesives for Enhanced Tissue Adhesion and Sustained Protein Drug Delivery
Seung Yun Yang 1 , Keum-Yong Seong 1 , Eoin O'Cearbhaill 2 , Jeffrey Karp 3
1 , Pusan National University, Miryang Korea (the Republic of), 2 , University College Dublin, Dublin Ireland, 3 , Brigham and Women's Hospital, Boston, Massachusetts, United States
Show AbstractTissue adhesive has received great attention due to an increasing clinical need. It has been widely used for wound closure, hemostasis, and fixation of tissues. Since tissue adhesion is essential for the application of drug delivery patches or medical devices, many research efforts have been devoted to the development of adhesives enabling long-term tissue adhesion in a clinical setting.
In this talk, I will present double-layered microneedle (MN) arrays consisting of water-swellable tips and non-swellable core designed to achieve mechanical interlocking with tissues. This highly engineered MN design was inspired by the ability of endoparasitic worms to firmly attach to the intestinal wall of their hosts through expanding a chamber following penetration. By harnessing reversible swelling/deswelling property of swellable tips, insulins used as a model protein drug can be loaded in the swellable tips by simple drop/dry procedure, thus minimizing loss of structural integrity. The insulin-loaded MN patch showed 60% release of insulin over the course of 12 hrs in PBS and ~ 70% of the released insulin preserved their biological activity. This swellable MN patch may reduce the potential for a chronic foreign body response and possess the advantage over dissolving MN systems of avoiding regulatory issues with delivery materials.
3:30 PM - *BM07.08.04
Selective In Vivo Cell Labeling Mediated Cancer Targeting
Jianjun Cheng 1
1 , University of Illinois at Urbana-Champaign, Urbana, Illinois, United States
Show AbstractNearly all current targeting strategies rely on specific recognition of the endogenous protein receptors; one such technology that has received great success is the antibody/antigen technology. Antibody/antigen technology for cancer targeting, however, has several drawbacks such as large size of both receptor and targeting ligand, limited number of endogenous receptors, immunogenicity, high cost and difficulty of handling of antibody, and synthetic challenges of structure-specific drug-antibody conjugates for immunotherapy. In this talk, I will present the development of an unprecedented in vivo targeting technology based on exogenous introduction of azido group, an artificial “antigen”, onto the surface of the target cancer cells followed by highly efficient in vivo Click chemistry to target the introduced azido group. This new targeting technology is essentially the small molecule version of antibody/antigen targeting technology and has several obvious advantages such as much reduced size of receptor/targeting moieties, substantially increased cell-surface receptor numbers, ease of handling and synthesis of both azido-sugars and targeting substrates, low cost, and non-immunogenicity. Substantially reduced toxicity was observed with the use of this small molecule mediated targeting technology followed by chemotherapy. We also successfully demonstrated the use of this technology for nanomedicine in vivo targeting.
4:00 PM - BM07.08.05
Magnetoelectric Microrobots for Biomedical Applications
Xiangzhong Chen 1 , Marcus Hoop 1 , Fajer Mushtaq 1 , Bradley Nelson 1 , Salvador Pane i Vidal 1
1 Multi-Scale Robotics Lab, Institute of Robotics and Intelligent Systems, ETH Zürich, Zurich, Zurich, Switzerland
Show AbstractMicrorobots are promising miniaturized devices for targeted therapeutic interventions and controlled drug delivery. The primary advantage of these microdevices over other small-scale therapeutic delivery systems is their controlled locomotion and thus enhanced targeting ability. Magnetoelectric materials, which can convert magnetic energy into electric output, have shown great promise in devices such as attenuators, filters, field probes and data recording devices, but they are seldom employed in mobile devices such as micro- and nanomachines. The implementation of magnetoelectric building blocks in microrobots can provide these devices not only the ability of being maneuvered with non-invasive magnetic fields, but also additional functionalities as magnetoelectric materials can wirelessly generate electric fields.
In this presentation, we will present different microrobots with magnetoelectric properties for biomedical applications. Devices with various architectures such as Janus microparticles, artificial bacteria flagella, and core-shell nanowires will be shown. Several materials ranging from metals and ceramics to polymeric materials are selected and combined with different fabrication techniques (e.g. template-assisted electrodeposition, photolithography, sputtering). Different actuation methods with a focus on magnetic manipulation are employed to maneuver the developed microrobots in aqueous environments. We will demonstrate that the same energy source (magnetic fields) can be used not only to steer these magnetoelectric microrobots but also to trigger other functionalities via magnetoelectric effect, such as on-demand delivery of drugs and cells, or inducing ocalized cell differentiation for tissue regeneration.
4:15 PM - BM07.08.06
Biocatalytic Carbon Nanotube Film—One-Step Method for Fabrication of Enzyme-Immobilized Membranes for Organophosphates Bioremediation
Guy Mechrez 1
1 Department of Food Quality & Safety, Volcani Institute, ARO, Rishon LeZion Israel
Show AbstractRecent world events have demonstrated the critical need for facile and miniaturized bioremediation technologies of organophosphates (OPs). These compounds are among the most toxic substances synthesized to date and are used as pesticides and nerve agents. Biotechnological methods based on the use of organophosphate hydrolase (OPH) for detoxification of OPs have drawn significant attention. This work presents a new ‘one-pot’ methodology for a rapid and straightforward fabrication of enzymatically-active carbon nanotubes (CNT) paper for OPs bioremediation. Carboxylated CNTs are ultrasonically dispersed in an aqueous surfactant solution followed by a microfiltration process, to generate a paper-like membrane, which is assembled from entangled nanotubes. Herein, OPH conjugation to the CNTs is carried out by carbodiimide chemistry during the microfiltration process. Successful covalent immobilization of the enzyme onto the nanotubes surface is confirmed by cryo-transmission electron microscopy and infrared spectroscopy. To study the potential of this platform for OPs bioremediation, an aqueous solution of methyl paraoxon (used as a model OP) is filtered by the resulting OPH-CNT membranes. Significant decrease of methyl paraoxon concentration is obtained, ascribed to its in situ hydrolysis by the immobilized OPH during the filtration process. These thin membranes allow performing many subsequent filtration cycles, while maintaining their enzymatic activity, owing to the unique combination of mechanically robust CNT scaffold and high OPH loading. This study presents a new generic approach for the design of bioactive paper-like scaffolds, which can be rationally tailored for a variety of applications.
4:30 PM - BM07.08.07
CarboNano-Tweezers for Twisting Tail of Cancer
Santosh Misra 1 2 , Indu Tripathi 1 2 , Fatemeh Ostadhossein 1 2 , Indrajit Srivastava 1 2 , Dipanjan Pan 1 2
1 , University of Illinois at Urbana Champaign, Urbana, Illinois, United States, 2 Biomedical Research Center, Mills Breast Cancer Institute, Urbana, Illinois, United States
Show AbstractWe are preparing and using novel trogers base decorated carbon nanoparticles "CarboNano-Tweezers" for breast cancer therapy applications considering these particles act as nano-tweezers to twist the genomic DNA and induce apoptosis cascade for cancer therapy. The unique tweezer shaped geometry of Troger's base makes it applicable as molecular receptors, in development of molecular torsion balances, in asymmetric catalysis, and as chiral solvating agent. In recent years its chemistry has been used for cancer therapy applications by development of DNA minor grove binders which can intervene in the regular replication and transcription processes to lead the host cell to death with or without triggering apoptosis cascades. Generally lack of aqueous solubility and complex synthetic procedures to make trogers base derivatives suitable for therapy applications masks its potential. Synthesized particles were found to be sub-100 nm in size and interacting with duplex plasmid DNA as seen in UV-Vis absorption studies. Gel electrophoretic patterns also supported the interaction patterns with plasmid DNA. Studies with circular diachroism (CD), FT-IR, Raman and XPS confirmed the presence of trogers base molecules on carbon nanoparticle surface and interaction with duplex polynucleotides. The prepared particles were tested in MCF-7 (ER (+)) and MDA-MB231 (ER (-)) breast cancer cells with improved efficiency of cell growth regression compared to small molecule itself with better selectivity against MCF-10A cells of same origin. These particles were found to be environmentally benign with one to four week degaradtion time in human and soil systems, respectively. Thus, these nano-tweezers could be developed for eventual use in in vivo and clinic setups after appropriate changes.
4:45 PM - BM07.08.08
Development of Thermostable Microneedle Patch for Polio Vaccination
Chandana Kolluru 1 , Yasmine Gomaa 1 , Jeongwoo Lee 1 , Mark Prausnitz 1
1 MSE/ ChBE, Georgia Institute of Technology, Atlanta, Georgia, United States
Show AbstractINTRODUCTION
Poliomyelitis is a highly infectious disease which can only be prevented by immunization. The two most common types of vaccine are inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Both of them have certain drawbacks. The development of a dissolvable microneedle patch for IPV administration could be very useful for better vaccine coverage in developing countries. Using this approach, patches are applied to the skin, which painlessly puncture micron-scale, solid microneedles into the skin, where they dissolve and release encapsulated vaccine.
EXPERIMENTAL METHODS
Trivalent IPV were generously provided by Bilthoven Biologicals (Netherlands). High throughput screening was performed by mixing concentrated IPV with different excipients (such as sugars and polyols) and testing IPV potency (D-antigen activity) by ELISA. The four best excipients were selected and microneedle patches were prepared. Tests such as thermogravimetric analysis (TGA), pig skin insertion and accelerated stabiity testing were performed on the final optimized microneedle patch.We hypothesized that a very low moisture content may retard vaccine mobility and thereby help maintain its native structure.
RESULTS AND CONCLUSIONS
The four best excipients found from primary screening were D-sorbitol, maltodextrin, sucrose and trehalose. Further optimization of the ratios of these excipients resulted in a final microneedle patch with a first cast of IPV mixed with D-sorbitol and maltodextrin, and a second cast of containing fish gelatin and D-sorbitol. Based on process optimization studies, the optimal drying time prior to demolding was 2 days at 25°C and 0% RH. The final RMC for the patch was below 6% based on TGA. Drying to an RMC of 3% did not show any improvement in ELISA values, implying that further removal of bound and unbound water did not help increase the thermal stability. The final MN patch was deemed to be mechanically strong based on successful insertion in pig skin. Examination of used microneedle patch after 15 min showed complete dissolution of the microneedles.
The microneedle patches were then stored at 5°C, 25°C and 40°C at 0% RH. Some patches were also stored at 40°C and 50% RH. ELISA and TGA testing at various time points showed that the greatest degradation in D-antigen activity was observed at 40°C. The patches were found to be stable at 25°C and 0% RH for at least 1 month. ELISA was correlated to the storage temperature, implying that the reduction in IPV potency is thermally driven. In contrast, IPV completely loses its potency if dried without any excipients, and conventional liquid IPV is recommended to be stored at 2°C to 8°C. Hence, achieving IPV thermostability of at least 1 month at 25°C is a significant advance. It is theorized that sorbitol prevents the IPV from unfolding during drying, thereby enabling IPV to maintain its potency even after completely drying.
ACKNOWLEDGEMENTS
This work was funded by the Bill and Melinda Gates Foundation.
BM07.09: Poster Session III
Session Chairs
Thursday AM, November 30, 2017
Hynes, Level 1, Hall B
8:00 PM - BM07.09.01
Three-Arm Junction RNA Nanostructures as a Dicer Substrate for Programmable Gene Silencing
Bora Jang 1 , Boyoung Kim 1 , Hyukjin Lee 1
1 , Ewha Womans University, Seoul Korea (the Republic of)
Show AbstractThree arm junction RNA nanostructures (Y-RNA) can provide dual roles of structural and biological functions such as RNA interference (RNAi) by inserting siRNA sequences (anti-GFP, RFP, and BFP) into the each arm of Y-RNA. Through the sequence and length optimization of Y-RNA, Y-RNA is carefully designed to be a Dicer substrate for enhanced RNAi potency as well as a prolonged effect. Interestingly, the Dicer processing and loading of the Y-RNA into the RNA Induced Silencing Complex (RISC) is highly dependent on the physical structure and sequence design of the three-arm junction. In addition, we enzymatically prepared Dicer substrate Y-RNA with a large scale using rolling circle transcription (RCT) and site-specific cleavage. Depend on the cleavage site, various Y-RNA samples can be produced from a single DNA template, inducing programmable RNAi. Our study can provide valuable information on how to design RNAi nanostructures for enhanced and programmable gene silencing in vitro and in vivo.
8:00 PM - BM07.09.02
Study of Cellular Behavior on Nanostructured Surfaces
Yi-Seul Park 1 , Jin Seok Lee 1
1 Chemistry, Sookmyung Women's University, Seoul Korea (the Republic of)
Show AbstractBiochemical cues ignore cellular microenvironments such as cell to cell interaction and extracellular matrix (ECM). Since the cells in vivo are communicated with ECM and cell-to-cell interaction, studies of topological cues are associated with cell in vivo. In recent year, various studies have shown that nanotopological cues regulate cell behavior such as cell adhesion, cell proliferation, migration, polarity, and differentiation. Although many previous studies have investigated cell behavior using various topographical cues, the results have meant topographical confinement by nanostructured architecture. That is, overall control of cellular response is carried out by cells isolation in the structure through geometry, and is not affected by surface topography. Therefore, it is essential for surface topography to be studied through controlling the size, shape and density of the nanostructure. In previous study, we described that hippocampal neuron on silica beads bigger than 200 nm in diameter accelerated neurite outgrowth and formed the lamellipodia and filopodia.
In this study, we investigated the effects of the hexagonally close-packed silica bead arrays on cellular behavior, especially adhesion. The silica beads were synthesized by stöber method by controlling the amount of reagents or injection rates. In addition, the hexagonally close-packed silica bead arrays can be achieved by rubbing method, which is simple and fast to obtain monolayer of silica beads. The different size of silica beads were examined to figure out the nanotopological effect on the cellular behavior, which are range of 300 nm to 1600 nm. The silica beads arrays classified by two groups, such as Group-I and Group-II, of nanotopological effect along with cell adhesion and morphology. The Group-II surface was long distance between contact points, resulting in increase of tension to cells.
8:00 PM - BM07.09.03
Neurite Outgrowth of Hippocampal Neurons on Patterned Silica Bead Arrays
Gyuri Kim 1 , Jin Seok Lee 1
1 , Sookmyung Women's University, Seoul Korea (the Republic of)
Show AbstractNeurite outgrowth and path-finding behaviors of neurons is an important preceding step for the development of nerve systems. These are governed by two protrusive, actin-based molecular structures, flopodia and lamellipodia, the diameter of which is generally in the rage of 100-300 nm. The dynamic stability of filopodia and lamellipodia allows neuronal cells to recognize the surrounding environments at the nanometer scale and to subsequently modify their cytoskeletal structures in response to stimuli/cues. Given that the in vivo environments of neurons consist of numerous hierarchical micro/nanotopographies, there have been many efforts to investigate the relationship between neuronal behaviors and surface topography.
In this study, we investigated the effects of the orientation of nanotopographies on the neuronal development and neurite outgrowth using patterned close-packed silica bead arrays. The silica beads were synthesized by stöber method and assembled by rubbing method. In particular, we can obtain the both the hexagonally and tetragonally close-packed silica bead arrays using template assisted rubbing method. The initial hippocampal neurons become to accelerate their neurite outgrowth on close-packed silica bead arrays due to tension-induced developmental acceleration. The neuronal networks were formed randomly on the hexagonally close-packed silica bead arrays, whereas they were aligned bidirectionally along to the orientation of tetragonally close-packed silica bead arrays.
8:00 PM - BM07.09.04
Characterization of the Use of UV Laser Ablation to Etch Polyvinylidene Fluoride for Heterogeneous Integration with CMOS
Jeffrey Elloian 1 , Ken Shepard 1
1 , Columbia University, New York, New York, United States
Show AbstractThe properties of piezoelectric beta-phase polyvinylidene fluoride (PVDF), in particular its acoustic impedance matching to tissue, have created considerable interest in its application for acoustic interfaces to biological systems. Being a chemically inert yet flexible polymer makes PVDF a popular choice for energy harvesting, tactile sensors, and ultrasonic transducers [1-3].
Microfabrication of devices using PVDF remains difficult because the loss of its piezoelectric properties above 90°C[1]. Although spin-coating and poling remain post-processing options, such methods are infeasible for integration of PVDF directly on complementary metal-oxide-semiconductor (CMOS) integrated circuits due to the required poling fields [1]. Although reactive ion etching (RIE) and wet chemical etching have been shown to be capable of patterning the material, both methods have difficulty achieving reasonable etch rates and selectivity without degrading piezoelectric coefficients. Moreover, these methods are impractical for etching more than 100 microns of material while maintaining vertical sidewalls, properties that are necessary for many applications [1,4].
In this study, the etching characteristics of PVDF are investigated using an ArF excimer laser (193 nm) [5]. In previous studies using laser ablation, devices were fabricated by etching completely through the polymer without etching to a specific depth [1]. Here, different energy fluence levels are applied across varying numbers of passes to determine the etch rate and sidewall profiles without the use of a stop layer. Furthermore, the intermediate liquid phase polymer that is ejected by the laser plume during ablation is investigated and its effect on surface debris is shown. This allows for precision micromachining of thick layers of PVDF and control of the sidewall characteristics.
References
[1] Ramadan, K. S., Sameoto, D., & Evoy, S. (2014). A review of piezoelectric polymers as functional materials for electromechanical transducers. Smart Materials and Structures, 23(3), 33001.
[2] Spanu, A., Pinna, L., Viola, F., Seminara, L., Valle, M., Bonfiglio, A., & Cosseddu, P. (2016). A high-sensitivity tactile sensor based on piezoelectric polymer PVDF coupled to an ultra-low voltage organic transistor. Organic Electronics: Physics, Materials, Applications, 36, 57–60.
[3] Kim, J., Lindsey, B. D., Li, S., Dayton, P. A., & Jiang, X. (2017). Dual-frequency transducer with a wideband PVDF receiver for contrast-enhanced, adjustable harmonic imaging. In T. Kundu
[4] Miki, H., Matsui, G., Kanda, M., & Tsuchitani, S. (2015). Fabrication of microstructure array directly on β -phase poly(vinylidene fluoride) thin film by O 2 reactive ion etching. Journal of Micromechanics and Microengineering, 25, 35026.
[5] Izumi, Y., Kawanishi, S., Hara, S., Yoshikawa, D., & Yamamoto, T. (1998). Irradiation Effects of Excimer Laser Light on Poly(vinylidene fluoride) (PVdF) Film. Bulletin of the Chemical Society of Japan, 71(11), 2721–2725.
8:00 PM - BM07.09.05
Piezoresistive Behavior of Polymeric Cantilevers Based on Integration of Nanowires
Hana Han 1 , Vincent Martinez 1 , Luca Hirt 1 , Flurin Stauffer 1 , Cathelijn van Nisselroy 1 , Tomaso Zambelli 1 , Janos Voros 1
1 Institute for Biomedical Engineering, ETH Zurich, Zurich Switzerland
Show AbstractAtomic force microscopy (AFM) is a widely used technique not only for material science at sub-micron scale but also for biology at the single-cell or single-molecule level. The optical beam deflection (OBD) is a commonly used detection method for measurement cantilevers angular changes caused by deflection. Although this method allows to obtain high spatial resolution images, it requires complex instrumentation and frequent re-adjustment of the laser position. This is not desirable especially for biological applications since particles or cells which are suspended in liquid can interfere with the laser signal. Piezoresistors represent an attractive alternative to OBD as a deflection sensor, since they offer the flexibility to work in different media. Addtionally they can be integrated on a chip for multiple cantilever arrays.
We have developed piezoresistive self-sensing SU-8 cantilevers using silver nanowires. SU-8 is an epoxy-based negative photoresist, which has proven to be a promising polymer for microfabrication, thanks to high transparency, thermal stability and chemical resistance. Due to the low SU-8 Young’s Modulus, higher force sensitivity can be obtained for a higher accuracy [1]. To integrate the piezoresistive materials in the SU-8 cantilevers, nanowires are transferred and patterned to form micro electrode structures, then embedded into the SU-8 layer. Gauge factor could be tailored by altering the geometry of the nanowire tracks [2] or coating the nanowires with an insulator. The piezoresistive behavior of the nanowires in SU-8 matrix were investigated for similar strains under uniaxial stretching and bending. Piezoresistive behavior was then compared with OBD method, to correlate mechanical deformation and bending, and investigate further the linear response of such cantilevers.
[1] Johansson, A., Calleja, M., Rasmussen, P. a. & Boisen, A. SU-8 cantilever sensor system with integrated readout. Sensors Actuators, A Phys. 123-124, 111–115 (2005).
[2] Martinez, V., Stauffer, F., Adagunodo, M., Forro, C., Vörös, J., Larmagnac, A. Stretchable Silver Nanowire-Elastomer Composite Microelectrodes with Tailored Electrical Properties. ACS Appl. Mater. Interfaces 7, 13467–13475 (2015).
8:00 PM - BM07.09.06
Microparticles with Different Surface Roughness for Efficient Delivery to Macrophages and Dendritic Cells
Jung Heesun 1
1 , Konkuk University, Seoul Korea (the Republic of)
Show AbstractThe immune response was evaluated as a signal of protection from pathogens. Among the various strategies for increasing the immune response, nano and micro particles were developed to encapsulate adjuvants and antigens. It is well-known that 2-3 μm sized microparticles exhibits maximal phagocytic properties for antigen presenting cells. In addition, increasing attachment of 2-3 μm particles is attributed to characteristic features of membrane ruffles in macrophage. In previous study, the ruffles present characteristic local curvature to the membrane and increase contact area between particles and the macrophage membrane. Accordingly, we hypothesized that intracellular delivery via phagocytosis will be enhanced by effect of surface roughness of microspheres. PLGA, a biocompatible and biodegradable material used in medical devices and drug delivery applications, has been extensively developed as nano and microcarriers by encapsulating proteins and antigens. In this study, 1 μm-sized PLGA microspheres were formulated by water-in-oil-in-water double emulsion method. To increase the surface roughness, cancer cell-derived vesicle with a size of 200 nm was adopted to deliver cancer-associated antigens to immune cells. After preparation of vesicle coated PLGA microparticles via covalent bond through click reaction, morphology and size of particles were measured by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The root mean square (RMS) was increased following to increasing molar ratio of amine group of vesicles. Also, PLGA microsphere and vesicles were stained with coumarin-6 and indocyanine green, respectively, to evaluate the amount of PLGA-ve internalized into cells including macrophage, cancer cells and fibroblasts. The amount of PLGA-ve taken up cells was measured by in vivo imaging system (IVIS) and confocal microscopy. To enhance the immune response to immune cells such as macrophages and dendritic cells, monophosphoryl lipid A (MPLA) was encapsulated into PLGA microspheres. Using macrophages and bone marrow derived dendritic cells, secreted two kinds of cytokines were measured with enzyme-linked immunosorbent assay (ELISA). This study demonstrated that intracellular uptake of microparticles could be determined by surface roughness, which could trigger sufficient cytokine induction for macrophages and dendritic cells.
8:00 PM - BM07.09.07
Curcumin-Based Nano-Complexes for Enhanced In Vivo Fluorescence Monitoring and ROS Detection
Kang Yoon-Young 1
1 , Konkuk University, Seoul Korea (the Republic of)
Show AbstractCurcumin, a bioactive phenolic compound from turmeric root, has shown high biological activity and therapeutic safety in a wide range of diseases, e.g. cancer, neurodegenerative diseases and inflammatory diseases. To better understand biological effects of curcumin in vivo, elucidation of the fate of curcumin after systemic administration is necessary. Previous study, biodistribution of curcumin was determined by high performance liquid chromatography, nuclear magnetic resonance, and mass spectrome-try after extraction of curcumin from tissues or organ. On the other hand, it is well known that curcumin has poor aqueous stability and is too prone to oxidation and enzymatic degradation in solution. Therefore procedure for extraction of curcumin from tissue or organ can affect quantification of such unstable compound in tissue or organ. Not only stability of curcumin but also varying extraction efficiency depending on tissues and extraction methods. In this study, we aimed to develop a method for elucidation of biodistribution of curcumin, via direct fluorescence imaging without any labeling and extraction procedures. We prepared curcumin nano-complexes with 2-Aminoethyl diphenyl borate(DPBA) for enhance curcumin stability. After intravenous injection, curcumin and prepared borate–curcumin complexes showed similar fluorescence intensities at 15 min. However, stabilized borate–curcumin complexes exhibited much stronger fluorescent signals at metabolically active sites such as liver tissues than native curcumin. After incubation for 1–3 h, native curcumin showed significantly rapid reduction of fluorescent signals, compared to borate–curcumin complexes, probably due to degradation and reduction. In addition, complicate extraction procedures inhibited precise fluorescent monitoring of unstable curcumin, which result in different biodistribution of curcumin before and after extraction. ROS-responsive curcumin was also successfully incorporated within PLGA/PLA scaffolds via noncovalent hydrophobic interaction. Curcumin-incorporated PLGA/PLA showed strong fluorescence intensities in PBS solution for 3 days. Fluorescence signals of curcumin-incorporated PLGA/PLA scaffolds were significantly decreased by radical molecules. We successfully determined distribution of curcumin via simple and fast fluorescence detection of curcumin complexes, which could be applied for in vivo monitoring and ROS detection in vivo.
8:00 PM - BM07.09.08
How Material Properties Dictate Magnetic Nanoparticle Heating
Fabian Starsich 1 , Christian Eberhardt 2 , Andreas Boss 2 , Ann Hirt 1 , Sotiris Pratsinis 1
1 , ETH Zurich, Zurich Switzerland, 2 , Universitätsspital Zürich, Zürich Switzerland
Show AbstractThe therapy of diseased cells via the heating of magnetic nanoparticles is hindered by the required high nanoparticle concentrations. A clear relationship between heating efficiency and magnetic properties of nanoparticles is therefore of great need. This has not been attained due to the limited versatility of nanoparticle compositions achievable by the typically applied synthesis methods. Here we use scalable flame aerosol technology for synthesis of magnetic single domain nanocrystals of varying composition, size and morphology for hyperthermia or thermoablation therapy [1]. In detail, the respective magnetic hysteresis and first order reversal curves of the produced systems are compared to the corresponding heating efficiencies. Most interestingly, we could show a clear connection between coercivity and heating properties of the magnetic nanoparticles. SiO2-coated non-stoichiometric Gd-Zn ferrite was revealed as the most promising system. Its in vitro performance for magnetic particle heating treatments of cancerous cells was closely investigated.
[1] Starsich, F. H. L.; Sotiriou, G. A.; Wurnig, M. C.; Eberhardt, C.; Hirt, A. M.; Boss, A.; Pratsinis, S. E. Silica-Coated Nonstoichiometric Nano Zn-Ferrites for Magnetic Resonance Imaging and Hyperthermia Treatment. Adv. Healthc. Mater. 5, 2698–2706 (2016).
8:00 PM - BM07.09.09
Suppression of Insulin Aggregation by Zwitterionic Polymers—Mechanistic Insights
Robin Rajan 1 , Yu Suzuki 2 , Kazuaki Matsumura 1
1 , JAIST, Nomi Japan, 2 , University of Fukui, Fukui Japan
Show AbstractIntroduction
Insulin regulates sugar in the bloodstream and is widely used in the treatment of diabetes. It is usually injected in the body, which is not always suitable for everyone. Insulin, like many proteins, is extremely prone to aggregation, which has hampered widespread development of alternate forms of its delivery (which can be more precise and convenient) and prevented long term storage of insulin.1 Due to the unusually high frequency of insulin related diseases, insulin-based formulations have attracted interest of many researchers worldwide. Many compounds have been developed previously to overcome this issue, however satisfactory results have not been achieved yet.2 Moreover, not much research has been done with synthetic polymers to tackle insulin aggregation. This is very surprising, considering the huge potential of synthetic polymers, as many parameters can be easily adjusted to increase their efficiency and meet clinical requirements. In this report, we developed a zwitterionic polymer (poly-sulfobetaine) and introduced varying amounts of hydrophobicity (butyl methacrylate; BuMA) and checked its propensity to suppress insulin aggregation. These polymers showed very high efficiency in the protection of insulin. Preliminary mechanistic investigations revealed that they enable insulin to preserve its higher order structures.
Results and Discussion
Molecular weight and introduction of hydrophobicity of the polymer was controlled by reversible addition fragmentation chain transfer polymerization. UV-Visible experiments clearly showed that insulin aggregates on incubation at 37 °C and addition of polymer markedly supresses aggregation. Fibrillation of insulin was investigated using Thioflavin T and the results unambiguously demonstrated that these polymers are extremely effective in supressing fibrillation. Incorporating hydrophobicity lead to a massive increase in its overall efficiency, and polymer containing 30% BuMA shows suppression of around 98% fibrillation. Kinetic study of fibrillation showed that lag times for aggregation were almost quadrupled when incubated in presence of these polymers, indicating that these polymers arrests the process of fibrillations and aggregation.
Mechanistic investigation was done using circular dichroism spectroscopy and 2D NMR. It was conclusively established that these polymers prevent any change in secondary structure of insulin. Protection is also due to their weak and reversible interaction with proteins, thus they act as molecular shields and reduces collisions between aggregating species.
Conclusion
Zwitterionic polymers have extremely high efficiency in inhibiting insulin aggregation. Hydrophobicity increases the efficiency by masking the hydrophobic surfaces of proteins. These polymers exhibit protection by preventing any destructive change in the higher order structure of insulin.
References
J. S. Thompson et al., World J. Surg. 25, 523 (2001).
W. Wang, Int. J. Pharm. 289, 1–30 (2005).
8:00 PM - BM07.09.10
A Self-Adherent, Bullet-Shaped Microneedle Patch for Controlled Transdermal Delivery of Insulin
Keum-Yong Seong 1 , Min-Soo Seo 2 , Dae Yeon Hwang 1 , Eoin O'Cearbhaill 3 , Seamus Screenan 4 , Jeffrey Karp 5 , Seung Yun Yang 1
1 , Pusan National University, Miryang Korea (the Republic of), 2 , Daegu-Gyeongbuk Medical Innovation Foundation, Daegu Korea (the Republic of), 3 , University College Dublin, Dublin Ireland, 4 , Connolly Hospital, Dublin Ireland, 5 , Harvard Medical School, Cambridge, Massachusetts, United States
Show AbstractProteins are important biologic therapeutics including antibodies, interferons, enzyme and vaccines used for the treatment of various diseases. However, owing to short half-time and fast clearance of the proteins in a body, long-term delivery of protein therapeutics poses a significant challenge. Recently, microneedles (MNs) based transdermal drug delivery has been studied for a sustained protein drug delivery. Here we present a bullet-shaped double-layered MN array with swellable tips prepared by polystyrene-block-poly(acrylic acid) (PS-b-PAA) block copolymer, which enable to mechanically interlock with soft tissues. This design enabled the MNs to mechanically interlock with soft tissues by selective distal swelling after skin insertion. Additionally, prolonged release of loaded proteins by passive diffusion through the swollen tips was obtained. When we analyzed the pull-off adhesion of a MN patch on rat skin tissue, this swellable MN patch achieved ~1.6 N/cm2 in maximum adhesion strength to a rat skin. By harnessing reversible swelling/deswelling property of swellabe tips, insulins used as a model protein drug can be loaded in the swellable tips by simple drop/drying procedures. The insulin-loaded MN patch showed 60% release of insulin over the course of 12 hours in PBS and more than 70% of the released insulin preserved structural integrity. Furthermore, when the efficacy of insulin-loaded MN was evaluated at normal experiment animals, the blood glucose levels of swellable MN group were gradually decreased, maximally at 4 hr point levels. To confirm the biological safety of swellable MNs to the live animal skin tissue, histopathological and clinical pathological assessments were conducted. There were no remarkable changes and inflammatory cell infiltration compared with normal mice skin. All items of biochemistry markers in blood also have no significant expression. Therefore, this transdermal delivery platform using self-adherent MN patches could be applied for versatile protein drugs requiring sustained release kinetics.
8:00 PM - BM07.09.11
Highly Efficient Mammalian Cell Engineering via Cas9 mRNA Transfection
Minjeong Kim 1 , Hyukjin Lee 1
1 , Ewha Womans University, Seoul Korea (the Republic of)
Show AbstractThe RNA-guided nuclease CRISPR (clustered regularly interspaced short palindromic repeat)/CRISPR-associated (Cas) protein 9 system is an emerging gene editing system in the field of gene therapy. Up to date, Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) were delivered into a variety of mammalian cells through an electroporation method with a significantly high gene editing efficiency. However, current methods for in vivo RNP delivery suffer from low tolerance for serum degradation, poor endosomal escape limiting their use in animal studies. In this study, we have compared the delivery and gene editing efficacy of two different CRISPR editing tools such as 1) Cas9 mRNA/sgRNA and 2) Cas9 RNPs through the transfection using electroporation and lipid carriers. It is found that the delivery of Cas9 mRNA and sgRNA enabled permanent genome editing with the use of low sgRNA doses. In addition, the genome editing efficacy of Cas9 mRNA/sgRNA was superior to that of the direct delivery of Cas9 RNPs.
8:00 PM - BM07.09.12
Porous Silicon Nanoparticle Delivery of Tandem Peptide Anti-Infectives for the Treatment of Bacterial Lung Infections
Alessandro Bertucci 2 1 , Ester Kwon 3 4 , Matthew Skalak 3 4 , Gary Braun 5 , Francesco Ricci 1 , Erkki Ruoslahti 5 , Michael Sailor 2 , Sangeeta Bhatia 3 4 6
2 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, United States, 1 Department of Chemistry, University of Rome Tor Vergata, Rome Italy, 3 Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 4 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 5 Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States, 6 Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractThe rise of antibiotic resistance combined with the paucity of new antibacterial agents entering the clinic has created an urgent need for new treatments for bacterial infections. Major obstacles facing existing and new antibiotic agents are poor localization of drug to bacteria and off-target toxicity.
Peptides are promising candidates to develop anti-infective agents, offering biological specificity and ease to scale-up production. Here, we show the development of a nanoparticle/peptide-based anti-infective that is designed to coordinate the action of two separate functions in a single agent: (1) a membrane-interacting peptide, and (2) a toxic peptide cargo. Our tandem peptide, selected upon screening of a library of 25 candidates, is engineered to feature two domains that specifically target and kill the gram-negative bacteria P. aeruginosa, yielding a greater efficacy than the sum of its individual components. In order to improve tissue distribution and to mitigate off-target toxicity, we formulated our tandem peptide into a biodegradable nanoparticle and applied it to a lung infection model. The nanoparticle skeleton is composed of biocompatible porous silicon. We investigated a set of surface chemistries to mediate physical interaction with the peptide cargo and found the porous silicon nanoparticles modified with phosphonate groups offer the best performances in terms of peptide loading. In vivo experiments showed that, when delivered to the lung, these nanoparticle-formulated tandem peptide anti-infectives exhibit improved safety profiles over free peptides based on blood cytokine levels and lung histology. Furthermore, treatment of the P. aeruginosa pneumonia leads to markedly improved survival and decreased lung bacterial titers by several orders of magnitude compared to untreated mice (a greater than 300-fold reduction in bacterial numbers). In summary, we present a therapeutic strategy based on an anti-infective agent designed as a bi-functional tandem peptide formulated into biodegradable porous silicon nanoparticles. We show that our formulation is effective in an animal model of bacterial pneumonia. We believe our approach based on a synthetic nanoparticle/peptide anti-infective may complement existing small molecule antibiotic treatments, and may include tissue targeting ligands and combination of therapeutics.
8:00 PM - BM07.09.13
Microfabrication of Organic Electrochemical Transistors for Biological Sensing
Naixiang Wang 1 , Feng Yan 1
1 Department of Applied Physics, Hong Kong Polytechnic University, Hong Kong Hong Kong
Show AbstractOrganic electrochemical transistors (OECT) are promising platforms for the design of chemical and biological sensing applications, based on their advantages in low-voltage operation, good biocompatibility and interaction with the aqueous medium. Furthermore, facile chemical modification of semiconductors and applicability on varied substrates including flexible plastics make it more convenient for integration into complex biological systems.
Considering for microfabrication of OECT device, previous reports normally need the dry etching processing to open up channel pattern, which may be expensive and not well controllable. To overcome this problem, here we develop a novel approach to miniaturize OECT by using only multilayer photolithographic and lift-off process. The channel of OECTs could be reduced to several microns therefore the speed of device response could be raised up to order of 10-5 s in aqueous medium.
Due to the improvement in transient response time of the micro OECT device, alternating current (AC) is possible to be introduced for device operation. Combined with lock-in amplifier, the small AC channel signal could be precisely extracted from an extremely noisy environment, which provides the possibility to enhance the sensing capability of the OECT devices in complex situations. A new detection method is then established based on AC signal driven micro OECT. The detection limit of dopamine, an important neuro transmitter within the central nervous system, is down to 10-9 M, indicating that the AC signal driven micro OECT could be a promising candidate for rapid sensing and diagnosis applications.
In conclusion, we introduce a simple and easy-to-fabricate process for miniaturization of OECT devices to the cellular dimensions. The advantage of fast transient response makes the AC-driven micro OECT capable of in-situ cell activity monitoring and associated chemical sensing applications.
8:00 PM - BM07.09.14
A New Visible Light Photodegradable Polymer Utilzing a Dinitro Derivative of Bisstyrlthiophene
Vincent Tran 1 , Sangeun Lee 1 , Alexandra Stubelius 1
1 , University of California, San Diego, La Jolla, California, United States
Show AbstractLight-responsive nanocarriers may serve as versatile tools to control the release of encapsulated drugs or imaging agents. In order to utilize light-responsive nanocarriers in vivo, they must respond to electromagnetic radiation that can harmlessly penetrate tissue. Currently, the majority of photochemical systems do not meet these requirements as they utilize UV-light activation, which can damage tissue. Other systems employ near-infrared (NIR) light, which requires an inefficient two-photon excitation approach.
In this research, a new visible light-responsive material was polymerized by using a dinitro derivative of bisstyrylthiophene. The polymer chromophore utilizes the o-nitrobenzyl photocleavage by absorbing in the biobenign violet-blue visible light range. Based on these excellent properties, the polymer displays practicality as a photodegradable nanocarrier. The polymer was formulated into nanoparticles and release studies of bioactive molecules was performed to further assess it's ability as a drug delivery vehicle.
8:00 PM - BM07.09.15
Photothermal Enhancement of Biocatalytic Activity of Polymer Encapsulated Enzyme
Sirimuvva Tadepalli 1 , Jieun Yim 1 , Rajesh Naik 2 , Srikanth Singamaneni 1
1 , Washington University in St. Louis, St. Louis, Missouri, United States, 2 , Air Force Research Laboratory (AFRL), Dayton, Ohio, United States
Show AbstractThe rational integration of biomolecules and functional nanostructures can enable remote-controlled biological processes such as molecular transport, catalysis and molecular recognition. Photothermal ability of plasmonic nanostructures is highly attractive to optically modulate biomolecular processes such as biocatalysis. However, the studies pertaining to the phothermal enhancement of enzyme activity are limited to thermophilic enzymes due to the thermal denaturation and loss in the activity of conventional enzymes at elevated temperatures. The lack of effective strategies for preserving the activity of immobilized enzymes at elevated temperatures hinders the potential use of plasmonic nanostructures as nanoheaters for the photothermal enhancement of enzyme activity. Here, we demonstrate a simple and highly effective strategy to stabilize enzymes immobilized on plasmonic nanostructures by encapsulating them through in situ polymerization. Apart from enhanced thermal and biological stability, the encapsulation strategy provides enhancement in the enzyme activity with an external optical trigger. The encapsulation strategy demonstrated here can be a highly attractive approach for designing remote-controlled biomolecular reactions.
8:00 PM - BM07.09.16
Fabrication of Functional Nanostructured Surfaces Based on Selective Vapor Deposition
Shih-Ting Chen 1 , Hsien-Yeh Chen 1
1 , National Taiwan University, Taipei, Taiwan Taiwan
Show AbstractAn electrically induced bottom-up process was introduced for the fabrication of multifunctional nanostructures of polymers. Without requiring complicated photolithography or printing techniques, the fabrication process first produced a conducting template by colloidal lithography to create an interconnected conduction pathway. By supplying an electrical charge to the conducting network, the conducting areas were enabled with a highly energized surface that generally deactivated the adsorbed reactive species and inhibited the vapor deposition of poly-p-xylylene polymers. However, the template allowed the deposition of ordered poly-p-xylylene nanostructures only on the confined and negative areas of the conducting template, in a relatively large centimeter-scale production. The wide selection of functionality and multifunctional capability of poly-p-xylylenes naturally rendered the synergistic and orthogonal chemical reactivity of the resulting nanostructures. With only a few steps, the construction of a nanometer topology with the functionalization of multiple chemical conducts can be achieved, and the selected deposition process represents a state-of-the-art nanostructure fabrication in a simple and versatile approach from the bottom up.
8:00 PM - BM07.09.17
Cytotoxicity Study of Eutectic Gallium-Indium (EGaIn) Liquid Metal in Aqueous Environment
Ji Hye Kim 1 , SungJun Kim 2 , Kyobum Kim 2 , Hyung-Jun Koo 1
1 , Seoul National University of Science and Technology, Seoul Korea (the Republic of), 2 Division of Bioengineering, Incheon National University, Incheon Korea (the Republic of)
Show AbstractLiquid metal is an emerging material, which flows like fluid at room temperature with high electrical and thermal conductivity. One of the most actively studied liquid metal materials is eutectic gallium-indium liquid metal (EGaIn, 75% Ga and 25% In by weight). Compared to mercury, another representative liquid metal with high surface tension, EGaIn is more moldable because EGaIn forms gallium oxide skin on the surface which drops surface tension. Such a moldable EGaIn could find many applications in the fields of flexible/stretchable electrodes, sensors, microfluidics, bio-embedded electronics and drug-delivery. Even though it has been known that EGaIn has relatively low toxicity compared to mercury, however, little systematic study about EGaIn cytotoxicity has been reported. Here, we present time-dependent release profiles of Ga and In ions from EGaIn droplet in aqueous environment and the effect of droplet surface area and sonication on the ion release. The amount of Ga and In ions were detected by inductively couples plasma mass spectrometry. To evaluate the toxicity of the elements released from EGaIn, we performed in vitro cytotoxicity test by WST-1 assay and live & dead staining. EGaIn releasates are generally non-toxic, but the cells tested in this study (HeLa cells, human adipose-derived stem cells and fibroblast) exhibited less than 50% proliferation after 3 days of culture in the presence of the EGaIn releasates by 20 min sonication. Our findings could provide a guideline in using EGaIn, especially for bio-related applications.
8:00 PM - BM07.09.18
A Simply Fabricated DEP Tweezer for Cell Patterning and Electroporation
Meera Punjiya 1 , Caleb Neufeld 1 , Hojatollah Rezaei Nejad 1 , Qiaobing Xu 1 , Sameer Sonkusale 1
1 , Tufts University, Medford, Massachusetts, United States
Show AbstractThe study of single cells necessitates a method for manipulation of these cells. Traditionally, a glass micropipette attached to an aspirator is used to impose a negative pressure on the cell sufficient manipulation. This technique can easily damage the cell membrane and underlying structures [1]. Thus various approaches have been adapted for single-cell manipulation including optical and optoelectronic tweezers which utilize often expensive optics and high power lasers, countless micro-fabricated platforms which require access to various lithographic techniques [2] and dielectrophoretic (DEP) tweezers. DEP tweezers fabricated using specialized equipment have been demonstrated for cell manipulation, but as of now, not for controlled cell electroporation [1,3].
We present a simple coaxial DEP tweezer for cell patterning and electroporation constructed from magnet wire. We interface this tweezer with a commercial microplotter for demonstration of cell capture and manipulation. Through application of DC voltages, we are further able to electroporate selected cells and demonstrate this function with transfection of Jurkat cells with propidium iodide. For fabrication, a 5 inch segment of commercially available magnet wire (58 AWG, Elektrisola) is cut and sputter coated with titanium and gold along the length of the wire. The edge of the tweezer is then cut with a microtome, yielding a coaxial cable with a flat cross section for interface with cells. This flat tweezer edge is then plated in gold to prevent corrosion in ionic cell media. The tweezer is attached to a commercially available microplotter (SonoPlot® Microplotter II) via a custom designed 3D printed fixture which simultaneously makes two electrical connections to the tweezer and holds it in place. The final tweezer has a central electrode with a 10um diameter, a 2um polyimide dielectric, and a 350nm Ti/Au outer electrode. FEA Analysis shows field strengths greater than 8 MV/m for a 10Vpp input at 1MHz.
[1] Hunt, T. P., and R. M. Westervelt. "Dielectrophoresis tweezers for single cell manipulation." Biomedical microdevices 8.3 (2006): 227-230.
[2] Van Vliet, K. J., G. Bao, and S. Suresh. "The biomechanics toolbox: experimental approaches for living cells and biomolecules." Acta materialia. 51.19 (2003): 5881-5905.
[3] Menachery, Anoop, et al. "Dielectrophoretic tweezer for isolating and manipulating target cells." IET nanobiotechnology 5.1 (2011): 1-7.
8:00 PM - BM07.09.19
Enzyme-Mimetic Luminescent Antioxidant Nanoparticles for H2O2 Biosensing
Anna Pratsinis 2 , Dorian Henning 1 , Georgios Kelesidis 2 , Jean-Christophe Leroux 2 , Georgios Sotiriou 1
2 , ETH Zürich, Zurich Switzerland, 1 , Karolinska Inst, Solna Sweden
Show AbstractHydrogen peroxide (H2O2) is an abundant molecule associated with biological implications and reacts with natural enzymes, such as catalase. Thus, H2O2 quantification constitutes a powerful tool for detection of disease biomarkers linked to enzyme-based assays such as the plasmonic ELISA. However, the optical H2O2 biosensing without organic-dyes in biological media and at low, submicromolar, concentrations has yet to be achieved. The target of this work is to design biomimetic artificial enzymes based on antioxidant CeO2 nanoparticles that become luminescent upon their Eu3+ doping [1,2]. CeO2 nanoparticles have received a lot of attention recently due to their antioxidant enzyme-like (nanozyme) properties. Here, europium-doped cerium oxide (CeO2:Eu3+) nanoparticles with well-controlled size (d = 4 – 16 nm) are prepared by flame aerosol technology and characterized in regards to H2O2 sensor response in physiologically-relevant solutions. Temporal stability was compared to a commercially available fluorescent dye in a peroxidase coupled reaction. The developed biosensors are coupled to enzyme-based assays that consume or generate H2O2 aiming the detection of other important bioanalytes, such as alcohol and glucose, using alcohol and glucose oxidase enzymes, respectively. Upon the addition of a luminescent material with no H2O2 sensitivity, ratiometric sensors can be made for the facile and fast H2O2 detection in vitro. The biomimetic artificial enzyme developed here could serve as a starting point of sophisticated in vitro assays towards highly sensitive detection of disease biomarkers.
[1] Sotiriou, G.A., Schneider, M., & Pratsinis, S.E. (2011) J. Phys. Chem. C 115, 1084-1089.
[2] Sotiriou, G.A., Franco, D., Poulikakos, D., & Ferrari, A. (2012) ACS Nano 6, 3888-3897.
Symposium Organizers
Yizhou Dong, The Ohio State University
Christopher Alabi, Cornell University
Daniel Anderson, Massachusetts Institute of Technology
Bozhi Tian, University of Chicago
Symposium Support
Alnylam Pharmaceuticals, Inc.
Nanobio Delivery Pharmaceutical Co., Ltd.
Precision NanoSystems Inc.
BM07.10: Nanomaterials for Probing Biology I
Session Chairs
Thursday AM, November 30, 2017
Sheraton, 2nd Floor, Grand Ballroom
8:00 AM - *BM07.10.01
Bioluminescnece Can Activate TiO2 Nanocomposites—Towards Single-Cell Targeted, Depth Independent Nanoparticle Based Phototherapy
Tijana Rajh 1 , Tamara Koritarov 2 , Fatima Rizvi 1 , Vani Konda 2 , Marc Bissonnette 2
1 , Argonne National Laboratory, Lemont, Illinois, United States, 2 Department of Medicine, The University of Chicago Medicine, Chicago, Illinois, United States
Show AbstractTiO2 nanoparticles exhibit exceptional photoreactivity, stability and biocompatibility. Their high surface-to-bulk ratio offers precise engineering of their surface and linking of multiple biological probes to the surface of each nanoparticle. Surface modification of TiO2 nanoparticles with enediol molecules extends their light absorption to the visible and near-infrared part of the light spectrum, while maintaining their ability to create ROS upon illumination with the visible light. However, the small penetration depth of light through the tissue remains the primary limitation of their use in photodynamic therapy, restricting the treatments to the targets near the skin and the lining of a lumen. In this work, we present a different approach; instead of delivering the light to the tumor we bring the light source to the place where light is needed. By covalent attachment of the light to the light-active biofunctionlized nanoparticles we develop localized therapy that is activated only within tumor tissue, leaving healthy cells intact.
8:30 AM - BM07.10.02
Using Multicolor Gold Nanoparticles to Achieve Specificity with Cross Reactive Antibodies in Immunoassays for Infectious Disease
Helena de Puig Guixe 2 , Marc Carré Camps 3 , Irene Bosch 2 , Lee Gehrke 2 , Kimberly Hamad-Schifferli 1 2
2 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 3 , Instituto Quimico de Sarria, Barcelona Spain, 1 , University of Massachusetts Boston, Boston, Massachusetts, United States
Show AbstractRapid point-of-care (POC) diagnostic devices are needed for field-forward screening of severe febrile illnesses such as Dengue, Zika, Ebola, Chikungunya, and others. Multiplexed rapid lateral flow diagnostics have the potential to distinguish among multiple pathogens, thereby facilitating diagnosis and improving patient care. We present a platform for multiplexed pathogen detection which uses Au nanoparticles (NPs) conjugated to antibodies to sense the presence of biomarkers for different infectious diseases. New capabilities for immunoassays are enabled by using the material and size dependent properties of the gold NPs that are used in the assay. We present a multiplexed immunoassay that exploits both the use of both specific and cross-reactive antibodies with gold nanoparticles of different colors. This allows us to provide a specific diagnostic for two related diseases, such as Dengue and Zika; or Marburg and Ebola by using antibodies raised against only one of the two related diseases. Because the biomarkers are related, cross reactivity can be observed, but using different colored NPs can ameliorate to enable distinguishing between single or mixed infections. Because positive test lines can be imaged by eye or a mobile phone camera, the approach is adaptable to low-resource, widely deployable settings. This design requires no external excitation source and permits multiplexed analysis in a single channel, facilitating integration and manufacturing.
8:45 AM - *BM07.10.03
pH Transistor Nanomedicine for Cancer Surgery and Immunotherapy
Jinming Gao 1
1 , UT Southwestern Medical Center, Dallas, Texas, United States
Show AbstractCancer exhibits profound genetic and histological heterogeneities therefore broad yet cancer-specific detection and therapy are challenging. Recently, my lab established a library of transistor-like pH threshold nanosensors with binary off/on reporters that are finely tunable in a broad range of physiological pH (4-7.4). The ultra pH-sensitive property is a unique nanoscale phenomenon that arises from the cooperative self-assembly of amphiphilic copolymers. The pH threshold nanosensors target dysregulated pH, a ubiquitous cancer hallmark, which allowed binary delineation of a broad set of tumors. Real-time, image-guided surgery on established tumors as well as small tumor foci (<1 mm3) resulted in significantly improved long-term survival over white light surgery in head/neck and breast cancers. Using the same nanoplatform, we also demonstrate the spatio-temporal orchestration of antigen delivery to dendritic cells with innate stimulation to produce robust T cell immunity against multiple cancer types. The cooperative nanomedicine platform opens up new opportunities for cancer diagnosis and therapy.
9:15 AM - BM07.10.04
Controlling the Bio-Nano Interactions and Enzyme Kinetics Using Size, Curvature and Charge Controlled Plasmonic Nanostructures
Sirimuvva Tadepalli 1 , Zheyu Wang 1 , Joseph Slocik 2 , Rajesh Naik 2 , Srikanth Singamaneni 1
1 , Washington University in St. Louis, Saint Louis, Missouri, United States, 2 , Air Force Research Laboratory (AFRL), Dayton, Ohio, United States
Show AbstractThe physicochemical properties of abiotic nanostructures determine the structure and function of biological counterparts in biotic-abiotic nanohybrids. A comprehensive understanding of the interfacial interactions and the predictive capability of their structure and function is paramount for virtually all fields of bionanotechnology. In this study, using gold nanostructures as a model abiotic system, we investigated the effect of surface charge, hydrodynamic diameter and surface curvature on the activity of enzymes adsorbed on the surface of the nanostructures. We found that the surface charge of nanostructures profoundly influences the biomolecular structure, orientation and activity of the bound enzyme. In contrast with the previous studies, we have employed a novel class of gold superstructures (gold nanoparticles on spheres) to deconvolute the effects of size and curvature of the abiotic nanostructures on the enzyme kinetics. We found that the size of the nanostructures determines the kcat while the surface curvature influences the KM values of the bionanoconjugates. Further, we show that we can regulate and enhance the enzyme activity and biological pathways using photothermal effect. This study improves our understanding of the bio-nano interface and the design of bioinorganic hybrids with potential applications in biomimetic and bioenabled sensors, energy harvesting, optoelectronic components and devices, responsive and autonomous materials.
10:00 AM - BM07.10.05
Electrochemically Molecularly Imprinted Polymer Chemical and Biosensors
Rigoberto Advincula 1
1 , Case Western Reserve University, Cleveland, Ohio, United States
Show AbstractThe early detection and diagnosis of diseases is crucial for health monitoring and prevention of epidemic and communicable diseases – especially tropical diseases that can easily be passed on and are classified as respiratory diseases. The detection of oligopeptide analytes, nerve agents, pollutants is a challenge especially when present in very low quantities. There are a number of methods to improve sensitivity and selectivity but usually, this requires more elaborate instrumentation methods. Lab-on-a-chip models are viable for simultaneous sample preparation and a high figure of merit detection. The key is the use of high-performance chemical and biosensor elements (films or coatings) with a highly efficient transducer element that can be put in a portable device to enable on-site usability. In this talk, we will focus on our work using electropolymerized molecularly imprinted polymers (E-MIP) for chemical and biosensing and its ability to utilized in transduction methods such as surface plasmon resonance (SPR) spectroscopy or quartz crystal microbalance (QCM) to enable high sensitivity and selectivity. This will be demonstrated in terms of drug detection and use of the epitope approach to detect disease biomarkers. The monomer and molecular design for optimized analyte interaction via E-MIPs enable effective templating protocols in a conducting polymer matrix with tunable oxidative states to enable a high volume of analyte-cavity site production. Optimized electropolymerization methods are important for film deposition and surface characterization. This robust artificial enzymes or receptors will be useful for disease diagnosis and early detection that can be adapted towards network distribution and personalized medicine
10:15 AM - *BM07.10.06
Imaging-Guided Nanopharmaceutical Evaluation In Vitro and In Vivo
Xing-Jie Liang 1 , Haihua Xiao 2
1 , Nat Center for Nanoscience and Technology of China, Haidian, Beijing China, 2 Institute of Chemistry, Chinese Academy of Science, Beijing China
Show AbstractChemotherapeutics are the primary options applicable to cancer treatment, however, most anti-cancer drugs have poor water solubility, rapid blood clearance and severe side effects to normal tissues. In an attempt to overcome these drawbacks, various nanoscaled drug delivery systems (NDDSs) such as, dendrimer, vesicles, liposomes, micelles and inorganic materials are fabricated and widely employed in cancer therapy owing to their improved pharmacokinetics and pharmacodynamics arising from the enhanced permeation and retention (EPR). Because NDDS-based nanopharmaceutics are difficult to be traced after they enter the cells and release the drugs, it is important to develop technique for evaluating their ADMET in vitro and in vivo before it is approved for clinical trials by CFDA.
Nanomedicine offers unprecedented opportunity to reach the objectives of promoting the precise treatment of cancers and mitigating undesired side effects. However, it remains challenging to illustrate the correlation of nanostructures with PK/PD profiles of nanopharmaceuticals. The nano-drug dynamics in living cells, including the cell penetration process, intracellular distribution, detailed drug releasing process, the destinations of drugs and carrier, are essential for sophisticated nanopharmaceutical development. Revealing the subcellular behaviors of nano-DDS can employ cell organelles better to precisely guide drugs into targets and controllably release drugs. Herein, self-assembly drug delivery nanosystems were developed with intrinsic fluorescence. By introducing supermolecular strategy, we developed aggregation-induced nanostructures to unveil the spatiotemporal release of pharmaceutical nanoformulations in living cells. By evaluating the specific fluorescence variations of the drugs and the carriers, the detailed spatiotemporal drug dynamics were visualized and analyzed. More than that, the all-in-one nanostructures also can improve the inhibiting effect and realize combining functionalities of cancer therapy and cell imaging.
10:45 AM - *BM07.10.07
Manipulating Cell Function with Magnetic Nanotransducers
Polina Anikeeva 1
1 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractThe complexity and vastness of the mammalian nervous system demands tools capable of modulating activity of specified cell types in arbitrary deep parts of the body. Magnetic fields with low magnitudes of tens of milli-Tesla provide convenient means to deliver stimuli into the biological tissue. The latter possesses negligible magnetic permeability and low conductivity allowing such fields to penetrate without attenuation and without eliciting unwanted side effects.
To enable magnetic control of biological processes, these fields must be transduced into stimuli, such as heat, force, torque, pH change, and chemicals, commonly received by receptors on cell membranes. This talk will discuss the design and synthesis of magnetic nanoparticles that exhibit hysteretic heat dissipation in alternating magnetic fields with frequencies in the range of 10-1000 kHz, as well as anisotropic nanomaterials that undergo physical rotation in slow-varying magnetic fields. These effects are then harnessed to achieve wireless and minimally invasive magnetothermal, magnetochemical, and magnetomechanical control of neuronal activity and protein aggregation in vitro and in vivo. Furthermore, combination of multiple independent stimuli may enable multiplexed magnetic control of several processes allowing for future studies of complex biological circuits.
BM07.11: Electronic, Photonic and Semiconductor-Based Biomaterials
Session Chairs
Thursday PM, November 30, 2017
Sheraton, 2nd Floor, Grand Ballroom
1:30 PM - *BM07.11.01
Emerging Materials and Devices for Engineering Biological Function and Dynamics
Molly Stevens 1
1 Department of Materials and Department of Bioengineering, Imperial College London, London United Kingdom
Show AbstractThis talk will provide an overview of our recent developments in the design of nanomaterials for ultrasensitive biosensing. Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering (1). Using enzyme-mediated signal readouts we have developed a suite of nanoparticle based ultrasensitive biosensing approaches as well as a cell/tissue-interfacing nanoneedle platform (2). We are applying these biosensing approaches both in high throughput drug screening and to diagnose diseases ranging from cancer to global health applications.
[1] Stevens MM, George JH, Exploring and engineering the cell surface interface., Science, 2005, Vol:310, Pages:1135-1138.
[2] Howes PD, Chandrawati R, Stevens MM, 2014, Colloidal nanoparticles as advanced biological sensors, Science, 2013, Vol:346, DOI: 10.1126/science.1247390.
2:00 PM - BM07.11.02
Antimicrobial Molecule Screening Using Organic Biphasic-Electrolyte Gated Transistors
Anna-Maria Pappa 1 , Charalampos Pitsalidis 1 , Duc Duong 2 , Gregorio Faria 2 , Mintu Porel 3 , Christine Artim 3 , Christopher Alabi 3 , Susan Daniel 3 , Alberto Salleo 2 , Roisin Owens 1
1 , CMP-EMSE, Gardanne France, 2 , Stanford University, Santa Clara, California, United States, 3 , Cornell University, Cornell, New York, United States
Show AbstractSince antibiotics have become widely available, they have been considered as miracle drugs, the “silver bullet” able to destroy any type of infection. As a consequence their broad overuse and misuse has fuelled the spread of multidrug-resistant bacteria inhibiting the treatment of healthcare-associated infections and thus posing major concerns in the human population. In this work we present a proof-of-concept in vitro toxicology assay based on organic electrochemical transistors (OECTs)that are operated through a biphasic (liquid:liquid) electrolyte, bearing a suspended lipid monolayer between the two immiscible fluids. Traditionally, the mechanism of the OECT relies on the penetration of ions from an electrolyte into an electrically-conducting polymer. Although an extensive library of organic semi-conductors (OSC) exists, these materials have been thus far compatible only with organic field effect transistors which allow shielding of the active material from the electrolyte. In the current study we have prepared liquid: liquid interfaces to maintain the OSC in the solvent phase, while allowing biological reactions to occur in the aqueous phase but relaying signals to the OSC channel via ion motion. A lipid monolayer was prepared at the interface thus partitioning the phases. Polar headgroups are assumed to orient upwards into the aqueous phase, while the hydrophobic tails of the lipids orient down into the organic solvent phase. We show that the presence of the lipid monolayer almost completely blocks gating of the transistor, which otherwise can gate efficiently. The presence of gramicidin A (gA), a bacterial pore protein, restores gating of the transistor in a dose dependent manner. Further, transient monitoring of gA in the lipid monolayer shows the ability of the transistor to capture the onset of gating due to ion movement through the defects induced by gA insertion. Additionally, our platform was used to test a bacterial lipid model monolayer with synthetic antimicrobial macromolecules that were recently proven to lyse cells via membrane permeabilization depending on their hydrophobicity and macromolecular conformation. Our findings strongly suggest that such bio-electronic platforms can serve as direct, easy-to-use and highly versatile toxicity testing assays of new compounds, essentially for drug development.
2:15 PM - BM07.11.03
A Bio-Electronic Membrane to Monitor Tissue Cultures
Pradeep Ramiah Rajasekaran 2 , David Quan 1 , William Bentley 1 , Reza Ghodssi 2
2 Institute of Systems Research, University of Maryland, College Park, Maryland, United States, 1 Bio-Engineering, University of Maryland, College Park, Maryland, United States
Show AbstractOrgan-on-chip systems are rapidly emerging due to their potential in revolutionizing personalized medicine, drug development and testing, and pathology modeling. Transendothelial electrical resistance (TEER) and fluorescence are the most commonly used methods for characterizing the progress of tissue culture in trans-well systems. Complementary methods that provide unprecedented access to chemical and physical events occurring at various phases of organ development will be an indispensable asset to organ-on-a-chip systems. We envision that the new bio-electronic membrane platform envisioned here, can acquire such information. This discovery platform consists of an impedemetric sensor-integrated tissue culture membrane that can monitor in real-time with great proximity, the ongoing physio-chemical transformations occurring during cell culture at the cellular/molecular level.
We have successfully cultured Caco-2 (human epithelial colorectal adenocarcinoma) cells on a customized 3D printed trans-well plate-like system, with integrated electronic sensors. This system consists of a nanoporous tissue culture membrane with interdigitated electrodes (IDEs) and built-in contact ports, on which cells are grown. Impedance spectroscopy is used to monitor the progress of cell growth and the subsequent formation of tight junctions. This impedimetric analysis, in addition to augmenting existing organ-on-a-chip systems, can provide very crucial real-time physio-chemical information on the progress of tissue culture.
The IDEs patterned on a track-etched PET membrane serves as a bio-electronic substrate for the cells. The electrodes are fabricated via e-beam evaporation of Ti/Au (20nm/200nm) with laser-cut shadow masks. Shadow-masking method is preferred over photolithography due to the potential membrane cytotoxicity imparted from the chemicals involved in the latter processing. This membrane is sandwiched and encased into a biocompatible (MED610), polyjet 3D-printed chamber. The bioelectronic membrane-encased 3D printed chamber is sterilized by autoclave. Caco-2 cells dispersed in 1X Dulbecco's modified eagle medium (DMEM) with 10% Fetal bovine serum (FBS), are suspended on this membrane and the device is placed in a 10%CO2 incubator at 37°C. The cell culture is impedimetrically tracked with the IDEs for 5 days at an interval of 24 hours in a biosafety cabinet. They were also monitored continuously inside an incubator. The impedance of bio-electronic membrane was ~ 32% lower, in comparison with a control (DMEM + 10% FBS). The decrease in impedance suggests that the cells have adhered to the surface via the formation of a conductive layer, adjacent to the IDEs. The conductivity of the film is attributed to the ionic nature of the proteins and other biomolecules present in the extracellular matrix. The impedance decreases as these cells grow to form tight junctions, which is verified independently by fluorescent staining.
2:30 PM - BM07.11.04
Design of Polymeric Microstructures Showing Active, Dynamic and Hierarchical Deformations
Yuxing Yao 1 , Jiaxi Cui 1 , Xiaoguang Wang 1 , C. Nadir Kaplan 1 , Joanna Aizenberg 1
1 , Harvard University, Cambridge, Massachusetts, United States
Show AbstractIn nature, adaptive microstructures are widely used to dynamically change the topography of a macroscopic object that results in a material with novel static and dynamic functions. Here we report a design of active and dynamic microactuation systems based on liquid crystalline elastomers (LCEs). Specifically, the deformation of LCE microactuators is driven by the anisotropic mechanical response parallel and perpendicular to the alignment of their mesogenic components, during nematic-isotropic phase transition. Interestingly, a rich palette of deformation types, including mechanically unfavored motions (e.g., in-plane bending) and their hierarchical assembly, can be achieved by programming the internal orientation of the mesogens. Overall, these results provide the basis of a versatile class of active microstructures that enable the deformations in ways that are not possible with passive systems, and they can be optimized for a range of potential applications, including self-regulated antenna, controlled release system, and information encryption.
2:45 PM - BM07.11.05
Designing Biocompatible Systems at the Materials-Biological Interface for CO2 and N2 Fixation
Chong Liu 1 , Kelsey Sakimoto 2
1 Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, United States, 2 , Harvard University, Cambridge, Massachusetts, United States
Show AbstractStudies at the materials-biological interface assume that the presence of materials themselves shall not perturb the biological systems significantly, i.e., the materials should be biocompatible. Here we present one example showing that the knowledge of inorganic chemistry can guide us to design biocompatible materials at the materials-biological interface. We designed a biocompatible inorganic catalyst system that split water into H2 and O2 at low driving voltages. Here the redox chemistry of these materials was carefully designed to minimize the generation of toxicants including reactive oxygen species and transition metal cations. When grown in contact with these catalysts, microorganisms consumed the produced H2 to fix CO2 or N2 into commodity chemicals with high energy efficiency.
3:30 PM - *BM07.11.06
Dynamic Materials Inspired by Cephalopods
Alon Gorodetsky 1
1 , University of California, Irvine, Irvine, California, United States
Show AbstractCephalopods (squid, octopuses, and cuttlefish) have captivated the imagination of both the general public and scientists for more than a century due to their visually stunning camouflage displays, sophisticated nervous systems, and complex behavioral patterns. Given their unique capabilities and characteristics, it is not surprising that these marine invertebrates have recently emerged as exciting models for novel materials and systems. Within this context, our laboratory has developed various cephalopod-derived and cephalopod-inspired materials with unique functionalities. Our findings hold implications for next-generation adaptive camouflage devices, sensitive bioelectronic platforms, and advanced renewable energy technologies.
4:00 PM - BM07.11.07
Sensing Metabolites with Accumulation Mode Organic Electrochemical Transistors
Anna-Maria Pappa 3 , Alexander Giovannitti 2 , David Ohayon 1 , Iain McCulloch 1 2 , Roisin Owens 3 , Sahika Inal 1
3 , Ecole des mines des Saint Etienne, Gardanne France, 2 , Imperial College London, London United Kingdom, 1 , King Abdullah University of Science and Technology, Thuwal Saudi Arabia
Show AbstractOrganic electrochemical transistors (OECTs) are electrolyte gated organic thin film transistors that can transduce, as well as amplify, ionic signals of biological origin into electronic ones. Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), an intrinsically doped organic semiconductor, represents to date the workhorse material of OECTs used in numerous biological applications including metabolite sensing. Such transistors that operate in the depletion regime, i.e., switch off with a gate voltage, pose specific disadvantages when it comes to biosensing as compared to accumulation mode devices that switch on upon recognition of a biological event. The accumulation mode transistors are identified with a large operation window, a high signal on/off response and a low-power operation. We herein show, for the first time, an accumulation mode electrochemical transistor comprising an n-type organic semiconductor in the channel for enzymatic lactate detection. In the absence of an electron-transfer mediator, the channel is doped as lactate is catalyzed by a specific enzyme in the electrolyte, leading to a biosensor with superior sensitivity and detection range compared to PEDOT:PSS analogues. This is due to the operation principle of this transistor type which allows for the bioelectrocatalytic reactions to occur at the channel. By tuning the morphology of the semiconductor film, we can detect lactate with high sensitivity and in a wide concentration range in physiologically relevant electrolytes such as blood and sweat. The device exhibits a linear detection range from 10 µM to 1mM and a very fast response for low metabolite concentrations (<0.3 mM). Highlighting the materials properties that enable biochemical sensing, this work provides an understanding of materials-device performance relations for the development of high-performance, simple-to-fabricate, low cost point-of-care sensors.
4:15 PM - BM07.11.08
Elucidating Morphology and Orientation of Biomolecules on 2D Nanomaterials for Flexible FET Biosensors Using Synchrotron X-Ray Techniques
Nicholas Bedford 1 , Li Xing 1 , Ming-Siao Hsiao 1 , Ahmad Islam 1 , Thomas Ferron 2 , Brian Collins 2 , Tiffany Walsh 3 , Jorge Chavez 1 , Steve Kim 1 , Lawrence Drummy 1
1 , Air Force Research Laboratory, Wright Patterson AFB, Ohio, United States, 2 , Washington State University, Pullman, Washington, United States, 3 , Deakin University, Melbourne, Victoria, Australia
Show AbstractFET-based biosensors have been identified as promising sensor platforms for real-time monitoring of biomarkers for various health/clinical diagnoses. To achieve the needed specificity for such sensors, antibodies-analyte interactions are most commonly used to trigger a sensor response, where small changes in biomolecular conformation upon binding induce a change in the FET gate voltage. Issues exist with larger biomolecules such as antibodies however, where eventual denaturing results in a loss of selectivity. As an alternative, our group and others have focused on smaller molecule peptide and nucleic acid biological recognition elements (BREs) that exhibit high sensitivity/selectivity properties despite their comparatively disordered structure. Though results to date have been promising, further improvement to sensor properties requires an understanding of molecular orientations and structures driving interactions between target biomarkers, corresponding BREs, and the inorganic device. Without such knowledge, sensor improvement is limited to trial and error approaches built upon biocombinatorial identification processes. To better understand these interactions, we have developed new in-situ and in-operando characterization techniques to correlate sensor properties to orientation and morphology at the biotic/abiotic surface. In particular, solution and grazing incidence SAXS and NEXAFS of BREs on graphene were used to determine biomolecular structure and orientation on the sensor surface, and these results were correlated with liquid cell electron microscopy and sensor performance data. This work focuses on BRE-biomarker interactions relevant to real-time monitoring of biomarkers correlated to stress, fatigue, and cognition. The methodology implemented herein, rooted in understanding fundamental structure/function relationships, is expected to be readily applicable to tackle similar issues across various health monitoring and clinical application areas of biosensors.
4:30 PM - BM07.11.09
Focal Manipulation of Neural Interstitial Ion Concentration Using Ion-Selective Membrane Electrodes
Matthew Flavin 1 2 , Daniel Freeman 2 , Jongyoon Han 1
1 Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 , Draper Laboratory, Cambridge, Massachusetts, United States
Show AbstractDeveloping precise and effective means of modulating the nervous system is a major challenge in neural prosthetics. Our approach is to perform localized chemical modulation by polarizing electrodes modified with ion-selective membranes (ISM), creating ion-concentration polarization (ICP). With this physical process, the interstitial concentrations of selected ions can be manipulated in a spatially and temporally precise manner. Here, we report the design and testing of a prototype peripheral nerve cuff electrode with ISM-modified contacts for the purposes of ICP-based neuromodulation. The operation of this device was evaluated by performing electrophysiological studies in an ex vivo frog sciatic nerve preparation.
Using the finite element method (FEM), we simulated the behavior of a characteristic cuff electrode system as a multidimensional Nernst-Planck-Poisson (NPP) model in COMSOL Multiphysics. This tool allowed us to evaluate the relative contributions of electrical and chemical modulation as well as identify key parameters. In addition, we fabricated, using rapid-prototyping equipment, a multi-contact cuff electrode that could be used for testing in an animal model. An ISM with either Ca2+ or K+ –selective formulations would then be drop-cast onto one of the contacts, creating a solid-contact ISM electrode. Electrode impedances of coated and uncoated contacts within the cuff were determined using electrochemical impedance spectroscopy (EIS). This allowed us to identify defects and evaluate long-term stability.
This ISM cuff electrode was ultimately used to test the impact of electrochemical neuromodulation in an ex vivo frog sciatic nerve model. In preliminary results, we see that cathodic current applied at the Ca2+–ISM contact resulted in lowering of excitation thresholds, consistent with neurons being subjected to depletion of extracellular Ca2+. The original excitation threshold was restored following a brief recovery period. Similar experiments with modulation of other ions (e.g. K+) will be investigated in the future.
As a fully realized technology, an ISM cuff electrode could potentially be used to lower the stimulus energy required to elicit muscle activation (via Ca2+ manipulation), or block aberrantly firing nerves that characterize neuropathic pain disorders (via K+ manipulation). Furthermore, ISM-based stimulation could eventually be applied beyond the peripheral nervous system to address neurological and psychiatric disorders in the brain. Advancements such as these will be necessary to provide improved intervention strategies for an aging population characterized by an increasing incidence of neurological disorders.
Symposium Organizers
Yizhou Dong, The Ohio State University
Christopher Alabi, Cornell University
Daniel Anderson, Massachusetts Institute of Technology
Bozhi Tian, University of Chicago
Symposium Support
Alnylam Pharmaceuticals, Inc.
Nanobio Delivery Pharmaceutical Co., Ltd.
Precision NanoSystems Inc.
BM07.12: Nanomaterials for Probing Biology II
Session Chairs
Yin Fang
Yuanwen Jiang
Bozhi Tian
Friday AM, December 01, 2017
Hynes, Level 2, Room 204
8:00 AM - BM07.12.01
Bioinspired Engineering of Artificial Heart Valve Constructs Using Natural Protein Fibers
Chang Liu 3 , Yun Bai 1 , Xing Zhang 1 2
3 , Northeastern University, Shenyang, Liaoning, China, 1 , Institute of Metal Research, Chinese Academy of Sciences, Shenyang China, 2 , University of Science and Technology of China, Hefei, Anhui, China
Show AbstractHeart valve disease with major symptoms of stenosis and regurgitation is prevalent worldwide. Surgical replacement of diseased heart valves at the end-stages has been widely performed with mechanical valves (MVs) or bioprosthetic heart valves (BHVs). All these current devices have significant limitations with risks of further morbidity and mortality. For example, MVs may cause hemorrhage and thromboembolism, and require anticoagulation for the lifetime of the patients. BHVs show better hemodynamic behavior due to the composition and structural similarity to native heart valves when compared to MVs, however, they do show limited durability because of calcification and progressive degeneration. Thus, polymeric heart valve (PHV) prostheses combining the advantages of MVs and BHVs with long-term durability and no necessity for permanent anticoagulation are of great interest and also show potential applications in advanced transcatheter devices.
In this study, novel PHVs were fabricated, consisting of natural protein fibers to mimic the fibrous networks in the fibrosa and ventricularis layers for stress bearing, as well as poly(ethylene glycol) (PEG) hydrogels to improve anti-fouling function. These layered constructs showed mechanical properties, i.e., elastic modulus and elongation percentage, close to those of human aortic valve leaflets. The hemodynamic property of these PHVs was obtained under the physiological condition using a pulse duplicator, which can meet the requirements of the ISO-5840 standard. Furthermore, the presence of PEG hydrogels in the composites improved the resistance to progressive calcification of the embedded protein fibers in vitro, likely due to prevention of large-size hydrated ions to pass through by the polymeric networks of the hydrogels. In addition, the fibrous structures retained in the PEG-protein fiber composites after subcutaneous implantation for four weeks, while those from natural protein samples showed early degradation to certain extent, suggesting the prevention of enzymatic degradation of protein fibers by the polymeric network of PEG hydrogels in vivo. Thus, this study lays down a basis for fabrication of novel PHVs to mimic the heterogenic structures, mechanical properties and biological functions of heart valve leaflets.
8:15 AM - BM07.12.02
Protein Corona Evolution in Tumor Microenvironment Defines the Fate and Theranostic Efficacy Of Nanocarriers
Cristina Rodriguez-Quijada 1 , Gwendolyn Cramer 1 , Ljubica Petrovic 1 , Jonathan Celli 1 , Kimberly Hamad-Schifferli 1
1 , University of Massachusetts Boston, Boston, Massachusetts, United States
Show AbstractGold nanoparticles have attracted attention in cancer research as theranostic platforms because their surface can be easily functionalized with biomolecules for drug delivery, gene therapy and specific accumulation at the tumor site. However, nanomaterials change their surface composition when exposed to biological fluids such as blood or serum, resulting in a protein corona which can hinder their function as therapeutic or diagnosis tools. Recently, researchers have put intense effort toward understanding protein corona formation when the nanoparticle is exposed to a static environment, such as a solution of human serum. Although it is well known that because of weak protein-NP bonds protein corona composition depends on the biological surrounding, protein corona behavior in the tumor microenvironment is still not completely understood. Tumor environments have increased protease activity and lower pH, which can affect the protein corona in a manner completely differently than other tissues. In cell culture experiments using PANC1 cells, we measure the time dependent degradation of protein coronas attributed to proteolytic activity of this aggressive pancreatic ductal adenocarcinoma (PDAC) cell line. In addition, we vary the protein corona composition to modulate the degradation rate. While there has been some work in designing coronas for their carrier properties, a study of how to optimize their properties for protease degradation has not yet been performed. Results can enable engineering protein corona properties for optimal biodistribution, stability, cell interaction, and uptake.
8:30 AM - BM07.12.03
A Biomimetic Hemodialysis Microfluidic Device
Connie Cheng 1 , Shirley Tang 1
1 , University of Waterloo, Waterloo, Ontario, Canada
Show AbstractHemodialysis is one of the three most commonly used renal replacement therapies, which takes use of extracorporeal dialysis to remove waste products such as creatinine, urea, and free water from blood and to balance electrolytes. Current hemodialysis procedure is very expensive, causes great inconvenience to patient’s life, and can lead to severe health complications. Here, we report our recent progress in creating a novel biomimetic hemodialysis microfluidic device. Combining 3D printing and soft lithography, a two-compartment device is fabricated with unique fluid-pathway network that mimics the function and shape of kidney nephrons. An ultra-thin nanoporous semi-permeable membrane is used to separate the blood and the dialysate, allowing highly efficient waste removal and ion exchange. Further, an monolayer antithrombogenic coating is applied to the semipermeable membrane and the inner-surface of the device that come into contact with blood. Results demonstrating efficient creatinine, urea, and water removal from spiked human blood samples, as well as effective inhibition of protein and platelet surface adhesion/aggregation will be presented. Our biomimetic hemodialysis device can potentially offer a more compact, efficient, and hemocompatible alternative to the existing hemodialysis machines.
8:45 AM - BM07.12.04
iPSC-Derived Brain Endothelial Cells Retain Biological Functionality Long-Term in Perfused Gelatin Channels
Shannon Faley 1 , Emma Hollmann 1 , Jason Wang 1 , Callie Weber 1 , Ethan Lippmann 1 , Leon Bellan 1
1 , Vanderbilt Univ, Nashville, Tennessee, United States
Show AbstractFunctional integrity of the multicellular complex known as the blood-brain barrier or neurovascular unit (NVU), comprised of brain microvascular endothelial cells (BMECs), pericytes, and glial cells, serves as the critical protective barrier that separates the nervous system from peripheral circulation. Not only are BBB dynamics central for designing therapeutics which target or avoid the neural compartment, but abnormal NVU function is associated with a broad spectrum of neurodegenerative and cerebrovascular diseases. Given the inherent difficulty of translating information gleaned from costly animal models of human disease to successful clinical intervention, development of a functional in vitro tissue model of the human NVU is key to a mechanistic understanding of disease progression and identification of future therapeutic agents. This work describes the initial phase of efforts to fabricate a biomimetic NVU, specifically focused upon establishing a functional endothelial layer lining continuously perfused channels in enzymatically crosslinked, 3D gelatin hydrogels using iPSC-derived BMECs, which have been shown to exhibit near in vivo level barrier properties1. iPSC-BMECs were seeded in arteriole-sized (~800 µm) gelatin channels and cultured for at least 14 days under static (no flow) or continual perfusion at 30 µl/min. Barrier strength of iPSC-BMECs evaluated at days 1, 7, and 14 was measured by confocal imaging of 3K MW fluorescent dextran diffusion. Samples were then fixed and stained with VE-cadherin and Claudin-5 antibodies to verify tight junction formation. iPSC-BMECs exhibited permeability to dextran diffusion two orders of magnitude lower than HUVEC controls in both perfused and non-perfused cohorts. Interestingly, while perfused BMEC-lined channels were highly resistant to dextran extravasation at all timepoints, the permeability of samples cultured for 14 days under perfusion did not exhibit the order of magnitude increase in permeability measured in those maintained without perfusion, suggesting that may promote resilience in barrier strength over time. Immunofluorescent staining of VE-cadherin and Claudin-5 provided further confirmation of tight junction formation contributing to establishment of robust barrier properties. Finally, evaluation of efflux transporter activity based upon cellular retention of rhodamine-123 in BMECs lining perfused channel for 14 days with and without cyclosporine inhibition, revealed a 68% increase in rhodamine fluorescence in inhibited cells. This provides strong evidence that the iPSC-BMECs retain biological functionality after 14 days in perfusion. In conclusion, iPSC-derived BMECs possess robust barrier properties in continuously perfused gelatin channels for 14 days, a significant step towards fabricating a clinically relevant NVU tissue model. Future work will also include integration of matched, iPSC-derived pericytes, astrocytes, and neurons.
1Lippmann, E.S., Sci.Rep. 2014. 4, 4160.
9:00 AM - BM07.12.05
Discrimination of Nano-Structural Features in Breast Cancer Biomarkers by Using Tailored Superhydrophobic Surfaces
Angelo Accardo 1 , Emmanuelle Trevisiol 1 , Jean-Francois Arnal 2 , Coralie Fontaine 2 , Christophe Vieu 1
1 , LAAS-CNRS, Toulouse France, 2 , INSERM U1048-I2MC, Toulouse France
Show AbstractBy exploiting the evaporation mechanisms of droplets drying in quasi contact-free conditions, biomimetic superhydrophobic substrates provide novel possibilities for studying a wide range of biological compounds such as neurodegenerative proteins [1] and cancer-related bio-compounds (e.g. exosomes [2]). ERα expression is the most important biomarker in breast cancer [3], as it provides the index for sensitivity to endocrine treatment. Here we report the fabrication of lotus-leaf-like tailored SU8 micropillars and their application in the context of a multitechnique characterization protocol for the investigation of the structural properties of two estrogen receptors (ERα66/ERα46) [4].
The superhydrophobic supports (contact angle of 158°) were developed by using a two-step approach including an optical lithography process and a plasma reactive ion-roughening one. The microstructures were grown either on CaF2 windows (for Raman and FTIR measurements) or on Si3N4 membranes (for XRD measurements). Upon drying on the micropillars, the biological samples resulted in stretched fibers, which were then characterized by synchrotron X-ray diffraction (XRD), Raman and Fourier-transform infrared spectroscopy (FTIR). We attribute the formation of such fibers to the stretching mechanism provoked by the shrinking of the contact line of the droplet while drying and displacing from one pillar to the adjacent one. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggested the presence of conformational changes between the two biomarkers. In particular, the three performed characterizations converge to the conclusion that, from a protein secondary structure point of view, while ERα46 features mainly β-sheet phases, ERα66 contains also some evident α-helical contributions, which we attribute, as a tentative hypothesis, to the absence of the flexible A/B domain in the structure of ERα46.
In conclusion, the proposed protocol is able to enhance the discrimination sensitivity of structural features of this class of biomarkers and pave the basis for the development of a novel spectroscopic tool aiming at the structural identification of bio-compounds linked to breast cancer disease.
[1] A. Accardo, V. Shalabaeva, E. Di Cola, M. Burghammer, R. Krahne, C. Riekel, S. Dante, ACS Appl. Mater. Interfaces. 7 (2015) 20875-20884.
[2] A. Accardo, L. Tirinato, D. Altamura, T. Sibillano, C. Giannini, C. Riekel, E. Di Fabrizio, Nanoscale 5 (2013) 2295-2299.
[3] E. Chantalat, F. Boudou, H. Laurell, G. Palierne, R. Houtman, D. Melchers, P. Rochaix, T. Filleron, A. Stella, O. Burlet-Schiltz, A. Brouchet, G. Flouriot, R. Métivier, J.-F. Arnal, C. Fontaine, F. Lenfant, Breast Cancer Res. 18 (2016) 123.
[4] A. Accardo, E. Trevisiol, A. Cerf, C. Thibault, H. Laurell, M. Buscato, F. Lenfant, J.-F. Arnal, C. Fontaine, C. Vieu, J. Vac. Sci. Technol. B 34 (2016) 06K201.
9:15 AM - BM07.12.06
Developing a Magnetic Nanoparticles for Multiplexed Magnetothermal Control of Biological Signaling
Junsang Moon 1 , Michael Christiansen 1 , Danijela Gregureć 1 , Polina Anikeeva 1
1 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractIron oxide magnetic nanoparticles (MNPs) have been widely used for biomedical applications due to their biocompatibility. The ability of MNPs to dissipate heat in the presence of alternating magnetic fields (AMFs) through hysteretic power loss has enabled their biological applications in cancer theranostics, drug delivery, and neuromodulation. The heat dissipating efficiency of MNPs, quantified by specific loss power (SLP measured in W/g), is a function of not only their materials properties but also the driving AMF conditions. Therefore, to maximize MNP heating, driving AMF conditions should be considered together with MNP materials properties.
In this work we synthesized a broad range of finely tuned MNP sets with linear dimensions ranging 15-30 nm and magnetocrystalline anisotropy varied through doping of CoxFe3-xO4 with cobalt at concentrations of x=0~0.25. Dynamic magnetization process of these MNPs under a range of applied AMF conditions was analyzed to explain their heat dissipating efficiencies. Furthermore, independent heating of two MNP types with two differing AMF conditions was demonstrated. Based on these findings, independent modulation of calcium signaling in two cell populations was demonstrated.
9:30 AM - BM07.12.07
A Turn-on Enzymatic Biosensor for Lactate Detection Based on FITC Embedded ZnO/Silica Nanocomposite for In Vitro Diagnosis
Shreeram Joglekar 1 , Prashant Pimpliskar 1 , Vedashree Sirdeshmukh 2 , Prashant Alegaonkar 1 , Anup Kale 2
1 , Defence Institute of Advanced Technology, Pune India, 2 Applied Sciences, College of Engineering Pune, Pune, Maharashtra, India
Show Abstract
L-lactate, a metabolite produced during anaerobic metabolism of glucose in muscles, has emerged as an important biomarker for clinical analysis. Lactate level increases significantly in some physiological diseases like cancer, metastasis and tumor recurrence, muscular dystrophy, congestive heart disease, renal failure, tissue hypoxia, myocardial infarction. Thus, monitoring lactate levels is of great significance for early diagnosis. A novel Fluorescence Resonance Energy Transfer (FRET) based turn “ON” enzyme biosensor has been developed using fluorescent ZnO/APTMS-FITC nanoparticles as sensing probe. Turn-on fluorescence biosensor is more desirable than Turn-off biosensor because in latter’s case the intensity of fluorescence from probe decreases in concentration dependent manner. There is always a chance of interference from background noise at lower intensity and it is not desired for developing a sensitive assay.
Herein, we report ZnO/Silica/FITC nanocomposites based enzymatic Lactate FRET biosensor. In the presence of analyte to be detected, the electron transfer is reduced leading to multiple fold increase in fluorescence. We chose 3-aminopropyltrimethoxy silane as a capping for ZnO nanoflakes to prevent agglomeration in the detection solution. APTMS capping also introduces amine group on the surface of ZnO nanoflakes, thus promoting their interaction with LDH. Fluorescein isothiocynate (FITC) is embedded in the APTMS capping around the nanoflakes to increase the inherent fluorescence of ZnO nanoflakes. In the presence of lactate, LDH catalyzed reaction occurs converting NAD into its reduced form NADH thus switching 'ON' the fluorescence of ZnO nanoparticles. The present study found that the fluorescence of these nanoflakes can be reversibly quenched by nicotinamide adenine dinucleotide (NAD) because of the electron transfer between ZnO nanoflakes and NAD. It was observed that fluorescence intensity was about 2-3 folds higher in the presence of ZnO/APTMS-FITC than bare ZnO nanoflakes. Moreover, NAD is an important cofactor for many enzymes. So, this detection system can be used to detect various substrates for NAD dependent enzymes.
10:30 AM - BM07.12.09
Functionalized Plasmonic Nanoarray—Towards Multidimensional Label-Free Surface Enhanced Raman Spectroscopy Signatures in Complex Biological Environments
Nayoung Kim 1 , Michael Thomas 1 , Mads Bergholt 1 , Molly Stevens 1
1 Department of Materials, Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, London United Kingdom
Show AbstractThe development of universal, reliable label-free platforms for the facile assessment of complex biological samples is of fundamental importance in bioanalytical technologies. Direct profiling of fingerprint label-free surface enhanced Raman spectroscopy (SERS) spectra, has been identified as a potential candidate for achieving this due to the broad spectral landscape of Raman fingerprints with the potential for single-molecule sensitivity. Despite its great sensing potential of SERS, reliable label-free SERS sensing has been a continuing challenge especially in liquid-phase biological environments. The practical difficulties are mainly attributed to the inherent chemical complexity and statistical binding of multiple analytes to the SERS active regions within liquid media, which usually result in complex and poorly reproducible SERS spectra.
This work proposes a chemical-nose approach to achieve enhanced precision in label-free SERS sensing by adding another dimensionality of data that is guided by the physical properties of the target analytes. This involves an arrayed sensing platform, termed functionalized arrayed SERS, consisting of 8 different self-assembled monolayers (SAMs) formed on plasmonic gold nanopillars. It capitalizes on the differing affinities of multiple components in liquid-phase complex biological samples via low-specificity physical interactions with SAMs of different molecular characteristics without introducing any targeting entity. The results for varied populations and extents of components in SERS active regions appear as subtle to distinguishable differences along the broad SERS spectral landscape. The resulting three-dimensional spectra are further analysed by statistical intrinsic 3-way analysis to identify and classify analytes/samples with higher selectivity and reproducibility. The rationale of using multiple SAM functionalization was systematically demonstrated with several analyte solutions of different molecular properties (e.g. aminated aromatic acid molecules), from which distinctive spectral differences were observed as a function of subtle molecular variations. The feasibility of employing functionalized array SERS substrates for identification of complex biological samples was successfully demonstrated with different healthy and cancerous cell lysates, which showed enhanced accuracy in classifying samples when compared to using a single unmodified platform. Within a broad range of potential biological samples, the universality and reliability of this arrayed label-free SERS platform holds great promise in biomedical applications for sensing and monitoring of complex biological environments.
10:45 AM - BM07.12.10
Metal Organic Framework Encapsulation for the Preservation and Photothermal Enhancement of Enzyme Activity
Sirimuvva Tadepalli 1 , Jieun Yim 1 , Rajesh Naik 2 , Srikanth Singamaneni 1
1 , Washington University in St. Louis, St. Louis, Missouri, United States, 2 , Air Force Research Laboratory (AFRL), Dayton, Ohio, United States
Show AbstractInterfacing biomolecules with functional materials is a key strategy towards achieving externally-triggered biological function. The rational integration of functional proteins, such as enzymes, with plasmonic nanostructures that exhibit unique optical properties such as photothermal effect provides a means to externally control the enzyme activity. However, due to the labile nature of enzymes, the photothermal effect of plasmonic nanostructures has only been utilized for the enhancement of the biocatalytic activity of thermophilic enzymes. In order to extend and utilize the photothermal effect to a broader class of enzymes, a means to stabilize the immobilized active protein is essential. Inspired by biomineralization for the encapsulation of bioactive molecules within protective exteriors in nature, we have utilized metal-organic framework to stabilize the enzyme. This strategy provides an effective route to enhance and externally modulate the biocatalytic activity of enzymes bound to functional nanostructures over a broad range of operating environments that are otherwise hostile to the biomolecules.
11:00 AM - BM07.12.11
Design of SERS Nanotags for the Multiplexed Detection of Dengue and Zika in a Lateral Flow Assay
Maria Sanchez-Purra 1 , Marc Carré Camps 2 , Biel Roig Solvas 3 , Alice Versinani 4 , Cristina Rodriguez-Quijada 1 , Helena de Puig Guixe 5 , Irene Bosch 6 , Lee Gehrke 7 8 , Kimberly Hamad-Schifferli 1
1 , University of Massachusetts Boston, Boston, Massachusetts, United States, 2 Bioengineering, Institut Químic de Sarrià, Barcelona Spain, 3 Electrical Engineering, Northeastern University, Boston, Massachusetts, United States, 4 Microbiology, Federal University of Minas Gerais, Belo Horizonte Brazil, 5 Mechanical Engineering, Massachusetts Institute of Technology, Boston, Massachusetts, United States, 6 Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Boston, Massachusetts, United States, 7 Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Boston, Massachusetts, United States, 8 Microbiology and Immunology, Harvard Medical School, Boston, Massachusetts, United States
Show AbstractA novel Surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) has been developed as a point-of-care device to distinguish between Dengue (DENV) and Zika (ZIKV) improving the sensitivity of regular LFA tests.
Zika and Dengue are mosquito-borne diseases that are currently a major global threat. They can co-circulate in endemic areas, as they are transmitted by the same vector and show similar non-specific symptoms with dramatically different outcomes. Because many outbreaks occur in areas that are resource-poor, assays that are easy to use, inexpensive, and require no power have become invaluable in patient treatment, quarantining, and surveillance. Paper-based sandwich immunoassays such as lateral flow assays (LFA) are attractive as point-of-care solutions as they have the potential for wider deployability than lab-based assays such as PCR. However, the low sensitivity of these assays imposes limitations on their ability to detect low biomarker levels, which is the case for Zika infections, co-infections with other viruses, and also for early diagnosis of any disease.
Here, we exploit the high sensitivity of surface-enhanced Raman spectroscopy (SERS) in a multiplexed assay that can distinguish between Zika and Dengue non-structural protein (NS1) biomarkers. SERS-encoded gold nanoparticles (GNP) were conjugated to specific antibodies for NS1 to distinguish between ZIKV and DENV without cross-reactivity in a dipstick immunoassay. Two different Raman reporters, BPE and 4-MBA, were used to differentiate between both viral biomarkers in a multiplexed assay by measuring the SERS signal on the test line of the LFA strip. Due to the gold nanoparticles, the Raman signal of a specific reporter adsorbed on the NP surface can be enhanced by several orders of magnitude due to SERS. Gold nanoparticles with different shapes were compared in terms of their SERS enhancement factor and diffusion through the LFA strip to increase the sensitivity of this approach. Using SERS allowed the detection of low concentrations of the viral biomarkers with a limit of detection of 15-fold and 7-fold, for ZIKV and DENV, lower than that of colorimetric LFA.
The combination of SERS with LFA can make a promising platform for a point-of-care device, as it can provide a much higher sensitivity than optical LFA devices, and the signal photobleaching is negligible compared to fluorescence detection. In addition, it is easily deployable as it does not require the use of expensive reagents or equipment and trained users, and portable Raman spectrometers are currently available on the market.
11:15 AM - BM07.12.12
Chemiluminescence-Assisted Cell Internalization Kinetics Assay
Di Wu 1 , Yunfeng Lu 1
1 , University of California, Los Angeles, Los Angeles, California, United States
Show AbstractIntracellular delivery of biomolecules has become a critical component of cell-based therapeutics, gene editing, induced pluripotent stem cells and other applications. While a growing list of nanocarriers with tailored physicochemical characteristics, such as cell penetration, control release, and targeting, are developed focusing on improvement of the delivery efficiency. How to precisely understand, predict and control the spatiotemporal distribution of as-delivered materials through the time course of internalization, kinetics in brief, is still challenging. Currently, the cell internalization kinetics are monitored by conjugation of imaging probes such as fluorescent agents. However, the fluorescence-based methods only provide a snap shot at specific time points instead of real-time evaluation. Moreover, due to self-quenching and photo bleaching, the quantification of cargo concentration from the fluorescent signal of imaging probes remains unclear. As stressed above, it’s critical to develop a novel approach to monitor the kinetics of cell internalization through the lens of both parameters: real time and quantification.
We report herein a chemiluminescence-assisted cell internalization kinetics assay (CIKA) using chemiluminescent nanocapsules. Firefly luciferase (FLuc), which catalyzes the emission of light in the presence of adenosine-5’-triphosphate (ATP), is used to optically track cellular uptake of nanoparticles. As the most commonly used phosphate donor within cells, ATP is present in much lower concentrations in extracellular environment (1-1000 nM) than that within cytosol (1-10 mM). The substantial difference of ATP levels restrains the bioluminescence signal only within the cytosol, making FLuc an ideal tool to track the process of cellular uptake. Despite the broad adaptations of FLuc, the poor stability of FLuc retards its utilization. Recently, our group developed a platform for protein delivery based on protein nanocapsules that endow enhanced stability and tunable surface properties. This strategy offers an novel approach to extend the half-life of FLuc for long-term studies. Since one photon is emitted in each reaction, bioluminescent intensity is directly proportional to the quantity of FLuc nanoparticles (denoted as nFLuc) delivered into the cytosol. Therefore, coalescing with the mathematical, real-time information for internalization process of nFLuc nanoparticles can be further quantified. Here internalization kinetics of nanoparticles with four different surface charges, RGD targeting ligand, and eight cell penetrating peptides were investigated in three cell lines. By realizing precisely spatiotemporal control over distribution and functions, this platform provides a simple and efficient approach for optimization of dosimetry, characterization of therapeutic efficiency and screening of novel medicine. It can be extended to evaluate the internalization kinetics profiles of other nanoparticle-based delivery systems.
11:30 AM - BM07.12.13
Rapid Immunochromatography Assays for Dengue Virus Serotypes and Zika Virus in Patient Serum
Helena de Puig Guixe 1 , Irene Bosch 1 , Marc Carré Camps 1 , Jose Gomez-Marquez 1 , Diana Fandos 1 , Kimberly Hamad-Schifferli 1 , Lee Gehrke 1
1 , MIT, Cambridge, Massachusetts, United States
Show AbstractThe recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross reactions and false results in molecular, antigenic, and serologic diagnostics. We report here the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes and ZIKV without cross reaction. To complement visual test analysis and remove user subjectivity in reading test results, image processing and data analysis are used for data capture and test result quantification, generating standardized objective data. Using a 30l serum sample, the sensitivity and specificity values of the DENV serotypes 1-4 tests and the pan DENV test (detects all four dengue serotypes) range from 0.76 to 1.00. Similarly, sensitivity/specificity for the ZIKV rapid test is 0.81/0.86 using a 150l serum input. Serum ZIKV NS1 protein concentrations are approximately tenfold lower than corresponding DENV NS1 levels in infected patients; moreover, ZIKV NS1 protein is not detected in PCR-positive patient urine samples. These new approaches and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases; moreover, the approach platform can be applied toward developing rapid antigen diagnostics for emerging viruses.
11:45 AM - BM07.12.14
Singled-Walled Carbon Nanotube Based Corona-Phase Molecular Recognition of Insulin
Gili Bisker 1 , Xun Gong 1 , Naveed Bakh 1 , Michael Lee 1 , Jiyoung Ahn 1 , Michael Strano 1
1 Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractInsulin is an important peptide hormone playing significant roles in metabolism and disease. Thus, its detection can potentially be an important component of discovery and therapy. Though the detection of insulin exists through molecular recognition elements such as antibodies and aptamers, these natural systems carry the inherent disadvantage of high cost and limited lifetime. Corona Phase Molecular Recognition (CoPhMoRe) is a technology that utilizes the structure of non-covalent conjugation heteropolymers as recognition elements for analyte detection. Fluorescent single-walled carbon nanotubes (SWCNTs) have been shown to be effective CoPhMoRe templates due to their signal transduction capabilities via optical signal modulation. In this work, high-throughput screening was performed on fluorescent SWCNTs wrapped with a library of poly(ethylene glycol) (PEG) – lipid conjugates. We demonstrate insulin recognition with C16-PEG(2000kDa)-Ceramide conjugation by a 62% fluorescent intensity decrease of the (10,2) SWCNT chirality upon introduction of insulin at 20 µg/ml. Isothermal titration calorimetry show that the insulin protein has no direct affinity towards the C16-PEG(2000kDa)-Ceramide molecules, indicating that the interaction of analyte adsorption occurs on the polymer conjugated SWCNT. To rule out non-selective mechanisms of binding such as molecular weight, isoelectric point, and hydrophobicity, a panel of proteins originating from human blood as well as fragments of the insulin peptide were screened. Though signal changes were observed in the case of longer fragments of the insulin chains, it was much decreased as compared to the recognition of insulin in its native form. Finally, insulin detection was demonstrated in buffer as well as in competitive environments such as serum. This work shows the potential for the engineering of non-biological molecular recognition elements for the specific detection of biological molecules in complex environments.