Symposium Organizers
Donglei (Emma) Fan, University of Texas at Austin
Jianping Fu, University of Michigan
Xingyu Jiang, National Center for Nanoscience and Technology
Matthias Lutolf, Ecole Polytechnique Federale de Lausanne
Symposium Support
Air Force Office of Scientific Research
Biomaterials Science
National Science Foundation
H3/B3: Joint Session: Biointerfaces
Session Chairs
Donglei Fan
Andreas Lendlein
Monday PM, December 01, 2014
Sheraton, 2nd Floor, Grand Ballroom
3:00 AM - *H3.01/B3.01
Hydrogel Microbeads and Microfibers for Biomedical Applications
Shoji Takeuchi 1 2
1The University of Tokyo Tokyo Japan2Japan Science and Technology Agency Tokyo Japan
Show AbstractIn this presentation, I am planning to talk about several MEMS/Microfluidic-based approaches for the rapid and reproducible construction of hydrogel microstructure. Hydrogels are attractive materials because of its excellent deformability, biocompatibility, and the ability to be chemically-modified. They are thus very useful for various biomedical applications including implantable monitoring and tissue engineering.
Fluorescent hydrogels hold great promise for in vivo continuous glucose monitoring with wireless transdermal transmission and long-lasting activity. We synthesized a highly-sensitive fluorescent monomer, and then fabricated injectable-sized fluorescent polyacrylamide hydrogel beads and fibers with high uniformity and high throughput. We find that the fluorescent beads provide sufficient intensity to transdermally monitor glucose concentrations in vivo.
Large-scale 3D tissue architectures that mimic microscopic tissue structures in vivo are very important for not only in tissue engineering but also drug development without animal experiments. We demonstrated a construction method of 3D tissue structures by using cell beads and cell fibers. To prepare the cellular beads, we used an axisymmetric flow focusing device (AFFD) that allows us to encapsulate HepG2 cells within monodisperse collagen beads. We then seeded 3T3 cells on the surface of the collagen beads. Finally HepG2 and 3T3 cells were successfully made contact with each other. Moreover, by putting these capsules in a 3D chamber and incubating them, we successfully established complicated and milli-sized 3D structures. We believe that altering the shape can be possible as simple as changing the mold, and will try to combine multiple types of cells to create more complex system that functions as a living organism. As the cell fibers, a cell-encapsulating core-shell hydrogel fiber was produced in a double coaxial laminar flow microfluidic device. When with myocytes, endothelial, and nerve cells, they showed the contractile motion of the myocyte cell fiber, the tube formation of the endothelial cell fibers and the synaptic connections of the nerve cell fiber, respectively. By reeling, weaving and folding the fibers using microfluidic handling, higher-order assembly of fiber-shaped 3D cellular constructs can be performed. Moreover, the fiber encapsulating beta-cells is used for the implantation of diabetic mice, and succeeded in normalizing the blood glucose level.
References
Yun Jung Heo , Hideaki Shibata , Teru Okitsu , Tetsuro Kawanishi, and Shoji Takeuchi: Long-term in vivo glucose monitoring using fluorescent hydrogel fibers, Proc. Natl. Acad. Sci. USA, vol. 108(33), pp. 13399-13403, 2011
Hideaki Shibata, Yun Jung Heo, Teru Okitsu, Yukiko Matsunaga, Tetsuro Kawanishi, and Shoji Takeuchi: Injectable hydrogel microbeads for fluorescence-based continuous glucose monitoring, Proc. Natl. Acad. Sci. USA, vol. 107, no. 42, pp. 17894-17898, 2010
Hiroaki Onoe, Teru Okitsu, Akane Itou, Midori Kato-Negishi, Riho Gojo, Daisuke Kiriya, Koji Sato, Shigenori Mirua, Shintaroh Iwanaga, Kaori Kuribayashi-Shigetomi, Yukiko Matsunaga, Yuto Shimoyama, and Shoji Takeuchi: Metre-long Cellular Microfibres Exhibiting Tissue Morphologies and Functions, Nature Materials, vol.12, pp. 584-590, 2013
3:30 AM - H3.02/B3.02
Bioactive and Cell-Laden Nanofibrous Scaffolds Fabricated through a One-Step Process
Qilong Zhao 1 Min Wang 1
1The University of Hong Kong Hong Kong Hong Kong
Show AbstractElectrospinning is a popular technique for making nanofibrous tissue engineering scaffolds. After cell incorporation, the cell-scaffold construct can be used to regenerate various human body tissues/organs. However, owing to the normally dense structures of electrospun scaffolds, cells can only be seeded in 2D on scaffold surface using the post-electrospinning cell seeding approach. For putting cells in 3D and directly inside electrospun scaffolds, we designed and investigated a facile method by combining cell seeding with scaffold fabrication. In this method, cell electrospraying was performed concurrently with electrospinning of the scaffold, placing cell-encapsulated microspheres into the matrix of nanofibrous scaffold, and the subsequent immersion treatment dissolved the shell of microspheres, releasing the cells for the cell-laden scaffold. In our experiments, a dual-source dual power setup was employed for conducting concurrent cell electrospraying and electrospinning. For cell electrospraying, a coaxial device was used. A cell suspension of human umbilical vein endothelial cells (HUVECs) and a gelatin/alginate blend solution were fed into the inner and outer concentric tubes, respectively. Crosslinked, core-shell structured microspheres containing HUVECs could be dissolved by immersing them in a cell culture medium, releasing the cells. The microsphere structure and cell viability of both encapsulated cells and released cells were studied using SEM, live/dead staining assessment assisted by fluorescent microscopy and laser scanning confocal microscopy. The optimal condition for cell electrospraying was investigated by modulating major processing parameters (composition of polymer blend, applied voltage, flow rate, etc.). For electrospinning, emulsions were made using PLGA solutions and vascular endothelial growth factor (VEGF)-containing PBS (or PBS alone). They were subsequently electrospun to make nanofibrous scaffolds with or without VEGF incorporation. Various experiments were conducted for studying the morphological and structural properties of nanofibrous scaffold, as well as the release behavior of VEGF. When concurrent cell electrospraying and emulsion electrospinning was performed, bioactive and cell-laden scaffolds were fabricated. The post-electrospinning immersion treatment could release encapsulated cells in the nanofibrous scaffolds. Furthermore, the space left by the dissolved microspheres provided the room for subsequent cell proliferation and infiltration. Cell culture experiments and comparative studies were then performed for evaluating cell functions in scaffolds with or without VEGF incorporation. Results indicated that cell proliferation and cell infiltration were enhanced in VEGF-loaded scaffolds. This new fabrication method can lead to breakthroughs in developing electrospun scaffolds for the regeneration of complex human body tissues.
3:45 AM - H3.03/B3.03
Design Complex Hydrogel Microparticles for High Throughput 3D Cell Culture, Co-Culture and Microtissue Production
Yen-Chun Lu 1 Wei Song 1 Duo An 1 Robert Schwartz 2 Minglin Ma 1
1Cornell University Ithaca USA2Weill Medical College of Cornell University NYC USA
Show AbstractCell encapsulation in hydrogel microparticles have been investigated for decades in various bioengineering applications including tissue engineering, and cell therapy. However, most of the time, the cells are encapsulated randomly in whatever material that forms the microparticles, most commonly alginate. The lack of control over the spatial organizations of the cells and the extracellular environment within the microparticles significantly limits for advanced applications. Here we report a novel, multi-fluidic cell microencapsulation approach where 1 or more types of cells are encapsulated in pre-assigned compartments in the microparticles with controlled extracellular matrix. These microparticles can be produced with controllable and nearly monondispersed sizes at rates of over 10,000 microparticles per min and therefore provide a promising platform for high throughput applications. We demonstrated the utilization of these extracellular matrix-supported microparticles for 3D culturing of cells that typically require specific microenvironment to survive such as human umbilical vein endothelial cells (HUVECs) and small intestine stem cells. By taking advantage of the confinement effect, we also showed robust and scalable productions of size-controlled multicellular microtissues. Lastly, to demonstrate the broad applications of these microparticles, we performed proof-of-concept studies on three different co-culture systems including cell segregations under 3D confined space, the supporting role of stromal cells in hepatocyte functions and the paracrine cell signaling in aggregation of endothelial cells, all in a high throughput manner.
4:30 AM - *H3.04/B3.04
Strategies for Creating Functions in Polymer-Based Materials by Combining Different Components
Andreas Lendlein 2 1
1University of Potsdam Potsdam Germany2Helmholtz-Zentrum Geesthacht Teltow Germany
Show AbstractA common strategy for the creation of functions in polymeric materials is the targeted combination of different polymers or polymers with inorganic components and the design of the interface between the phases [1]. Hereby the different components can be physically mixed or joined. Alternatively they can be covalently linked.
Several examples for the design of functions are presented, each illustrating a different concept to gain a function. The multivalent binding of polyglycerols on micro porous polyetherimide membranes combines a separation capability with hemocompatibility [2]. The covalent integration of nanoparticles as netpoints in a polymer network matrix enables a magneto sensitive reversible movement of the resulting hybrid material [3]. The pore morphology of polymeric foams strongly influences their shape-memory capability [4]. The internal geometry of a magnetic, active phase in a polymer matrix results in a triple shape effect, whereby the series of the shape changes can be determined by the applied stimulus [5].
The fundamental principles demonstrated in these material systems might stimulate further research in the field of multifunctional materials.
[1] M. Behl, M. Razzaq, A. Lendlein, Adv. Mater. 2010, 22, 3388-3410.
[2] A.T. Neffe, M. von Ruesten-Lange, S. Braune, K. Lützow, T. Roch, K. Richau, A. Krüger, T. Becherer, A.F. Thünemann, F. Jung, R.
Haag, A. Lendlein, J. Mater Chem B, 2014, 2, 3626-3635.
[3] M.Y. Razzaq, M. Behl, K. Kratz, A. Lendlein, Adv. Mater. 2013, 25, 5730-5733.
[4] T. Sauter, K. Kratz, A. Lendlein, Macromol. Chem. Phys. 2013, 214 (11), 1184-1188.
[5] M.Y. Razzaq, M. Behl, K. Kratz, A. Lendlein, Adv. Mater. 2013, 25, 5514-5518.
5:00 AM - H3.05/B3.05
Multifunctional Nerve Guidance Channels for Improved Neural Regeneration and Prosthetic Interfaces
Ryan Koppes 1 2 Xiaoting Jia 1 2 Seongjun Park 3 Christina Tringedes 1 Polina Anikeeva 1 2
1Massachusetts Institute of Technology Charlestown USA2Massachusetts Institute of Technology Cambridge USA3Massachusetts Institute of Technology Cambridge USA
Show AbstractThere is currently no effective treatment strategy following traumatic injury to the peripheral nervous system (PNS) in either partial or full loss of extremity function. Recent work has demonstrated neural recording and electrical stimulation devices that allow for neural-motor control of prosthetic limbs. However, much improvement is required to reach the resolution of neural interfacing needed for physiological functionality. In addition to neural interfacing, tissue engineering strategies are potential means to restore functionality after traumatic injury to the PNS. However, current interventions are years from being effective in the clinic. Therefore, our goal is to engineer material platforms that both promote nerve regeneration and provide an electrical interface for prosthetic integration that is clinically relevant now.
To date, the influence of nerve channel geometry and dimensions of sub-200 mu;m scale on neural regeneration has been poorly investigated due to material processing. For interfacing with either the motor or sensory axons of the PNS, geometric constraints may provide a means for selectively regenerating axons to intimately interface electrodes with sensory or motor nerve fibers, respectively. Furthermore, topography robustly influences the orientation and length of neural growth. However, no technique currently exists to fabricate mu;m topography features on the interior surface of nerve guidance channels without the inclusion of films or rolling.
Herein, we present a new method for engineering polymeric nerve guidance channels with intrinsic topography or recording electrodes. Utilizing a thermal drawing process (TDP), macro-scale preforms of biocompatible polyetherimide were made with rectangular and cylindrical channels. Topographical features or electrodes composed of conductive polyethylene were machined and added to the preforms. TDP reduced the cross-sectional dimensions by up to 200 times while maintaining the original geometries. Rectangular, rectangular with microgrooves, and cyclindrical neural growth channels with dimensions 30-200 mu;m were evaluated in vitro for their influence on neurite outgrowth from primary dorsal root ganglia (DRGs). Total distance of neurite outgrowth into the channel as well as the orientation of neurite extension and cell nuclei within the channel were measured with respect to the geometry and dimensions of the growth channel. Preliminary data suggests that narrower channels (40-60 mu;m) enhance the orientation of DRG outgrowth compared to larger channels (>100 mu;m), but very limited growth is observed in small channels (<40 mu;m). However, inclusion of microgrooves within the large channel increases neurite orientation. These results demonstrate our ability to utilize the TDP to design new polymeric nerve guidance channels as a strategy for PNS regeneration and neural interfacing.
5:30 AM - H3.07/B3.07
How Architecturally and Functionally Complex Polymers Can Optimize Therapeutic Proteins In Vivo
Mi Liu 1 Gregor Fuhrmann 1 Pamp;#229;l Johansen 3 Jean-Christophe Leroux 1 Marc A Gauthier 2
1Swiss Federal Institute of Technology Zurich Zurich Switzerland2INRS Varennes Canada3University Hospital of Zamp;#252;rich Zurich Switzerland
Show AbstractIn comparison to neutral linear polymers, functional and architecturally complex (i.e., non-linear) polymers offer distinct opportunities for enhancing the properties and performance of therapeutic proteins. However, understanding how to harness these parameters is challenging, and studies that capitalize on them in vivo are scarce. This presentation will cover this important topic with emphasis on two types of therapeutic proteins: ones for which long circulation in the bloodstream is desired, and ones for which retention and/or stabilization in the gastrointestinal tract is desired.
We will first present how the modification of an enzyme with a polymer of appropriate architecture can impart exceptionally low immunogenicity (e.g., generation/recognition of antibodies in vivo), with a commensurably low loss of therapeutic activity.[1,2] Secondly, we will also discuss how the modification of an enzyme with a polymer bearing appropriate functional groups can promote its stability (and thus therapeutic activity) at different locations in the gastrointestinal tract. Furthermore, functional polymers that interact with mucin will be shown to promote retention in the upper part of the gastrointestinal tract, and thus enhance the therapeutic activity of enzymes at this location.[3] Overall, the importance of the findings will be framed with context to selected relevant diseases that stand to benefit most from the presented concepts. This work was supported by the Swiss National Science Foundation (310030_135732) and the Sassella Stiftung.
[1] Liu, Tirino, Radivojevic, Phillips, Gibson, Leroux, Gauthier. Advanced Functional Materials. 2013, 23, 2007
[2] Liu, Johansen, Zabel, Leroux, Gauthier. Submitted
[3] Fuhrmann, Grotzky, Lukicacute;, Matoori, Yu, Luciani, Walde, Schlüter, Gauthier, Leroux. Nature Chemistry, 2013, 5, 582
5:45 AM - H3.08/B3.08
Cellular and Biomolecule Isolation on Biodegradable Nanostructured Coatings
Eduardo Reategui 1 2 Nicola Aceto 3 James Sullivan 3 Anne Jensen 1 Eugene Lim 4 Mahnaz Zeinali 1 A. J. Aranyosi 1 Wei Li 5 Steven Castleberry 5 Aditya Bardia 3 Lecia Sequist 3 Daniel Haber 3 Paula Hammond 5 Mehmet Toner 1 Shannon Stott 3
1Massachusetts General Hospital Charlestown USA2Harvard Medical School Charlestown USA3Massachusetts General Hospital Cancer Center Charlestown USA4Massachusetts Institute of Technology Cambridge USA5Massachusetts Institute of Technology Cambridge USA
Show AbstractNanostructured materials have been used as substrates for sensitive cellular or biomolecule recognition due to their high surface-area to volume ratio and biocompatibility. Whereas the deposition of these nanomaterials on surfaces is often irreversible, the analysis of the isolated biological samples is often limited to on-device microscopic imaging and spectroscopy applications. Therefore, biodegradable nanostructure substrates will facilitate the recovery of cells or biomolecules for downstream analysis (e.g., DNA, RNA, or proteomic analysis) and cell culture. Here, we describe a biodegradable nanostructured coating that allows for either temperature-responsive or mechano-sensitive degradation. The ultrathin coating (135.2 nm ± 8.6 nm) was formed by a layer-by-layer (LBL) deposition of biotinylated gelatin and neutravidin. Nanoroughness on the coating (30.7 nm ± 6.1 nm) was achieved by the incorporation of 70 nm streptavidin nanoparticles that were physisorbed directly on the gelatin coating. Temperature degradation of the applied coating to a glass or PDMS surface was achieved by raising the temperature to 370C; allowing their complete removal after 10 min. For local degradation of the coating, a normal force was applied through a frequency-controlled 80 µm microtip to dislodge partial regions of the coating, mimicking thixotropic hydrogel behaviors.
To demonstrate the biocompatibility and extremely sensitivity of the nanocoating, we used it for circulating tumor cells (CTCs) isolation and recovery. CTCs are extremely rare cells present in the blood stream of metastatic cancer patients (1 CTC per 109 blood cells) and their isolation and processing constitutes a technological challenge. We incorporated the nanocoating on our microfluidic HBCTC-Chip1 with tumor-cell specific antibodies at its surface (anti-EpCAM, anti-EGFR, anti-HER2). The clinical value of the nanocoating-microfluidic system was established when CTCs were detected in 87.5 % of patients with metastatic breast and lung cancer. The temperature degradation mechanisms of the nanocoating allowed recovery of 98.3 % ± 3.5 % of target cells with viabilities up to 92.03 % ± 4.5 %. Additionally, the frequency-controlled microtip allowed the recovery of individual CTCs from cancer patients that were analyzed for the presence of driver mutations in the PIK3CA (H1047R) and EGFR (exon 19 deletion and L858R) oncogenes.
In summary, our nanoscale, reversible biomaterial would enable and/or improve downstream assays through the release of any surface (e.g. beads, glass surfaces) that was initially employed to selectively isolate cells, proteins or DNA from a biological specimen.
References
1 Stott, S. L. et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences107, 18392-18397, doi:10.1073/pnas.1012539107 (2010).
H4: Poster Session I: Inorganic Biodevices
Session Chairs
Monday PM, December 01, 2014
Hynes, Level 1, Hall B
9:00 AM - H4.01
Purification of Microvesicles by Standing Surface Acoustic Wave (SSAW)
Kyungheon Lee 1 Huilin Shao 1 Ralph Weissleder 1 Hakho Lee 1
1Massachusetts General Hospital Boston USA
Show AbstractCirculating microvesicles (MVs) have emerged as a promising surrogate for tissue-based markers, enabling non-invasive, real-time disease monitoring. Purifying MVs for downstream analyses, however, still remains a challenging task, which often involves time-consuming and extensive procedures (e.g., ultracentrifugation, multiple filtration). We herein present a new microfluidic platform for MV isolation and enrichment from clinical samples. The system utilizes acoustophoresis to size-selectively separate MVs. Interdigitated electrodes, patterned on LiNbO3 substrate, were used to generate standing surface acoustic wave (SSAW) inside a microfluidic channel, and the resulting acoustic radiation force separated MVs according to their size and density. The design and operation of the device was optimized through numerical simulation. When applied to sort nanobeads, the system achieved > 90% sorting yields. We further used the system to collect MVs from pRBC (packed red blood cell) samples as well as from cell culture media. The microfluidic-SSAW device successfully isolated and enriched pure MV population, which was confirmed by downstream molecular analyses (Western blotting). Based on label-free and continuous in-flow separation, the developed platform could be an ideal tool for fast preparation of intact MVs.
9:00 AM - H4.02
Importance of Diode Circuit Element in Electrolyte-Oxide Interface for Nanopore Ion-Transistors
Sung-Wook Nam 1 Binquan Luan 1 Eduard A Cartier 1 Marinus Hopstaken 1 Ajay K Royyuru 1 Gustavo A Stolovitzky 1
1IBM T.J. Watson Research Center Yorktown Heights USA
Show AbstractNanopore ion transistors are electrofluidic elements conceived to manipulate the transport of molecular species through nanopores using electric fields. In this work, we report on a newly discovered diode circuit element existing in the electrolyte-oxide interface, and its importance for electrofluidic gating in ion transistors. We built sub-20 nm nanopore ion transistor devices and characterized ionic transport of KCl electrolytes. Simultaneous monitoring of electric currents of source (Is), drain (Id) and gate probes (Ig), allowed us to characterize both ionic and interfacial transports, as a function of gate voltage (Vg). Ionic transport through the nanopore was modulated such that negative (-) gate voltage bias induced an increase of ionic current, representing p-type transport, suggesting that the majority carrier contributing to ionic transport is positively charged potassium ion (K+) which screens the surface charges of pore wall. Interestingly, a characterization of Ig showed the presence of a diode circuit element in the electrolyte-oxide interface: The electric field created by negative (-) gate voltage bias reaches the electrolyte more effectively than that created by a positive (+) gate voltage bias, thus leading more efficient electrical-control over ionic transport. This diode functionality in electrolyte-oxide interface results in the unipolar transport of ion transistor. Based on the analysis of secondary ion mass spectrometry (SIMS) of the gate oxide layer, we suggest that the diode functionality is attributed to the diffusion of permeable potassium ions into the gate oxide, driven by negative (-) gate voltage biases. Our interpretation of the electrolyte-oxide interfacial effect clarifies electrofluidic gating behavior in ion transistors.
9:00 AM - H4.03
Electrochemical Etching of Silicon: High-Aspect-Ratio Nanopore Arrays on Membranes
Torsten Schmidt 1 Miao Zhang 1 Fatemeh Sangghaleh 1 Jan Linnros 1
1KTH Royal Institute of Technology Kista Sweden
Show AbstractElectrochemical etching (EE) of silicon in hydrofluoric acid has been shown to be an outstanding tool to realize a large variety of structures addressing a broad range of applications. Examples are the well-known luminescent porous silicon1 and well-defined homogenous pore arrays in the micrometer as well as sub-µm range.2-5
Nowadays, for demanding applications, such as molecular filtering and possibly sequencing of binary encoded DNA strands, solid-state membranes featuring an array of well-separated nanopores with diameters of about 2 nm are desirable. To meet these extraordinary requirements, light-assisted EE on structured silicon can be applied. The formation of nanopores, either in bulk silicon or on silicon membranes, is based on the local dissolution of surface atoms in pre-defined etching pits. Pore growth and pore diameter are, respectively, driven and controlled by the supply of positive charge carriers.
Performing EE on moderately-doped n-type bulk silicon, arrays with sub-100 nm wide pores were fabricated. In particular, straight nanopores with aspect ratios above 1000 (~19 µm deep and ~15 nm pore tip diameter) were achieved. However, inherent to the formation of such narrow pores is a radius of curvature of a few nanometers at the pore tip, which favors electrical breakdown resulting in rough pore wall morphologies.6
The Si membranes, used in our study, were fabricated on silicon-on-insulator (SOI) wafers using the Bosch process of inductively-coupled plasma etching. Array patterns in the sub-micro scale were defined on the membrane front side by optical or e-beam lithography. Electrochemical etching was then carried out individually on free-standing Si membranes, which are 100 µm in diameter and between 300 nm and 5 µm thick. So far, nanopores with diameters from 7 nm to 15 nm have been obtained on 300 nm thick membranes. To verify that pores have been etched through the whole membrane, the nanopore arrays were subjected to simple translocation experiments with fluorophore-tagged oligonucleotides. Based on optical detection, well-distinguishable single translocation events could be observed simultaneously for several nanopores (pore pitch distance 2 µm).
References
L. T. Canham, Appl. Phys. Lett. 57 (10), 1046-1048 (1990)
V. Lehmann et al., Materials Science and Engineering B69-70, 11-22 (2000)
P. Kleimann et al., Mater. Sci. Eng. B 69-70, 29-33 (2000)
J. Linnros et al., Physica Scripta 126, 72 (2006)
G. Laffite et al., Journal of the Electrochemical Society 158, 1 D10-D14 (2011)
T. Schmidt et al., submitted (2014)
9:00 AM - H4.04
Hybrid Fabrication of Controlled TiO2 Nanowire Arrays for Cellular Analysis
Young-Shik Yun 1 2 Jong-Souk Yeo 1 2
1Yonsei University Incheon Korea (the Republic of)2Yonsei University Incheon Korea (the Republic of)
Show AbstractAccording to advanced nanotechnologies in the field of biomedical engineering, an understanding of cellular responses with nanostructures becomes increasingly important. It requires a fabrication of nanostructures with a precise control of their size and positions in order to investigate the interactions between nanostructures and cellular responses. Electron beam lithography (EBL) as a top-down approach has been considered as one of the most powerful processes to fabricate and control nano-scale patterns. In this work, we fabricate Ti-based nanowires based on both top-down and bottom-up approaches using EBL and vapor-liquid-solid (VLS) method for the nanoscale resolution control of the nanowires. The size and the position of TiO2 nanowire arrays are controlled by EBL. Au-nanodot arrays are patterned on a substrate by the EBL as a seed pattern of TiO2 nanowire arrays. The nanodot arrays play an important role for catalysts using VLS method as bottom-up approach. In order to control the spacing between nanodots, we optimize the EBL process with Poly(methyl methacrylate) (PMMA) as an electron beam resist. Metal lift-off is used to transfer the spacing-controlled nanodots. The sample is then placed in a tube furnace and heated at a synthesis temperature of 850 °C. After the heat-treatment, TiO2 nanowire arrays grow from the nanodots through the VLS method. The controlled growth of TiO2 will be used to the study of interactions between nanostructures and cellular responses. We will examine the cellular response of osteoblasts as a function of the size and spacing of nanowires.
Acknowledgements
This research was supported by the MSIP(Ministry of Science, ICT and Future Planning), Korea, under the “IT Consilience Creative Program” (NIPA-2014-H0201-14-1002) supervised by the NIPA(National IT Industry Promotion Agency)
9:00 AM - H4.05
Generating and Trapping Device for Size-Controlled Microbubbles Using Patterned Carbon Nanotubes
Hiroshi Nishimura 1 Kaori Hirahara 3 2
1Osaka University Suita Japan2Osaka University Suita Japan3Osaka University Suita Japan
Show AbstractThe extraordinary electrochemical properties of micrometer and nanometer sized fine bubbles, which are so-called microbubbles and nanobubbles, respectively, make them attractive for application in medical science and agriculture fields. Recently many application studies, such as bioactivation, clinical tests for bactericidal activities and wound cleaning, cleansing of seawater pipes, and so on., are already well underway, showing its potential. In addition, a method for trapping individual protein molecules using the gas-liquid interfaces on bubbles is also being developed. For promoting further applications utilizing such fine bubbles, detailed fundamental understanding is essential, especially regarding the correlation between size-dependent characteristics and resulting effects. Here we propose a novel electrochemical device using carbon nanotubes (CNTs), which can generate microbubbles with a uniform size and trap them individually to the specific addresses of the device. This device has a potential to enable us to single bubble level investigations with well-controlled sizes. It will also applicable to the manipulation or trapping of protein molecules and cells for their fundamental studies at single molecular or single cell level.
The devices proposed in this study employ patterned CNTs as the electrode for bubble generation by water electrolysis. Hole-patterned Fe catalyst was firstly fabricated at a pitch of 30#65374;200mu;m on a conductive Si substrate by photolithography process. Surface of the substrate at individual holes were insulated by 20nm thickness layer. Patterned CNTs were then fabricated on this substrate by chemical vapor deposition. After wiring the substrate to facilitate electricity from a power source, the electrode configuration was finally obtained in which only patterned CNTs exposed by insulating around the Si substrate and conductive wire.
Water electrolysis was conducted in a 10% NaOH aqueous solution using the fabricated electrode as a cathode. We found that hydrogen microbubbles generated on individual holes with a uniform size, at an applied voltage of 5 V. The experimental results showed that the bubble size can be controlled precisely by the reaction time and variation in the applied voltage. Furthermore, the bubbles still persist even after cutting off voltage. It demonstrates that the device can also function as a reservoir for bubbles with fixed diameter. Minimum diameter of the bubbles depended on the size of CNT hole pattern. It is expected to be possible to operate smaller bubbles less than 30mu;m in further study, by developing the CNT electrode with finer patterning.
This work was supported by JSPS KAKENHI Grant No. 26286024. Photolithography process was supported by the Nanofabrication Platform in Nanotechnology Open Facilities, Osaka University.
9:00 AM - H4.06
Use of Selenium Nanoparticles to Treat Head and Neck Squamous Cell Carcinoma (HNSCC)
Christopher Edward Hassan 1
1Northeastern University Boston USA
Show AbstractSelenium nanoparticles have been found to protect cells with normal p53 genes from cancer medications such as cisplatin and paclitaxel, while simultaneously leaving cells with mutant p53 proteins vulnerable to such drugs. This has allowed for higher doses than would otherwise be safe to administer. These observations will be applied to head and neck squamous cell carcinoma (HNSCC) cells. The protective selectivity of elemental selenium nanoparticles on these cells, as well as the ambient mucus which can also carry the mutated p53 protein, will be analyzed. Selenium functionalized with PEG will also be explored as a possible option. Additionally, the mechanism for converting glutathione and selenite to glutathione disulfide and elemental selenium will be analyzed.
9:00 AM - H4.07
Dextran Coated Cerium Oxide Nanoparticles as Antioxidants
Ece Alpaslan 1 Amit K. Roy 1 Thomas J. Webster 2
1Northestern University Boston USA2Northestern University Boston USA
Show AbstractThe presence of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) contribute to the progression of several human diseases. In clinical applications, antioxidants as Vitamin C and E and other free radical scavengers have only achieved limited success due to a lack of targeted delivery. However, recent advancements in nanotechnology have enabled targeted delivery, as well as the elimination and control of oxidation reactions, as smaller sizes induce greater oxidative stress. Some of the most studied nanomaterials, which may act as potential antioxidants, are rare earth oxide nanoparticles, fullerenes and carbon nanotubes. With recent reports on cerium oxide nanoparticles (CNP) being neuroprotective, radioprotective, and anti-inflammatory, CNP may be a novel potential free radical scavenger. Considering these facts, CNP may promote cell survival under oxidative stress to limit ROS and RNS damage.
The objective of this study was to determine whether human dermal fibroblast (HDF) cells are able to recover from cytotoxic reagents as hydrogen peroxide (H2O2) or hydroquinone (HQY) in the presence of CNP.
Sub 5 nm dextran coated CNPs were synthesized from 5 mL aqueous solutions of 1 M cerium nitrate (Sigma Aldrich, St Louis, MO) and 10 mL of 0.1 M dextran T-10 (Pharmacosmos, Holback, Denmark) and these solutions were added drop wise to 30 mL of a 30% ammonium hydroxide (Sigma Aldrich, St. Louis, MO) solution while stirring for 24 hours at 25 omicron;C.
TEM results revealed that the particles were around 3 nm and Dynamic Light Scattering results confirmed that there was no significant change in the size of the particles after storing them for a month.
Cell proliferation was assessed using MTS kit per the manufacturer&’s instructions. HDF (ATCC® PCS-201-010trade;) cells were cultured using DMEM (ATCC® 30-2003trade;), 10% FBS (ATCC® SCRR-30-2020trade;) and a 1% penicillin-streptomycin solution (ATCC® 30-2300trade;). The cells were seeded at 5,000 cells /well, allowed to adhere for 24 hours and the following day, the culture was treated with cytotoxic agents like H2O2 or HQY with the concentration range from 100 to 800µM. After 24hrs of incubation, the MTS reagent was added and was determined. A dose of 400µM for both cytotoxic agents was found effective in killing 50% of the cells. In order to determine the cyto-protective function of CNP, some of these cells were preincubated with ceria at 500 µg/mL concentration for 24hrs followed by the addition of cytotoxic agents. The culture was incubated for 24hrs before MTS assays. Results were compared with only 400 µM of H2O2 and HQY, but not CNP treated cells.
Sub 5 nm dextran coated CNP were synthesized and cultured with HDF cells, which were then treated by cytotoxic reagents. The results showed that CNP treated cells were able to recover from the oxidative damage /cytotoxicity exerted by the drugs, suggesting that CNP may act as antioxidants within the body.
9:00 AM - H4.08
A Mucus- and Biofilm-Resistant Modification on Endotracheal Tubes
Jun Li 1 Daniel Donahue 1 Gregory Brotske 1 Phu Nguyen 1 Michael Bouchard 1 Zheng Zhang 1
1Teleflex Medical Cambridge USA
Show AbstractMucus accumulation on medical devices such as the endotracheal tubes (ETTs) can cause serious occlusion and infection issues. Our research focused on understanding the interaction between mucin and different types of surfaces, and led to the development of a betaine-modified PVC substrate that can resist both mucus/sputum accumulation and bacterial attachment. A full-sized ETT tube was modified and exhibited a significant mucus reduction in an animal model.
Both hydrophobic and hydrophilic polymeric substrates were prepared, with their contact angles ranging from nearly zero to 130o. PVC surfaces were applied with positive, negative ,zwitterionic, and uncharged modifications. It was found that some highly hydrophobic and highly hydrophilic surfaces can resist mucus adsorption. The surfaces of betaine-modified, full size, PVC ETT exhibited a 48-65% reduction in mucus attachment in vitro, as measured by radiolabeled porcine mucin. The betaine-modified PVC ETT surfaces also reduced the attachment of S. aureus (MRSA) and C. albicans by nearly 99% and reduced P.aeruginosa by 76%. A process was developed to modify a full-sized ETT on both outside and inside surfaces. In a 5-hour sheep study betaine-modified ETTs showed a significant reduction in mucus/sputum attachment.
9:00 AM - H4.09
Microtopographic Control of Nanostructure Self-Assembly
Laith Kadem 1 Julia Purtov 1 Constanze Lamprecht 1 Christine Selhuber-Unkel 1
1Christian-Albrechts-University of Kiel Kiel Germany
Show AbstractHere we report the use of diblock-copolymer micelle nanolithography (BCML) to create gold nanoparticle arrays on microtopographic substrates. BCML is a widely used technique to generate quasi-hexagonally arranged patterns of metal nanoparticles on surfaces. Lateral spacing between nanoparticles is controlled by the molecular weight of the diblock copolymers, polymer concentration and surface coating parameters. Application to microtopographic substrates results in a superposition of nano- and microstructures, where particle nanospacings are additionally controlled by the microtopography. Our simple self-assembly method yields neighboring microdomains that display different interparticle nanospacings on a single substrate by a single BMCL step. It therefore provides a novel and high-throughput strategy to generate micro-nanostructured surfaces. Furthermore, these micro-nanostructured surfaces are of high interest in cell biology, as both nanostructures and microstructures are known to control cell behavior. In particular we will use this new platform to study cellular adhesion processes that are influenced by parameters such as ligand spacing and density.
9:00 AM - H4.10
The Development of Screw-Sense Responsive Fluorescent Probes
Francis Lister 1 Jonathan Clayden 1
1University of Manchester Manchester United Kingdom
Show AbstractIntroduction
The controlled inversion between a left-handed (M) and right-handed (P) screw-sense preference of an achiral helix can create a binary switch with a signal relay achievable over the length of the helix.1
Peptides of achiral α-aminoisobutyric acid have proven to be effective at relaying stereochemical information from one terminus to the other via control of their screw sense preference.2 The helical fidelity of (Aib)n helices has proven to be exceptionally high,3 with a recent publication highlighting their use in the control of an achiral reaction site 60 bonds away from the nearest chiral centre.4 This equates to a chiral relay through an achiral helix over a distance of 4 nm, roughly the thickness of a cell membrane.
Other recent work, which has made use of diastereotopic NMR probes 5 has highlighted the potential use of these (Aib)n helices in the development of a synthetic analogue of a G-protein coupled receptor,6 which transmits a signal through a conformational change in its trans-membrane domain.
While successful, the developed NMR probes are incompatible with a membrane environment and novel probe types are required to develop these systems further. Fluorescence techniques are highly sensitive, non-invasive and compatible with the translucent membrane.
The central aim of this project is to develop a chiral fluorescent probe using two pyrene chromophores that responds to changes in helical environment with a change in the excimer/monomer (E/M) ratio observed in the pyrene fluorescence region.7
Discussion
A variety of probe structures containing two pyrene units were synthesised. These were attached to the C-terminus of N-terminally Cbz-L-Phe or Cbz-D-Phe controlled (Aib)4 helices, which have opposing screw-sense preferences. Successful probes were identified as those exhibiting different E/M ratios for the L- and the D- controlled helices.
The earlier designs of the probe structures were found to overpower the control delivered by the Phe residue when attached to the (Aib)4 scaffolds. The most recent generation of probes however exhibit both reduced control and an observable difference in E/M ratio when comparing the L-Phe and D-Phe controlled species.
Conclusion
It would appear that the development of these probes, which need to be enantiomerically pure to respond to changes in helicity, centres around the fine balance of control from both termini. Work is on-going to investigate new probe designs, looking particularly at ways to reduce control and improve probe response.
References
1) Clayden, J. et al., Nature (London), 2004, 431, 966-971
2) Solagrave;, J. et al., J. Am. Chem. Soc., 2011, 133, 3712-3715.
3) Clayden, J. et al., Angew. Chemie Int. Ed. 2009, 48, 5962-5965.
4) Byrne, L. et al., Angew. Chemie Int. Ed. 2014 53, 151-155.
5) Brown, R. A. et al., Nat. Chem., 2013, 5, 853-860.
6) Rasmussen S. G. F. et al., Nature, 2011, 477, 549-555.
7) Bains G. et al., Molecules, 2011, 16, 7909-7935.
9:00 AM - H4.11
Reconfigurable Photonic Microcapsules for Building Blocks of Photonic Devices
Shin-Hyun Kim 1 Tae Min Choi 1 Jin-Gyu Park 2 Vinnothan Manoharan 2
1KAIST Daejeon Korea (the Republic of)2Harvard University Cambridge USA
Show AbstractColloidal crystals possess photonic stop band and exhibit selective reflection of light at the band wavelength. This is promising properties for many photonic applications. For example, the colloidal crystals can be used for structural color pigments and ultraviolet- or infrared-screening materials depending on position of the stop band. However, most colloidal crystals have been prepared in a film form on solid substrate, which severely limits the ease of further processing and structural reconfigurability. Although emulsion drops have been used as templates to produce closely packed spherical crystals, the resultant structures are still not reconfigurable and the dynamic control is lost.
One way to overcome such limitations is to encapsulate crystalline colloidal arrays in flexible microcapsules. High mobility of colloids in the liquid core of microcapsule allows their rapid rearrangement and flexible membrane enables the deformation of photonic microcapsules into desired shape. To achieve this, we prepare double-emulsion drops with a capillary microfluidic device and use them as templates to produce microcapsules. An aqueous suspension of polystyrene particles is used to form an innermost phase of the double-emulsion drops, whereas monomer resin, containing small amount of photoinitiator, is used to form a middle phase. The polystyrene particles in the aqueous core are negatively charged, which provides electrostatic repulsion between particles, leading to the crystallization of the particles into face-centered cubic (fcc) lattice. During crystallization, (111) plane of fcc lattice, a hexagonal array of colloids, is formed along the spherical inner wall and this provides rotation-independent stop band. The monomer resin is solidified into a highly flexible membrane by in-situ photo-polymerization, making the photonic structure highly stable and reconfigurable. The resultant photonic microcapsules can be employed as building block to construct various photonic devices. For example, the microcapsules are compressed between two parallel glass slides and densely packed to form a 2D photonic sheet with negligible voids; each microcapsule is deformed to a hexagonal disk. Upon the compression, particles in the capsules rearrange by aligning their (111) plane along the inner surface of the deformed membrane. Therefore, the sheet, composed of arrays of #64258;attened microcapsules, exhibits uniform re#64258;ection along whole the surface. The photonic microcapsules can be further assembled to make photonic structures with 3D shape, which is otherwise difficult to achieve. High reconfigurability, ease of processing, and potential controllability of stop band position of photonic microcapsules will provide new opportunity as building blocks for practical photonic devices.
9:00 AM - H4.12
The Use of MRI Technology for Studying the Adhesion of Microparticles
Nina Sarvasova 1
1Institute of Chemical Technology, Prague Prague Czech Republic
Show AbstractIn every development of functional particles, there is an important part consisting of the study of their behaviour in conditions simulating their end use. For such measurements, there are several methods available, though only few of them can yield any results without damaging or irreversibly changing the studied material. Magnetic Resonance Imaging (MRI) is a method widely acknowledged as a suitable one for such purposes, as it is non-destructive, non-invasive and provides the opportunity to observe the experiment in “real-time” arrangement. In this work, MRI was used as a means to observe the behaviour, mainly adhesion, of selected microparticles in 3D differentiated media.
As a part of this work, porous media with different pore sizes were created from Polydimethylsiloxane (PDMS) using solid template method. Each of the porous layers was placed within the flow cell connected to the peristaltic pump enabling the flow through the system. Thus, such setting allowed for both, stationary and flow measurements in the MRI scanner. Resulting scans were evaluated and processed using graphic software ImageJ. Regarding the microparticles used in this work, two types of composite particles containing magnetic nanoparticles were used, one with the size around 1.5 mm and the other with the size of approximately 100 mm. Magnetic nanoparticles contained in composite microparticles belonged to the crucial requirements for these experiments, since it acted as a contrast agent thus allowing for particle observation in MRI scans. In addition, the loss of the particles after the experiment was estimated indirectly using UV/VIS spectroscopy as the loss of Fe3+ in the circulating solution.
In summary, 3D complex PDMS media were created with the porosity estimated using MRI. These were successfully used in the flow experiments to study the adhesion of SiO2/PNIPAM/Fe3O4 and Alginate/SiO2/Fe3O4 microparticles. In both cases, there was some adhesion observed and it was also backed up with the concentration loss of Fe3+ ions in inlet and outlet streams during the experiments.
9:00 AM - H4.13
Acoustic Levitation for Neural Tissue Engineering
Charlene Bouyer 1 2 3 Pu Chen 1 Utkan Demirci 1
1School of Medicine, Stanford University Palo Alto USA2Inserm Lyon France3Universitamp;#233; Lyon 1 Lyon France
Show AbstractEngineering of multilayered cell sheets is motivated by the needs to understand cell-cell communication and to reconstruct brain tissues in vivo. For example, engineering of cortex tissue is demanded for broad applications ranging from understanding of neuron mapping to brain drug discovery. Current technologies for generation of cortex tissue are based on fabrication and assembly of neuron-encapsulating hydrogels. However, these methods are limited by problems including large time budget, incapability for manipulating large amount of cells and low cell viability. Here, we demonstrate an acoustofluidic technology platform allowing fabrication of bulk hydrogels containing multilayers of neurons in a rapid and cost-effective manner. As an initial concept validation, we illustrated levitation of microbeads with densities from 1.025 g/cm3 to 1.13 g/cm3 and sizes from 8 µm to 500 µm, in fluid densities from 1 g/cm3 to 1.205 g/cm3. We then explored acoustic standing waves for trapping cells in either negative or positive pressure regions of acoustic standing waves according to their acoustic impedance. Multilayered cell sheets were created in the acoustic waves in less than 10 seconds. The engineered cortex included 6 layers of neurons surrounding by soma cells. The levitated cell structures is stabilized by rapid UV crosslinking of hydrogel within 30 seconds. Cells viability assays indicated cytocompatibility of this method. We expect that these in vitro engineered artificial cortex can be used as an alternative choice for brain slides for neuroscience and brain study.
9:00 AM - H4.14
Barcoding Cells Using Magnetic Nanowires for Multiplexed Detection
Anirudh Sharma 1 Gregory M Orlowski 2 Seung Yeon Kim 3 Allison Hubel 4 Bethanie J.H. Stadler 1
1University of Minnesota Minneapolis USA2UMass Medical School Worcester USA3Georgia Institute of Technology Atlanta USA4University of Minnesota Minneapolis USA
Show AbstractMagnetic multi-layered barcode nanowires composed of gold/nickel multilayers with various surface coatings were used as agents for multiplexed detection in a heterogeneous cell population. Cell sorting and identification using magnetic beads, quantum dots and fluorescent probes is limited by single antibody-labeled magnetic beads or by the number of spectrally resolvable fluorophores (wavelengths). The ability of barcode nanowires to label populations of cells with unique magnetic signatures would enable identification and separation of multiple cell populations. Here, selective targeting of A549 human lung carcinoma and human THP-1 cells (monocytic leukemia) was followed by magnetic detection and demultiplexing using first order reversal curve diagrams (FORC diagrams) and coercivity values obtained from magnetic hysteresis curves through Vibrating Sample Magnetometer (VSM).Various imaging techniques will be shown to illustrate the barcoding concept when applied to a heterogeneous cell population - including differential interference contrast (DIC), reflectance and fluorescence. A combination of these techniques and SEM/TEM imaging was used to study dependence of cellular uptake on nanowire concentrations, lengths and surface coatings. Toxicity analysis included exposing peritoneal macrophages from C57 Bl/6 mice to the barcodes nanowires and then monitoring IL-1b and TNF-α levels, and metabolic activity (MTS assay). Almost no cell death was induced and minimal quantities of IL-1b and TNF-α were induced over a six hour incubation period. In short, barcode nanowires hold much promise for multiplexed diagnosis and therapy.
9:00 AM - H4.15
Decarboxylation and the Following Adduct to Alkenes Using TiO2 Photocatalyst
Kento Shimaoka 1 Yuki Taleda 1 Makoto Yamashita 1 Shota Kuwahara 1 Kenji Katayama 1
1Chuo university Tokyo Japan
Show AbstractCarboxylic acids are abundant in nature and are also produced industrially on a large scale, and organic reactions employing them have received much attention. Recently, several decarboxylation reactions using carboxylic acids have been reported using photosensitizers, which utilize a photoinduced single electron transfer [1]. Although this reaction is proceeded under a mild condition, it is difficult to remove the photosensitizers from the solution after the reaction. On the one hand, semiconductor photocatalyst has attracted attention because it is nontoxic and it can be easily separated from the solution. We have developed various photocatalytic reactions using microreactors inside which titanium oxide was coated as a photocatalyst. By using this reactor, we could proceeds photocatalytic reactions without any electric power sources by using the capillary force and diffusion of chemicals driven by the concentration gradient. [2] In this study, we applied decarboxylation reactions by using the photocatalytic microreactor instead of the molecular photosensitizers. Capillaries coated with a photocatalytic material were put into a test tube, where 1 mL of a reactant solution was added and UV-LED (365 nm) were iiradiated for photoreaction. As a result, we could successfully observe the decarboxylation reactions of carboxylic acids, which was confirmed from the dimer molecules generated from the radicals. Furthermore, the adduct reactions of the radicals generated from the carboxylic acids to various electron-deficient alkene were confirmed. [1]Y. Yoshimi, T. Itou, M, Hatanaka, Chem. Commun. 2007, 5244-5246 [2] K. Katayama, Y. Takeda, K. Kuwabara, Chem. Commun, 48, 7368-7370 (2012)
9:00 AM - H4.16
Mechanically Stimulated Release from Layered Drug Delivery Systems with Electrosprayed Micro-/ Nano- Superhydrophobic Coatings
Julia Wang 1 Jonah A Kaplan 1 Yolonda L Colson 2 Mark W Grinstaff 1
1Boston University Boston USA2Brigham and Women's Hospital Boston USA
Show AbstractTissue expanders are medical devices commonly used to grow autologous soft tissue for transplantation to eliminate tissue rejection and minimize scarring. For the over 2.9 million women in the US living with breast cancer, the primary course of treatment is removal of the tumor by surgery and subsequent breast reconstruction with tissue expanders, during which saline is injected every week in order to provide space for a permanent breast implant. However, the success of tissue expansion is compromised by increased fibrosis from necessary radiation therapy for reduced cancer recurrence. In order to provide better breast reconstruction after radiation therapy, we have incorporated the strain mechanics of the tissue expander to develop a biocompatible, multi-layered system that delivers cytokines and drugs based on applied strain. In the absence of tensile strain, the electrosprayed superhydrophobic barrier coating (contact angle >167°) consisting of interconnected micro- and nano-sized particles of biocompatible polymers, poly(ε-caprolactone) [PCL] and poly(glycerol stearate-co-ε-caprolactone), maintains a stable air layer to prevent drug release from the core. The absorbent core (polyester/cellulose) is tolerant of a variety of solvents, allowing versatile drug loading. When tension is applied, the mechanical mismatch between the coating and core material leads to strain-dependent crack propagation of these coatings, allowing water to infiltrate and subsequently release adsorbed agents. We demonstrate strain-dependent release (ε = 0%, 30%, or 100%) of Yellow 5/ Blue 1 and FITC-BSA in phosphate buffered saline and serum, and apply this system to the strain-dependent release of cisplatin (cytotoxic) and SN-38 (cytostatic). This delivery system is further evaluated with 3T3 fibroblasts, where strain-dependent amounts of TNF-α is delivered to decrease collagen production. Incorporating the mechanics of the tissue expander to trigger drug and cytokine release from this strain-dependent drug delivery system may improve breast reconstruction after radiation therapy while providing a continued reduction in cancer recurrence. The ease in fabricating these coatings and versatility in core loading affords a simple, tunable mechanoresponsive drug delivery system to apply to medical devices, such as tissue expanders.
9:00 AM - H4.17
Microwave Synthesis and Application of N Doped Nano TiO2 for Aromatic Wastewater Treatment
Mahdi Fathizadeh 1
1Ilam University Ilam Iran (the Islamic Republic of)
Show AbstractTiO2, as a photocatalyst, has been extensively used for wastewater treatment and air purification. Recently, many researches have been done aiming to improve the efficiency of photocatalysts under visible light by modification of TiO2. The N-doped TiO2 especially shows a significant photocatalytic activity under visible light irradiation. Two different methods are used to dope nitrogen in TiO2; the first one is sol-gel synthesis and the second one is physical method (ion implantation, magnetron sputtering). In this work, the microwave heating methods was used for fabrication of the N-doped TiO2. Then, the wastewater was treated by N-doped TiO2 under the visible light.
First, the microemulsion phase was prepared by adding aqueous phase (5 mol/L of tetrabutyltitanate in nitric acid) into oil phase (the mixture of Triton X-100, 1-hexanol, cyclohexane) with Urea as nitrogen source. Then, the mixture was transferred to a 250 mL polyethylene autoclave. Heat treatments were done at temperatures between 60 to 120°C and duration of 45 to 75 min. After the microwave heat treatment, the mixture was cooled down to room temperature and washed with deionized water several times to remove the oil from N-doped nano TiO2 powder. The photcatalytic property of N-doped nano TiO2 was tested by its efficiency in elimination of aromatic compounds in wastewater. A 1000 W halogen lamp with glass optical filter as sun light was used in the 250 mL batch reactor which was cooled by water circulation though jacket side.
The XRD patterns of synthesized N-doped nano TiO2 showed that 60 °C is not suitable for the synthesis. The anatase phase has been appeared without any phase change after nitrogen doping using microwave heating at 60 0C and 75 min. Increasing the microwave heating temperature caused peak broadening for N-TiO2 samples compared to the undoped ones. The distribution (PDI) and particle size of N-doped nano TiO2 were measured by DLS which showed increasing the temperature from 60 to 120 °C raise both the particles size and PDI. Increasing the exposure time amplified the particle size with PDI remaining unchanged at a constant temperature. As can be seen from the SEM images at 90 0C and 75 min, the N-doped TiO2 sample has uniform nanoparticles with average diameter of 25-30 nm.
XPS analysis was done to examine the states of the doped nitrogen samples. There are Ti, O, and N elements on the surface of the samples with different peak intensities. The optical absorption range of the doped samples was shifted to the lower energy region, from 380 to 500 nm.
The results of aromatic decomposition showed that the optimal heat treatment condition is at 90 0C and 75 min. At the highest temperature and time, the potocatalytic activity of N-doped nano TiO2decreased as a result of an increase in the ratio of Ti to N suggesting that when the mole ratio of Ti to N is higher than the optimal value, the photocatalytic activities will decrease rapidly under visible light.
9:00 AM - H4.18
Inorganic-Derived Iron Oxide/Silica Nanoparticles for Magnetic Isolation of Nucleic Acid
Valentin Natarov 1 Dzmitry Kotsikau 1 Vladimir Survilo 2 Vladimir Pankov 1
1Belarusian State University Minsk Belarus2Ltd. "Vega", Group of Companies "Alcor Bio" St. Petersburg Russian Federation
Show AbstractIn recent 10 years, there has been an intense interest in the preparation of modified magnetic nanoparticles for biomedical applications. Magnetic separation is a well-proven method for isolation of nucleic acids from biological samples. Facile synthesis of iron oxide/silica nanoparticles via non-expensive inorganic precursors is presented in this paper.
The synthesis is based on a three-step process, involving i) synthesis of magnetite nanoparticles by combined hydrolysis of Fe2+ and Fe3+ salts with aqueous ammonia at room temperature; ii) silica sol formation by the addition of necessary quantity of hydrochloric acid into sodium silicate solution containing the magnetite nanoparticles; iii) oxidation of the obtained Fe3O4#8210;SiO2 mixed sol with water solution of hydrogen peroxide. Size, shape and colloidal stability of the nanoparticles were found to be strongly dependent on various processes. In order to optimize the synthesis conditions, the effect of pH, nature of precursors, concentrations of solutions, steering intensity and temperature has been studied. All operations were carried out in water solutions under constant stirring without the addition of any stabilizers. In order to adjust the size of the agglomerates and to prevent their further aggregation, the particles were redispersed in physiologic saline.
Structural studies have been performed by XRD, TEM and IR-spectroscopy. Colloidal stability, magnetic properties, chemical composition, specific surface area and extraction efficiency of DNA molecules have been also studied.
The content of silica component in the prepared Fe3O4#8210;SiO2 composite was estimated by chemical analysis to be about 35-40 wt. %. According to XRD characterization of powdered samples, the composite consists of well-crystalline Fe3O4 nanoparticles mixed with X-ray amorphous silica. The occurrence of silica phase was proved by IR spectroscopy. The diameter of the obtained spherical nanoparticles varies from 40 to 80 nm for the Fe3O4#8210;SiO2 composite, and from 8 to 10 nm for the initial Fe3O4 grains.
Specific saturation magnetization of the powdered Fe3O4#8210;SiO2 sample was measured to be about 30-35 A#8729;m2kg-1. This value provides a complete and fast magnetic separation of the oxide particles from a solution using a standard magnetic holder.
The DNA isolation has been tested by a standard protocol in the presence of a chaotropic agent using salmon sperm DNA as a test sample. Extraction was measured by real-time PCR and found to be comparatively higher than efficiency of other commercially available nanoparticles. Thus, the synthesized magnetic nanoparticles meet the basic requirements as a component of DNA isolation kit.
9:00 AM - H4.19
Development of Multifunctional Silica Nanoparticles as Carrier for Cellular Delivery
Bhavana Deore 1 Michael L Barnes 1 Arnold Kell 1 Pankaj Bhowmik 2 Patricia Polowick 2
1National Research Council Ottawa Canada2National Research Council Saskatoon Canada
Show AbstractThe delivery of biomolecules into cells is crucial in modern biological systems. Most naked biomolecules are poorly delivered to cells owing to poor stability, potential toxicity and incompatible charges. Therefore, various natural and synthetic vectors are being investigated as cellular delivery vehicles. Silica nanocarriers are promising candidates for such cellular delivery due to their multifunctionality, stability, tunable size and charge and biocompatibility. Here, we present the synthesis and characterization of multifunctional luminescent silica nanoparticles and discuss their subsequent use as cell delivery vehicles and biomolecule sensors.
9:00 AM - H4.20
Conservative and Dissipative Interactions in Multimodal Force Microscopy Driving Multiple Higher Eigenmodes
Sangmin An 1 2 Christian J. Long 1 2 Vladimir Aksyuk 1 Santiago D. Solares 2 3
1National Institute of Standards and Technology Germantown USA2University of Maryland College Park USA3University of Maryland College Park USA
Show AbstractIn multifrequency atomic force microscopy (AFM) the cantilever probe is often excited at two or more resonance frequencies simultaneously and the respective dynamic responses are detected and analyzed, enabling simultaneous measurement of multiple surface properties. While the large-amplitude oscillation of the first eigenmode is used for topographical measurement, the smaller-amplitude oscillation of the higher eigenmodes is used for mapping surface stiffness, average dissipative behavior or other surface properties during the scan [1]. Recently, a trimodal AFM technique has been developed, which uses two higher eigenmodes (for example, the second and third) for compositional mapping and subsurface visualization of soft matter, respectively [2]. More recently a 4-eigenmode multimodal technique has been introduced [S.D. Solares, S. An and C.J. Long, “Multifrequency Tapping-Mode Atomic Force Microscopy Beyond Three Eigenmodes in Ambient Air,” submitted]. In this presentation we describe the dynamics of multimodal AFM based on experimental and computational results, focusing on the variation of the conservative and dissipative interactions during imaging and spectroscopy as the free oscillation amplitude of individual eigenmodes changes with respect to the rest of the active eigenmodes. We quantify the conservative and dissipative interactions for each eigenmode in terms of its respective virial and average dissipative power per oscillation cycle, in order to extract compositional information at different measurement frequencies. This type of multimodal characterization opens new avenues for more comprehensive material property measurement than what is currently possible.
< References >
[1] R. Garcia and E.T. Herruzo, Nat. Nanotech.7, 217 (2012)
[2] D. Ebeling, B. Eslami and S.D. Solares, ACS Nano7, 10387 (2013)
9:00 AM - H4.21
Microfabrication of Controllable Submicron Period Structures Employing Direct Femtosecond Laser Holographic Lithography System
Tomas Tamulevicius 1 Tadas Kudrius 2 Vytautas Stockus 2 Gintas Slekys 2 Dainius Virganavicius 1 Linas Simatonis 1 Asta Tamuleviciene 1 Ausrine Jurkeviciute 1 Nerijus Armakavicius 1 Sigitas Tamulevicius 1
1Kaunas University of Technology Kaunas Lithuania2Altechna Ramp;D Vilnius Lithuania
Show AbstractUltrafast pulsed laser holographic lithography (HL) setup for controllable period direct patterning employing a custom made diffraction optical element (DOE) has been assembled, tested and applied for production of different submicron period microstructures. Controllable period direct patterning HL system was designed in such a way that the laser beam was divided into two equal intensity beams that were collimated and then focused employing two lens 4f imaging system on the sample placed on a computer controlled XYZ translation stage. Second harmonic beam from the 1030 nm wavelength Yb:KGW femtosecond laser (Light Conversion) and XYZ translation stage (Aerotech) were employed. The DOE based beam splitter was designed from the set of 15 different period diffraction gratings arranged in one circle. In this DOE each grating diffracted the laser beam at different angles. Two beams imaged by the lens system were merged on the sample surface at different angles producing different period interference fringes. The setup was applied for machining of different type of microstructures including photomasks in chromium and iron oxide films on a glass substrate, point by point direct diffractive metallic surface inscription, direct laser ablation of diamond like carbon as well as silver - diamond like carbon (Ag:DLC) based nanocomposite thin films and submicron biosensor chips on fused silica. 1-D periodic structures with variable period were produced employing interference of the second harmonic (515 nm) of femtosecond laser. The morphology of periodic structures was analyzed by scanning electron microscopy and atomic force microscopy, mapping of the surface composition was done by energy dispersive x-ray spectroscopy, optical properties were studied by registering far field distribution of the diffracted light by the structures. Typical applications of such structures as templates in capillary assisted self assembly of nanoparticles as well leaky wave biosensor are provided. Reflection spectra as well as spectral composition of the resonant guided mode were registered in case of produced DLC and Ag:DLC nanocomposite gratings on fused silica. It is shown that high throughput submicron patterning technique is efficient tool in production of periodic structures in different material including biocompatible and chemically inert material such as diamond like carbon. Moreover it is demonstrated that direct HL laser ablation technique enables production of periodic structures in nanocomposite materials avoiding surface segregation processes that is typical for plasma based technologies. This kind of submicron structures in nanocomposites, combining resonant plasmonic properties of the nanoparticles as well as resonant response of the periodic structure, presents a novel type of biosensors operating in different liquid analytes without any functionalization of the surface.
9:00 AM - H4.22
Wireless Neural Excitation via Transcranial Magnetothermal Stimulation
Ritchie Chen 1 Gabriela Romero 1 Michael Christiansen 1 Polina Anikeeva 1
1MIT Cambridge USA
Show AbstractCurrent technologies to stimulate intact brain circuits suffer from several limitations. For example, pharmacological agents lack specificity and lead to unwanted side effects, while electrical stimulation is highly invasive. Herein, we demonstrate that wireless neural excitation can be evoked by remotely controlled heat dissipation via magnetic nanoparticle (MNP) transducers. By sensitizing cells to heat through the expression of the thermosensitive cation channel TRPV1, robust neural excitation is demonstrated in close proximity to ferrofluids in vitro and in vivo . We find that trains of action potentials can be controlled with repeated thermal cycles, and that subpopulations of neurons can be stimulated in deep brain structures. Our MNPs, optimized to dissipate heat efficiently and to elicit low immunoreactive response, may potentially serve as a new paradigm to achieve deep brain stimulation.
9:00 AM - H4.23
Alignment of Collagen Fiber in Pneumatic Soft Micromold (PSMM) Device
Po-Jung Huang 1 Jun Kameoka 2 1 Alvin Yeh 3
1Texas Aamp;M University College Station USA2Texas Aamp;M University College Station USA3Texas Aamp;M University College Station USA
Show AbstractWe have demonstrated the fabrication of fiber oriented collagen microparitlces by pneumatically actuated soft micro-mold (SMM) device. Collagen fiber alignment has been considerably important recently in tissue engineering because fiber scaforld in real tissue, including tendon, bone, blood vessels, and skin are aligned to constract the specific shapes and structures. Collagen orientation also plays a crucial role in cell proliferation, migration, and tumor metastasis and they are highly demanded by may fields. Currently, strain-induction, magnetical, electrochemical and microfluidic approach are reported to orient collagen fibers for generating artificial tissue. However, the collagen orientation established by these approaches are highly limited, complicated and far from the practical level to generate specific shape of tissue. Therefore, we present the SMM device approach to orient collagen fibers in microparticles that pneumatically deform the template and create force inside micropatterns at which collagen fibers are orientated along the direction of the expansion of mold. Second Harmonic Generation (SHG) is the useful tool to investigate the alignment index (AI) of collagen fiber in microparticles. Alignment index (AI) is the range from 1.0 for random alignment to 4.55 for strong alignment. The alignment index of fibers in microparticles made by the SMM device increases 40%.
9:00 AM - H4.24
Energetics of Formation and Hydration of Functionalized Silica Nanoparticles: An Atomistic Computational Study
Vagner Alexandre Rigo 1 Lucas Stori de Lara 2 Caetano Rodrigues Miranda 3
1Universidade Tecnolamp;#243;gica Federal do Paranamp;#225; (UTFPR) Cornamp;#233;lio Procamp;#243;pio Brazil2Universidade Estadual de Ponta Grossa Ponta Grossa Brazil3Universidade Federal do ABC Santo Andramp;#233; Brazil
Show AbstractSilica based nanomaterials have received great attention for nanomedicine and biological applications [1] due to their biocompatible properties [2]. SiO2 nanoparticles can be used for drug delivery and for applications in imaging diagnostic [3]. In many of these applications, nanoparticles are suspended in a fluid and a common way to maintain the suspension consists to functionalize the nanoparticle with either hydrophobic [4] and hydrophilic [5] groups. The functional form, chain length [4], graft density and distribution of groups at surface can be tuned to kept the suspension and improve the nanoparticle functionality. Using a combination of First-Principles calculations based on Density Functional Theory with van der Waals dispersion correction and Molecular Dynamics, the energetics of formation and hydration of functionalized silica nanoparticles were studied. The energetics and effects of group density were evaluated in both; hydrophilic (ethylene-glycol) and hydrophobic (sulfonic) organosilane functional groups, and the optimum group density were obtained in vacuum and aqueous environment [6]. In vacuum, an optimum graft density of 4.2 and 4.5 groups/nm2 was obtained for hydrophobic and hydrophilic coverage, based on Molecular Dynamics calculations. Interestingly, a double well energy profile is obtained when functionalized nanoparticles are placed within aqueous media, and those minima for hydrophilic groups appear at lower coverage compared to hydrophobic one. The double energy minima is explained by the H2O molecules arrangement as function of the group density on nanoparticles surface.
References
[1] I.I. Slowing, J.L. Vivero-Escoto, C.-W. Wu, V.S.-Y. Lin, Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers, Adv.Drug.Deliv.Rev. 60, 1278-1288 (2008)
[2] V. Chin, B.E. Collins, M.J. Sailor, S.N. Bhatia, Compatibility of Primary Hepatocytes with Oxidized Nanoporous Silicon, Adv.Mater. 13, 1877-1880 (2001)
[3] M. Liong, J. Lu, M. Kovochich, T. Xia, S.G. Ruehm, A.E. Nel, F. Tamanoi, J.I. Zink, Multifunctional Inorganic Nanoparticles for Imaging, Targeting, and Drug Delivery, ACSNano 2, 889-896 (2008)
[4] Y-L. Lee, Z.-C. Du, W.-X. Lin, Y.-M. Yang, Monolayer behavior of silica particles at air/water interface: A comparison between chemical and physical modifications of surface, J. of Colloid and Interface Sci. 296, 233-241 (2006)
[5] Z. Zhang, A.E. Berns, S. Willbold, J. Buitenhuis, Synthesis of poly(ethylene glycol) (PEG)-grafted colloidal silica particles with improved stability in aqueous solvents, J. of Colloid and Interface Sci. 310, 446-455 (2007)
[6] V. A. Rigo, C.R. Miranda, L.S. de Lara, Energetics of formation and hydration of functionalized silica nanoparticles: an atomistic computational study, Appl. Surf. Sci. 292, 742-749 (2014)
9:00 AM - H4.25
Decreased Macrophage Responses on Nanoscale Hydroxyapatite: Experimental Results
Garima Bhardwaj 1 Thomas Jay Webster 1
1Northeastern University Boston USA
Show AbstractIntroduction: To assess the immunological response on implants, the host response to materials must be determined prior to implantation. This response is highly dominated by macrophages.Macrophages not only fuse to become multinucleated foreign body giant cells, but they also activate T lymphocytes by expressing co-stimulatory molecules (e.g., CD86 and CD80) and surface antigens (e.g., MHC II).This current study modified the surface of titanium by coating it with nanoscale hydroxyapatite, in a range of sizes, using electrophoretic deposition (EPD) with both AC and DC current. Macrophages were then seeded onto this surface and their behavior observed.
Materials and Methods: Nanoscale hydroxyapatite (HA) was synthesized using a wet chemical synthesis process to possess an array of different sizes in the nanometer scale.HA was then coated onto a titanium mesh purchased from Alpha Aesar (Catalog no.7440-32-6) by electrophoretic deposition with AC and DC current.Surface characterization was done using SEM,contact angle analysis and AFM. Macrophages purchased from ATCC (RAW 264.7 (ATCC® TIB-71trade;)) were cultured using EMEM (ATCC® 30-2003trade;), 10% FBS (ATCC® SCRR-30-2020trade;) and a 1% penicillin-streptomycin solution (ATCC® 30-2300trade;). Cell adhesion and proliferation was observed using MTS assays after 1, 3, 5 and 7 days. Levels of TNF-α, IL-1, IL-6 and nitrite released by the macrophages were studied. Bacterial assays also were conducted using Staphylococcus aureus (ATCC® 29740trade;) and Pseudomonas aeruginosa (ATCC® 39324trade;) strains of bacteria. 0.03% tryptic soy broth (TSB) and agar plates (Sigma-Aldrich) were used as the media.A dilution of 108 bacteria/mL was then prepared using 0.03% TSB and the samples were treated with 2 mL of the 108 bacteria/mL solution and incubated for 24 hours. The bacteria solution was removed and the samples were rinsed twice with PBS.The number of bacterial colonies formed on each sample was counted and using these values, the number of bacteria/mL was found.
Results and Discussion: Results of the present study demonstrated that the change in the HA surface topography affected the adhesion and proliferation of macrophages leading to a reduced activation of macrophages with increased nanometer HA roughness. The level of TNF-α, IL-1, IL-6 and nitrite released by macrophages decreased with increasing HA nanometer surface roughness. Bacterial activity also decreased with increased HA nanometer surface roughness.
Conclusions: Results of the present study demonstrated that the present coating procedure and its parameters, when used to synthesize nanometer HA roughness, reduced macrophage attachment and activation (TNF-α, IL-1, IL-6 and nitrite release). Bacterial activity also reduced on nanometer rough HA coatings.
Acknowledgements: The authors would like to thank Northeastern University for funding.
9:00 AM - H4.26
WITHDRAWN 11/29/2014 In Vitro and In Vivo Performance of an Antioxidant Coating for Chronic Neural Electrodes
Noah Robert Snyder 1 2 Jenna Hanner 1 Xinyan Tracy Cui 1 2 3
1University of Pittsburgh Pittsburgh USA2Carnegie Mellon Pittsburgh USA3University of Pittsburgh Pittsburgh USA
Show AbstractUnderstanding the direct mechanism behind neuronal loss near chronically implanted electrodes is essential for the development of treatment paradigms that can improve the abiotic/biotic interface. Recent research has demonstrated neuronal degeneration and inflammatory gliosis at the vicinity of neural electrodes. Gliosis, is characterized by the activation of glial cell types, both microglia and astrocytes, and results in the formation of glial scar tissue and the production of a variety of neurotoxic factors. Activated microglia secrete pro-inflammatory cytokines, initiate the recruitment of additional macrophages/microglia, and produce various cytotoxic factors including reactive oxygen/nitrogen species (RONS). Given the proximity of microglia to implanted electrodes, nearby produced RONS may directly result in an environment promoting neurodegeneration, especially considering that microglia up-regulate RONS when presented with insoluble promoters of activation. Through the enzymatic production of superoxide and nitric oxide by phagocytic NADPH oxidase (PHOX) and NO synthase, respectively, the oxidative stress produced by activated microglia results in neuron death. In recent years, the development of low molecular weight synthetic molecules that mimic the function of the endogenous protein radical scavenger superoxide dismutase (SOD) has provided potential pharmaceutical agents for treating oxidative stress. One SOD mimic (SODm) in particular, manganese(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin (MnIIITE-2-Pyp5+), has proven to be neuroprotective in in vitro models of Alzheimer&’s disease as well as in in vivo models of stroke. Furthermore, MnIIITE-2-Pyp5+ is an equally capable peroxynitrite scavenger, a byproduct of the superoxide and nitric oxide released by microglia making it an ideal compound for treatment of neural electrode probes. Functionalizing silicon-based neural probes by covalent attachment of biomolecules that promote neuronal adhesion or reduce inflammation has shown great promise. Therefore, we have developed a neural implant coating by the immobilization of a newly synthesized derivative of the SODm MnIIITE-2-Pyp5+. The new compound was characterized via NMR and surface chemistry following immobilization was verified with XPS. To test the efficacy of the coating, an immortalized microglia cell line was plated onto silicon substrates coated with the immobilized SODm. Following activation of the cells, decreases in several RONS were observed including superoxide, peroxynitrite, hydrogen peroxide, and nitric oxide. In vivo effectiveness of the coating is demonstrated by implanting silicon based probes with the SODm coating into the cortex of Sprague Dawley rats and examining neuron survival (neuronal density and cell death) as well as degree of inflammatory responses.
9:00 AM - H4.27
The Morphology Modification of Limestone by Different Forms of Carbon Additives for CO2 Sequestration
Binglu Meng 2 Hui Li 2 Jiangfeng Li 2 Youhai Yu 1 Dewei Wang 1 Yidong Liu 3 Yong Min 2 1 3
1Xiamp;#8217;an Institute of Optics and Precision Mechanics of Chinese Academy of Science Xian China2Xiamp;#8216;an University of Architecture and Technology Xian China3Nanjing University of Posts and Telecommunications Nanjing China
Show AbstractThe limestone was used to sequestrate the CO2 in cement industry had been widely reported. The efficiency was dramatically reduced after several cycles due to the “sintering” of limestone. Here we found that we can improve the CO2 sequestration capability of limestone by adding different carbon additives to modify the limestone morphology. We have compared the influence of different forms of carbon and found that we can improve the efficiency over 18% and 8% by adding carbon black and bamboo carbon respectively. However, it will decrease by adding layered graphite type materials such as, graphite and graphene. The experimental results were discussed and characterized by SEM, particle distribution, XRD, optical microscope, and its corresponding mechanism was proposed in the paper.
9:00 AM - H4.28
Antibacterial and Osteogenic Stem Cell Differentiation Properties of Photoinduced- TiO2 Nanoparticles Decorated TiO2 Nanotubes
Wenwen Liu 2 1 3 Thomas Webster 1
1Chemical Engineering MALDEN USA2School of Stomatology, Capital Medical University Beijing China3School of Materials Science and Engineering, Beijing University of Technology Beijing China
Show Abstract
Introduction:
To avoid antibacterial drug resistance, a good strategy is to reduce bacteria attachment and growth by modifying implant surface without relying on antibiotics. Thus, in order to build upon the success of TNT (titania nanotube) for orthopedic and dental applications, and to obtain a photosensitive material for antibacterial properties during and after implant surgery, in this study, a hydrothermal method was used to impregnate TiO2 nanoparticles into TNT (TNT- TiO2). Materials and methods
Pure titanium sheets (Aldrich, 10×10×0.3 mm3) were treated according to a previous study. [1] TiO2 nanoparticles were impregnated into the TNTs after hydrothermal treatment. All samples were treated with UV light (30W UV lamp, lambda;>=365nm, 3mW/cm2 at a distance of 5 cm) for 3h before biological testing.
The ability of the proposed materials to reduce bacterial growth was evaluated using Streptococcus mutans (S. mutans, UA159) and Porphyromonas gingivalis (P. gingivialis, ATCC33277). Bacterial colonies were counted after 2, 4, 6 and 8 days. The expression of S. mutans adhesion-associated genes (specifically, gtf B, C and D) were determined after 24 h of culture on different samples.
Six week old male SD rats were used to extract bone marrow for bone mesenchymal stem cells (BMSCs). BMSCs proliferation and ALP, Collagen-I (Col-I), Osteocalcin (OC) and Osterix (Osx) genes were determined following previously reported protocols.[2]
Result and discussion
Results of this study showed that for S. mutans and P. gingivalis, there were fewer bacteria on the TNT and TNT-TiO2 surfaces than pure Ti. For the gene expression assays, all of the three bacteria adhesion - associated genes were lower on the TNT and TNT-TiO2 samples than for pure Ti. For the stem cell proliferation studies, the cells on pure Ti had higher proliferation rates than TNT and TNT-TiO2. All the gene expression results demonstrate that TNT-TiO2 have better osteogenic properties than TNT and Ti.
Conclusions
TiO2 nanotubes were coated with uniform TiO2 nanoparticles. Base on the higher surface to volume ratio and photocatalytic activity of TiO2 nanoparticles, TNT- TiO2 had a higher antibacterial effect. For stem cell culture, due to the nanotopography of the TNT- TiO2 and its high surface energy, TNT- TiO2 showed improved cell compatibility.
References
1. Liu, W. et al. The synthesis of TiO2 nanotubes with ZnO nanoparticles to possess antibacterial properties with no stem cell toxicity. Nanoscale, in press (2014).
2. Wang, N. et al. Study on the anticorrosion, biocompatibility, and osteoinductivity of tantalum decorated with tantalum oxide nanotube array films. ACS Appl Mater Interfaces 4, 4516-4523 (2012).
9:00 AM - H4.29
In Situ Surface Modification for Patterned Superhydrophobicity with Reversible Wettability and Adhesion
Yuekun Lai 1 Jianying Huang 1
1National Engineering Laboratory of Modern Silk Suzhou China
Show AbstractWe described a facile high surface energy oil-based ink-regulating approach to rapidly achieve the reversible water wettability and adhesion transition in a great contrast on the TiO2 nanostructure film. Oil-based ink printing and solvent erasion were used to change the surface composition, and this tunability of the surface composition and roughness, gave rise to the site-selectively switchable wettability varying from superhydrophobicity to superhydrophilicity in micro-scale, and reversible water adhesion between sliding superhydrophobicity and sticky superhydrophobicity for many times. It is immediate ink to realize the wettability and adhesion transition with the ink printing and removing. Additionally, positive and negative micropattern can be achieved by taking advantage of the inherent photocatalytic property of TiO2 with the assistance of ink mask. Finally, the potential application (microdroplets manipulation, specific gas sensing, wettability template for positive and negative patterning, and site-selective cell immobilization)of the site-selectively sticky superhydrophobic surface was demonstrated. This study represents an important addition to the field of functional superhydrophobic materials.
9:00 AM - H4.30
Colloidal Particle Assembly in Microchanneled, Bioactive Hydrogel for Guided Tubular Network Construction
Min Kyung Kyung Lee 1 Max H. Rich 1 Artem Shkumatov 2 Jonghwi Lee 3 Hyunjoon Kong 1
1University of Illinois at Urbana-Champaign Urbana USA2University of Illinois at Urbana-Champaign Urbana USA3Chung-Ang University Seoul Korea (the Republic of)
Show AbstractControlling spatial organization of bioactive microparticles in a three-dimensional matrix has been long sought to guide the growth direction and spacing of vascular and neural networks; however, it still remains a grand challenge. This study demonstrates that a new method to align microparticles releasing bioactive molecules in microchannels of a hydrogel and guide growth direction and spacing of vascular and neural networks. An uniaxial freeze drying of a cell adherent hydrogel loaded with vascular endothelial growth factor-releasing microparticles temporally increases freezing-induced shear stress on and subsequently aligns microparticles in resulting microchannels, similar to the glacier-induced moraine formation. The resulting device stimulated vascular growth into microchannels and further improved neovascularization. Additionally, coupled with microfabrication, a resulting microchanneled hydrogel guides neuronal growth exclusively along the microchannels functionalized to activate cell adhesion. This resulting platform would be broadly useful to better understanding and modulating emergent behavior of cells relevant to development and regeneration.
9:00 AM - H4.31
PLLA Nanocomposites for Altered Cell Behavior and Inflammation Responses
Michelle Stolzoff 1 Thomas J Webster 1
1Northeastern University Boston USA
Show AbstractCancer recurrence at the site of tumor resection is a common complication resulting from today&’s efforts to treat the disease. To combat this, we have previously investigated the effects of nanofeatured surfaces on poly(lactic-co-glycolic acid) (PLGA) as well as selenium-coated substrates. In both studies, cancerous cells were less able to proliferate on the nanostructured surfaces while non-cancerous cells were unaffected, if not more capable of growth. Additionally, nanofeatured surfaces have been found to decrease the activity of stromal cells that support tumor growth, as well as exhibiting antibacterial properties. Selenium has become a popular dietary supplement for its anti-cancer, anti-inflammatory and other effects, and nano-selenium has been demonstrated to be cytotoxic to cancer cells and bacteria when applied directly. Specifically, selenium is a naturally-occurring, major component of several anti-oxidant proteins. Here, we describe the synthesis, characterization and efficacies of different selenium nanocomposites within poly(l-lactic acid) (PLLA) films for changes in cytotoxicity, reactive oxygen species (ROS) generation and gene expression. Specifically, with higher concentrations of selenium nanoparticles (~14 nanoparticles /mu;m3), cancer proliferation (MG063 osteosarcoma cells) was reduced significantly (p < 0.01) more than non-treated PLLA films. Isolation of the effects of nano-selenium dosages and nanoroughness will further characterize the cell-material interactions we have demonstrated here.
H1: Microfluidics
Session Chairs
Monday AM, December 01, 2014
Sheraton, 2nd Floor, Back Bay A
9:30 AM - *H1.01
Nano- and Quantum-Biodevices for Cancer Diagnosis, Cancer Therapy, and iPS Cell Based Regenerative Medicine
Yoshinobu Baba 1
1Nagoya University Nagoya Japan
Show AbstractNano-/quantum-biodevice is a piece of contrivance, equipment, machine, or component, which is created by the overlapping multidisciplinary activities associated with nano-/quantum-technology and biotechnology, intended for biological, medical, and clinical purposes. During the past decade, nano-/quantum-biodevice has progressively begun to focus on the establishment of main four fields of biomedical applications of nanotechnology, including 1) diagnostic devices, 2) molecular imaging, 3) regenerative medicine, and 4) drug delivery systems.
In this lecture, I will describe the development of nano-/quantum-biodevices for biomedical applications, including single cancer cell diagnosis for cancer metastasis, circulating tumor cell (CTC) detection by microfluidic devices, nanopillar devices for ultrafast analysis of genomic DNA and microRNA, nanopore devices for single DNA and microRNA sequencing, nanowire devices for exosome analysis, single-molecular epigenetic analysis, quantum switching in vivo imaging of iPS cells and stem cells, and quantum technology-based cancer theranostics [1-19].
[1] N. Kaji, Y. Baba, et al., Chem. Soc. Rev., 39, 948 (2010).
[2] T. Yasui, N. Kaji, Y. Baba, Annual Rev. Anal. Chem., 6, 83 (2013).
[3] M. Tabuchi, Y. Baba, et al., Nature Biotech., 22, 337 (2004).
[4] R. Bakalova, Y. Baba, et al., Nature Biotech., 22, 1360 (2004).
[5] Y.S. Park, Y. Baba, et al., ACS Nano., 4, 121 (2010).
[6] H. Yukawa, Y. Baba, et al., Biomaterials, 31, 4094 (2010).
[7] M. F. Serag, Y. Baba, et al., ACS Nano., 5, 493, (2011).
[8] T. Yasui, Y. Baba, et al., ACS Nano, 5, 7775 (2011).
[9] M.F. Serag, Y. Baba, et al., ACS Nano, 5, 9264 (2011).
[10] H. Hatakeyama, Y. Baba, et al., Biomaterials, 32, 4306 (2011).
[11] H. Hatakeayama, Y. Baba, et al., Mol. Therapy, 19, 1487 (2011).
[12] M.F. Serag, Y. Baba, et al., Nano Lett., 12, 6145 (2012).
[13] K. Hirano, Y. Baba, et al., Nucleic Acids Res., 40, 284 (2012).
[14] H. Yukawa, Y. Baba, et al., Biomaterials, 33, 2177 (2012).
[15] T. Yasui, Y. Baba, et al., ACS Nano, 7, 3029 (2013).
[16] K. Hirano, Y. Baba, et al., Nano Lett., 13, 1877 (2013).
[17] H. Akita, Y. Baba, et al., Biomaterials, 34, 8979 (2013).
[18] H. Hayashi, Y. Baba, et al., Mol. Therapy-Nucleic Acids, 3, e154 (2014).
[19] S. Rahong, Y. Baba, et al., Sci. Rep. (Nature Pub. Group), 4, Article number: 5252 (2014).
10:00 AM - H1.02
Chemoenvironmental Modulators of Fluidity in the Suspended Biological Cell
John M Maloney 2 Krystyn J Van Vliet 2 1
1MIT Cambridge USA2MIT Cambridge USA
Show AbstractBiological cells can be classified as "active soft matter," with mechanical characteristics modulated by external cues such as pharmaceutical dosage or fever temperature. Quantifying the effects of chemical and physical stimuli on a cell's mechanical response informs models of living cells as complex materials. We discuss the mechanical behavior of single cells in terms of fluidity, or hysteresivity normalized to the extremes of an elastic solid or a viscous liquid. This parameter, which complements stiffness when describing whole-cell viscoelastic response, can be determined for a suspended cell within subsecond times. Questions remain, however, about the origin of fluidity as a conserved parameter across timescales, the physical interpretation of its magnitude, and its potential use for high-throughput sorting and separation of interesting cells by mechanical means. We have selected optical stretching, a non-contact tool that deforms cells in the suspended state via photonic pressure, to explore the linear power-law-rheology regime (1% strain, 1 s timescale) of single cells. Cells were probed for several seconds each by applying an oscillatory photonic stress, and machine vision was used to extract time-dependent elongation and phase lag from phase-contrast images. Our experiments employing various chemoenvironmental conditions---temperature, pharmacological agents, pH, and osmolarity---extend familiarity with suspended-cell mechanics in the context of both soft-matter physics and mechanical flow cytometry development. The strong influence of both osmotic volume changes and the cytoskeleton-disassembling drug latrunculin supports the interpretation of fluidity as a reciprocal friction within the actin cytoskeleton, with implications both for cytoskeletal models and for expectations when separating interesting subpopulations by mechanical means in the suspended state.
10:15 AM - H1.03
Ordered Packing of Emulsion Droplets towards the Preparation of Adjustable Photomask
Ju Hyeon Kim 1 Jae-Hoon Choi 2 Jae Young Sim 1 Woong Chan Jeong 2 Seung-Man Yang 1 Shin-Hyun Kim 1
1KAIST Daejeon Korea (the Republic of)2LG Chem. Ltd. CRD Center Daejeon Korea (the Republic of)
Show AbstractPhotolithography has been widely used for preparing two-dimensional micropatterns. The selective exposure of ultraviolet (UV) light passing through photomask provides local crosslinking or degradation of the photoresist molecules. After the removal of the relatively small molecules, designed micropatterns following photomask are obtained. Although photolithography is still powerful tool for preparing micropatterns, it faces several technical limitations: Only two-dimensional micropatterns may be obtained and a conventional photomask pattern has the low reconfigurability. To overcome these limitations, grayscale photomask using microfluidic devices have been developed. However, such devices have pre-fixed pattern shapes and only thickness of micropatterns is tunable. Therefore, the development of photomask which provide on-demand control of micropattern size and periodicity still remains an important challenge.
Here, we report a novel microfluidic approach to prepare adjustable photomask using crystalline phases of monodisperse emulsion droplets. To prepare regular array of drops, the microfluidic device is designed to have three distinct functional parts which are a flow-focusing junction, side channels, and a reservoir in series. At flow-focusing junction, transparent oil droplets are generated in a dye-containing continuous aqueous phase. The droplets are then concentrated as they pass through side channels where continuous phase is selectively removed. This concentration leads to to the formation of a regular array of droplets in the reservoir. The configuration of droplet array depends on the relative height of the reservoir to the diameter of droplet. Through varying the relative height, one of a single-layered hexagonal array, a bi-layered square array, or a bi-layered hexagonal array is selected. The droplet arrays with unique configuration serve as a photomask to create hexagonal or square arrays of microdots. Transparent spherical drops in a light-absorbing continuous phase provide a parabolic profile of the UV transmittance, which enables the adjustment of microdot size through controlling the UV irradiation time; this is difficult to achieve with conventional photomasks. The array periodicity is determined by droplet size which is adjustable through a control of flow rates in a drop maker. Therefore, the dot size and array periodicity could be independently adjusted through combined control of droplet size and the UV irradiation time. Using a single microfluidic device, we prepare microarrays with a wide spectrum of array parameters.
H2: Smart Organic-Based Biodevices
Session Chairs
Monday AM, December 01, 2014
Sheraton, 2nd Floor, Back Bay A
11:15 AM - *H2.01
Reversible Protein Patterning of 3D Hydrogels via Bioorthogonal Photochemistry
Cole A DeForest 1 2 David A Tirrell 2
1University of Washington Seattle USA2California Institute of Technology Pasadena USA
Show AbstractSynthetic hydrogels have emerged as a unique class of biomaterials that enable stem cells to be cultured in three-dimensions within near-physiological, synthetic microenvironments. Recent strategies have been developed that permit bioepitopes (e.g., peptides, full-length proteins) to be introduced at any point in time and space to affect cell function spatiotemporally within user-defined subvolumes of the bulk material. While these techniques have been successfully utilized to direct a variety of basic cellular functions, advanced platforms that permit biological cues to be both introduced and subsequently removed would be beneficial in recapitulating the dynamic abundance of signaling biomolecules in the native, temporally-variable niche and in modulating complex cellular behavior. In this work, we demonstrate that the combination of two bioorthogonal light-based chemistries provides for the reversible immobilization of protein cues spatially within a hydrogel. The highlighted approach enables precise control over 4D biochemical functionalization of a synthetic polymer network in response to user-defined photonic stimuli. Results further illustrate the versatility of such dynamic biomacromolecular signal presentation in directing 4D progenitor cell fate.
11:45 AM - H2.02
pH-Responsive Hydrogel Cubes for Release of Doxorubicin in Cancer Cells
Jun Chen 1 Veronika Kozlovskaya 1 Eugenia Kharlampieva 1
1University of Alabama at Birmingham Birmingham USA
Show AbstractWe report on a novel type of shaped hydrogel microparticles, which undergo large, rapid, and reversible volume changes in response to solution pH. The cubic hydrogels are produced as interconnected poly(methacrylic acid) (PMAA) network replicas of mesoporous manganese oxide templates by sequential infiltration of (PMAA) and poly(N-vinylpyrrolidone) (PVPON), followed by cross-linking of PMAA and template dissolution. The integrated advantages of the porous cubic sacrificial templates and responsive PMAA matrix enable synthesis of monodisperse and pH-sensitive hydrogel cubes in a rapid, facile, and reproducible manner. These hydrogel cubes display a reversible 2-fold change in size while maintaining their shape in response to pH variations. The swelling behavior of cubic and spherical hydrogel particles is controlled by the network structure that is regulated by the PMAA molecular weight. These networks maintain their three-dimensional shapes in the dry state. No cytotoxicity is found for cubic and spherical hydrogels upon their interactions with human cancer cells for various time intervals. Finally, pH-triggered loading and release of doxorubicin to and from the cubic hydrogels is shown and their anticancer effect is demonstrated. The viability of A549 and HeLa cancer cells was significantly decreased upon interaction with doxorubicin-loaded cubic hydrogels. The approach presented here provides a new platform of multi-functional particles with highly-controlled geometry, size, composition, and responsive properties to be potentially used in targeted drug.
12:00 PM - H2.03
Bio-Inspired Vascular Architecture Promotes Smooth Muscle Differentiation of Adult Stem Cells on Micropatterned Hydrogel
Chor Yong Tay 1 Yun-Long Wu 3 Pingqiang Cai 2 David Tai Leong 1 Subbu S Venkatraman 2 Xiaodong Chen 2 Lay Poh Tan 2
1National University of Singapore Singapore Singapore2Nanyang Technological University Singapore Singapore3Xiamen University Xiamen China
Show AbstractSmooth muscle cells (SMCs) plays a pivotal role in the development and maintenance of vascular homeostasis. Due to its ability to differentiate into contractile SMCs, adult mesenchymal stem cells (MSCs) has long viewed to be a valuable cell source for vascular tissue engineering applications. However, the key to unlock MSCs full potential is to devise novel strategies, to direct the commitment of MSCs along the SMCs lineage. Using a bio-inspired micropatterned polyacrylamide hydrogel platform, MSCs were systematically stretched to adopt shapes with varying degree of polarization, that mimics the elongated form of native SMCs with single cell resolution. To examine how cellular polarization could potentially influence SMCs differentiation of MSCs, the micropatterned cells were exposed to transforming growth factor- beta1 (TGF-β1) to initiate the SMCs differentiation process. This effort was complemented with in vitro techniques such as cell traction microscopy, real time polymerase chain reaction and immunofluorescence staining. It was observed that shapes with an aspect ratio (AR) of 5:1 and 10:1 to be the most conducive for SMCs differentiation as indicated by robust expression of filamentous alpha smooth muscle actin (α-SMA) protein, a hall mark marker of SMCs phenotype. However, excessive stretching of the cellular body negated the pro-SMCs effects due to drastic perturbation in the cellular contractile machinery, preventing the cells to generate sufficient contractile force to promote adoption of SMCs phenotype. Conversely, shapes with high degree of isotropy such as circle and square displayed high occurrence of dorsal and transverse stress fibers that were unable to support intracellular tension for the proper subcellular localization of α-SMA. Supporting this architectural driven phenomenon, it was shown that the cells with the “optimal” shapes expressed higher level of diphosphorylated form of myosin regulatory light chain, suggestive of a higher level of basal contractile force that could serve as a biophysical impetus to drive SMCs differentiation in MSCs. Collectively, our results revealed that for effective expression of α-SMA, two criteria must be met, namely, (i) the ability for the cells to produce sufficient intracellular tensile force and (ii) proper expression of stress fibers subtypes as the structural basis to encourage adoption of SMCs phenotype. This study provided critical information that will aid in the development of functional vascular graft for regenerative medicine.
12:15 PM - H2.04
Oxygen Delivery Scaffolds for Tissue Engineering and Tissue Preservation
Huaifa Zhang 1 Faleh Tamimi Marino 1 Svetlana Komarova 1 Jake Barralet 1 2
1McGill University Montreal Canada2McGill University Montreal Canada
Show AbstractOxygen (O2) concentration has great influence on cell proliferation, differentiation, and gene expression and is essential for the normal activities of mammalian tissues and cells. However, hypoxia and even anoxia can occur during tissue engineering and tissue preservation, because of the extremely low solubility of O2. We developed an O2 delivery system (ODS) made from biodegradable materials. CaO2 was used as the O2-generating agent since it decomposes in H2O and that its O2 release rate can be controlled by adjusting the availability of H2O. We chose a hydrophobic and biodegradable biopolymer to decrease the decomposition rate of CaO2. Moreover, biodegradable and biocompatible alginate hydrogel was incorporated into our system to further control the decomposition rate of CaO2. The O2 delivery efficiency of ODS was first assessed under anoxia with primary human fibroblasts. The material system was co-cultured with primary human fibroblasts under anoxia to evaluate its ability to support cell survival. On the basis of the preliminary experiments, we further refined the design of ODS and tested its O2 delivery capacity using extremely high cell-density chinese hamster ovary (CHO) cells, rat aortas and mouse kidney. The experimental results show the fibroblasts cultured with our ODS under anoxia presented identical viability compared with the cells cultured under normoxia. The terminal deoxynucleotidyl transferees (TdT)-mediated dUTP nick end labeling (TUNEL) staining, BrdU immunohistochemistry staining and RT-qPCR results reveal that O2 release material eliminated DNA damage of the cells from anoxia, maintained normal cellular dividing activities and restored the expression of glycolysis, apoptosis and angiogenesis related genes to the normal levels under anoxia. High cell-density culture of CHO cells is very valuable for the commercial production of biomolecules, however, O2 deficiency happens as cell density increases and inhibits the productivity of the cells. Endothelial cells facing the lumen of aorta as well as cells in kidney are extremely O2-sensitive and die fast in ischemia. Normally, kidney begins to lose its function after twenty five minutes of warm ischemia. Our refined ODS was able to culture CHO cells at the density of 2×108 cells/ml, maintaining high cell viability of >98%. In addition, the ODS successfully preserved rat aortas at 37 #8451; for up to seven days with very high endothelial cell viability (>98%) while the counterpart preserved at 4 #8451; suffered from severe cell damage after being rewarmed to 37 #8451; (endothelial cell viability around 50%). Moreover, our ODS was able to preserve mouse kidney at 37 #8451; for at least twenty four hours. In summary, we developed a novel biodegradable O2 delivery system that was capable of supporting the survival of cells under anoxia and at an extremely high cell density, and exhibited great potential for tissue preservation.
Symposium Organizers
Donglei (Emma) Fan, University of Texas at Austin
Jianping Fu, University of Michigan
Xingyu Jiang, National Center for Nanoscience and Technology
Matthias Lutolf, Ecole Polytechnique Federale de Lausanne
Symposium Support
Air Force Office of Scientific Research
Biomaterials Science
National Science Foundation
H7: Inorganic Nanoparticles for Bioapplications
Session Chairs
Tuesday PM, December 02, 2014
Sheraton, 2nd Floor, Back Bay A
2:30 AM - *H7.01
Micro- and Nanostructures Based on Phase-Change Materials for Biochemical Delivery Applications
Younan Xia 1
1Georgia Tech Atlanta USA
Show AbstractWe are developing a novel platform based on phase-change materials (PCMs) -- materials with sharp melting points near the temperature of interest -- for drug delivery applications. Below its melting point, the PCM in the solid state can serve as a barrier to prevent the encapsulated drug(s) from being released. When heated to its melting point, the PCM will response quickly and transform into the liquid state, allowing the release of pre-loaded drug(s). The PCM-based platform can be used in a number of different ways for drug delivery applications. In one approach, the PCM is used as a matrix material to host nanoparticles of biodegradable polymers that contain the drug. While the nanoparticles serve as a barrier for controlling the release kinetic of the drugs, the PCM serves as a container of the nanoparticles and a “stopper” for the drug release. As soon as the drug-loaded nanoparticles have escaped from the PCM matrix, the drug release will be controlled by the diffusion of drug molecules through the nanoparticles or the degradation profile of the nanoparticles. In another approach, the drug will be directly encapsulated in nanoparticles made of a PCM, together with an FDA-approved organic dye (e.g., ICG with an absorption peak at 800 nm). When the system is irradiated with a near-infrared light, the irradiation energy will be absorbed by the dye and converted into heat, causing the temperature of the system to rise beyond the melting point of the PCM.
3:00 AM - *H7.02
DNA Smart Materials
Dongsheng Liu 1
1Tsinghua University Beijing China
Show AbstractThe reversible responsiveness of DNA secondary structures to environmental stimuli has enable to facilitate responsive devices and materials based on pure DNA or hybrid systems. Based on sequence and structure design, we have prepared kinds of pure or hybrid DNA supramolecular hydrogels, which could be formed under physiological condition within a minute at room temperature and without using any organic solvents. By tailoring the length of “sticky ends” of DNA linker, mechanical property of the hydrogel could be varied from hundreds to thousands Pa (G&’, storage modulus); we also found that the viability of cell in a 4 mm diameter hydrogel is nearly 100% after 24 hours incubation from top in plastic tubes. Additionally, the hydrogels show an excellent multiple responsiveness including pH, DNA restriction enzymes, protease digesting, temperature etc., which enable easy removal after cell culture.
We believe these hydrogels have great potential in tissue engineering, especially for 3D cell printing.
References:
1. J. Jin, S. Wang and D. Liu Advanced Materials,2013, DOI: 10.1002/adma.201301175.
2. D. Liu, E. Cheng and Z. Yang, NPG Asia Mater., 2011, 3(10), 109.
3. Y. Xing, D. Liu, et al. Advanced Materials, 2011, 23, 1117.
4. E. Cheng, D. Liu, et al.Angew. Chem., Int. Ed.2009, 48, 7660.
3:30 AM - H7.03
Spatial Regulation of Cellular Uptake and Stimulation by Janus Particles
Yan Yu 1 Yuan Gao 1 Yi Yi 1 Bo Chen 1 Yilong Jia 1
1Indiana University Bloomington Bloomington USA
Show AbstractJanus particles possess functional asymmetry and directionality within a single entity and thus are predicted to enable many promising biomedical applications that are not offered by homogeneous particles. As the interactions of particles with cells determine the therapeutic efficacy and cytotoxicity, understanding how properties of Janus particles influence the particle-cell interactions becomes crucial. It is, however, a challenging task due to the complexity of Janus particles-cell interactions. This presentation will focus on our recent progress on understanding how Janus particle interact with cells. In particular, we will show how anisotropic presentation of ligands affects cellular uptake of Janus particles as well as cell stimulation, from investigations using fluorescence imaging and biophysical characterization methods.
3:45 AM - H7.04
Characterizing the Effect of Nanoparticle Size on Nanoconjugation Enhanced EGF-Induced Apoptosis
Linxi Wu 1 Bjoern Reinhard 1
1Boston University Boston USA
Show AbstractWe investigated the effect of nanoconjugation on the apoptotic efficacy of the epidermal growth factor (EGF). We found that the covalent attachment of EGF to 40nm gold nanoparticles (EGF-NP) enhances apoptosis in EGF receptor (EGFR) overexpressing A431 cells as well as in HeLa cells that have physiological EGFR expression levels. We next validated the impact of nanoparticle size and EGF surface density on the efficacy to induce apoptosis in cancer cells. Cells were treated with EGF-NPs of different sizes for 2.5h. After an additional incubation for 24h or 48h in fresh growth medium, caspase-3 activity was quantified as a measure of the apoptosis level. Our data show that 80nm EGF-NPs have a greater apoptotic enhancement compared with 40nm EGF-NPs. To elucidate the underlying mechanism, we have investigated the location of EGF-NP as function of time to determine the impact of EGF-NP tracking on apoptosis to validate potential differences in the trafficking as origin for the observed size dependence of the EGF-induced apoptosis effect. The characterization of the size-dependence of the nanoconjugation-enhanced EGF-induced apoptosis improves the current understanding of nanoparticle-cell interactions and provides new opportunities for overcoming apoptosis evasion in cancer cells.
H8: Manufacturing of Biodevices
Session Chairs
Tuesday PM, December 02, 2014
Sheraton, 2nd Floor, Back Bay A
4:30 AM - *H8.01
3-D Printing of Biological Systems for Tissue Engineering and Biological Soft Robotics
Ritu Raman 1 5 Caroline Cvetkovic 2 5 Hyunjoon Kong 3 5 6 Rashid Bashir 2 4 5
1University of Illinois at Urbana-Champaign Urbana USA2University of Illinois at Urbana-Champaign Urbana USA3University of Illinois at Urbana-Champaign Urbana USA4University of Illinois at Urbana-Champaign Urbana USA5University of Illinois at Urbana-Champaign Urbana USA6University of Illinois at Urbana-Champaign Urbana USA
Show AbstractThe integration of living cells with soft scaffolds can enable the fabrication of biological machines for tissue engineering and soft robotics. These cell-based biological machines can be defined as a set of sub-components consisting of living cells and cell-instructive micro-environments that could eventually perform a range of prescribed tasks. The realization of biological machines and their sub-components will require a number of suitable cell sources, biomaterials, and enabling technologies. Here, we review our group&’s recent efforts towards this goal and of developing cell based biological machines. We have fabricated locomotive ‘‘bio-bots&’&’ using a 3D printer with hydrogels and living cells. The multi-material bio-bot consisted of a ‘biological bimorph&’ cantilever structure as the actuator to power the bio-bot, and a base structure to define the asymmetric shape for locomotion. The cantilever structure was seeded with a sheet of contractile cardiomyocytes. We will also describe the development of a 3D-printed electrically paced skeletal muscle based ‘bio-bot&’ devices where skeletal myoblasts embedded in ECM proteins compacted around a hydrogel structure were used to create the power source of the biological walking machine. While the specific applications are yet to be defined, these devices could have potential applications in drug delivery, power generation, and other biomimetic systems. We will also present the use of 3D printing to fabricate cell-laden hydrogel patches that allows the spatial release of pro-angiogenic growth factors and formation of new blood vessels when placed in contact with live tissue.
5:00 AM - H8.02
Table-Top Production of Multimaterial Fibers for Scalable Fluid-Instability-Based Fabrication of Nanoparticles
Joshua Kaufman 1 Felix Tan 1 Ayman Abouraddy 1
1University of Central Florida Orlando USA
Show AbstractWe have recently developed a novel methodology for the fabrication of structured, multimaterial, functionalized particles via an in-fiber Plateau-Rayleigh capillary instability (PRI) [1,2]. The starting point of the process is the preparation of a macroscopic preform - a scaled-up model of the desired fiber structure - from which a fiber is thermally drawn in the viscous state. The ‘core&’ of the preform - and hence the fiber - encases the materials from which the particles are produced. Thermal treatment of the drawn fibers induces the PRI along the whole fiber length, and the resulting particles inherit the structure of the core in the preform. Several unique aspects distinguish this approach: it is applicable to a multiplicity of materials, particle formation has been confirmed over a range of diameters from 1 mm down to 20 nm, large-scale particle production is potentially achievable, and complex internal structure within the particle is readily achieved by judicious structuring of the macroscopic preform. A potential drawback of this approach is that it appropriates the fiber drawing technology used in producing the lengths of fibers used in telecommunications. This procedure, while conceptually simple, requires specialized equipment that is not commonly available in materials and chemical research laboratories dedicated to nanoparticle synthesis. Specifically, equipment for extrusion of preforms and a fiber draw tower designed for research in fiber optics represent entry barriers - in terms of equipment cost, space, and specialized training.
Here, we present open-source designs for two machines that are miniaturized versions of a draw tower and extrusion system and may be readily constructed and utilized on a table top. Each machine has a footprint area of 1x1 ft2 at the base and is no taller than 2.5 ft, making them suitable tabletop instruments. Additionally, the total cost of assembling both the fiber drawing station and the extrusion system is less than $25,000. We present guidelines for processing a wide range of materials into multimaterial fibers using these instruments and production of structured micro- and nanoparticles for applications in biotechnology and nanophotonics. Our goal is to remove the entry barriers to researchers in chemistry and materials science who may benefit from the rich opportunities afforded by particle synthesis via in-fiber fluid instabilities with respect to the control over the materials composition and internal particle architecture.
[1] J. J. Kaufman, G. Tao, S. Shabahang, E.-H. Banaei, D. S. Deng, X. Liang, S. G. Johnson, Y. Fink, and A. F. Abouraddy, “Structured spheres generated by an in-fibre fluid instability,” Nature 487, 463 (2012).
[2] J. J. Kaufman, R. Ottman, G. Tao, S. Shabahang, E.-H. Banaei, X. Liang, S. G. Johnson, Y. Fink, R. Chakrabarti, and A. F. Abouraddy, “In-fiber production of polymeric particles for biosensing and encapsulation,” Proc. Natl. Acad. Science (USA) 110, 15549 (2013).
5:15 AM - H8.03
Paper-Based Biomolecule Preconcentrator by Ion Concentration Polarization
Sung Il Han 2 Kyo Seon Hwang 1 Rhokyun Kwak 1 Jeong Hoon Lee 2
1Korea Institute of Science and Technology (KIST) Seoul Korea (the Republic of)2Kwangwoon University Seoul Korea (the Republic of)
Show AbstractMicrofluidic paper-based analytical devices (µPAD) for molecular detection hold a great potential in point-of-care diagnostics field with well-documented advantages (e.g. low cost, robustness, instrument-free). Currently, one of the most tantalizing problems in µPAD is improving detection sensitivity; colorimetric detection by naked eye frequently suffers from low sensitivity and poor accuracy. Various amplification processes have been developed, including enzyme/metal-based signal enhancement and colored particle aggregation; however, they are still limited in nucleotide targets or enhancement factor (O(10)-fold). Here, to address this issue, we demonstrate a paper-based preconcentrator for generic biomolecules, based on ion concentration polarization (ICP) initiated within a paper-channel coupled with cation selective membrane (i.e. Nafion). ICP phenomenon is a dynamic ion concentration change driven by cation or anion-biased transfer on ion selective membrane. Under electric field, such ion concentration change generates a strong electrostatic barrier, resulting the accumulation of charged biomolecules on the barrier. Microfluidic ICP preconcentrator has been already used to preconcentrate various biomolecules and cells up to million-fold.
To integrate ICP preconcentrator into paper-based systems, Nafion is line-patterned by filling the resin (20wt% Nafion perfluorinated ion-exchange resin) between reversely bonded silanized glass and poly(dimethylsiloxane)(PDMS) microchannel. After detaching the PDMS channel and drying solvent in the resin, the line-patterned Nafion on a silanized glass is transferred to a double-side tape (ACE plastic carpet tape). This Nafion-transferred tape is then attached to a paper microchannel layer (Whatman cellulose chromatography paper); this paper has two wax-printed microchannels (by Xerox ColorQube 8870DN), which are connected by the Nafion line; this line-pattern is relatively thin (thickness~100nm)), so the tape and the paper can contact tightly. Once voltage is applied at the two paper channels across the cation selective membrane, ICP and corresponding electric barrier is generated near the membrane. Previous ICP preconcentrators in microfluidic channel push biomolecules to the barrier by pressure-driven flow or electroosmotic flow. In the paper-based preconcentrator, however, passive capillary flow delivers biomolecules, and allows us to eliminate a pumping action. Here, we visualize ICP and biomolecule preconcentration with fluorescent dye (Alexa Fluor 488) and color dye (Orange G). Preconcentration speed is proportional to the sample delivery speed, i.e. capillary flow rate of the paper. The applied voltage is adjusted to generate ICP to block all charged targets, but its power consumption is small enough to be operated by commercial battery. As such, this paper-based ICP preconcentrator is well suited to use as a front-end preconcentration interface for various µPAD systems.
5:30 AM - H8.04
Bacterial Microarrays Obtained by Spontaneous Adhesion of Single Bacteria on Predefined Spots on Chemically Patterned Surfaces
Nina Bjamp;#248;rk Arnfinnsdottir 1 Vegar Ottesen 1 Rahmi Lale 2 Marit Sletmoen 1
1The Norwegian University of Science and Technology Trondheim Norway2The Norwegian University of Science and Technology Trondheim Norway
Show AbstractIn the biological community, the awareness of the challenges connected to population averages, i.e. their inherent masking of the behavior of minority subpopulations, explains why single-cell analysis is increasingly applied to ask biological questions. This awareness is accompanied by a growing demand for sensitivity and throughput in single cell studies. The study of a population of bacteria immobilized onto microarrays may be a solution to the above mentioned challenges. Preparation of such arrays can be obtained by controlling bacterial adhesion on patterned surfaces. Such microarrays allow microscopy based mapping of the behavior of individual cells in populations of bacteria in an efficient manner, while strictly controlling the environment of the bacteria.
We propose a procedure of making bacterial arrays that is fast, easy and applicable in a standard biology lab. Micro contact printing is used to deposit bacterial adhering chemicals on predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion in order to selectively immobilize bacteria onto the substrate. Different chemical regimes and the design features of the PDMS stamps used for deposition of chemicals have been evaluated in order to optimize the probability of attaching single bacteria to each deposited island of bacterial adhering chemical. By using Polyethylene Glycol (PEG) or Poly(vinyl) Alcohol (PVA) coated glass slides with micro contact printed islands of polydopamine (PD) bacteria were successfully immobilized onto predefined spots resulting in highly ordered arrays. When using PEG coated glass slides, bacteria were attached to 97 - 100 % of the PD islands, 21 to 62 % of which were occupied by a single bacterium. When exchanging PEG for PVA, bacteria were attached to 89 % of the islands, and 77% of the islands were occupied by a single bacterium. A viability test revealed that 99 % of the bacteria were alive after being immobilized onto patterned surfaces.
5:45 AM - H8.05
Liposomal Drug Screening Microarrays
Aubrey Emmanuel Kusi-Appiah 1 Steven John Lenhert 1 2
1Florida State University Tallahassee USA2Florida State University Tallahassee USA
Show AbstractThe need to shorten the time and reduce the cost of the process of drug discovery has necessitated the exploration of other methods apart from the conventional microtiter plate method for high throughput screening of new molecular entities for pharmaceutical applications[1, 2]. Furthermore, current high throughput screens are initially done at single concentrations thereby potentially missing out on doses that elicit useful biological responses[3]. The already existing application of lipids as carrier vectors in materials delivery to cells coupled with their lyotropic nature allows them to be useful in a novel way of materials delivery to cells via the encapsulation of small molecules in patterned surface supported multilayers. We produce patterned lipid multilayers consisting of more than a single bilayer on surfaces by using dip-pen nanolithography[4] and nanointaglio [5, 6] from a single surface at a demonstrated density of 625 tests per cm2 with the potential to go even higher. The height of the multilayers patterned determines the amount of material taken up by cells in culture [7, 8]. In addition, due to the low miscibility of these lipids in water, little to no mixing occurs between neighboring lipid arrays[7]. These properties show the potential for simultaneous dose dependent delivery of multiple materials to cells from a single surface for application in the high throughput drug screening industry.
1. Yang, J.Y. and M.Q. Yang, Transforming medicine: functional informatics, drug design, and personalised healthcare. International Journal of Functional Informatics and Personalised Medicine, 2008. 1(1): p. 1-7.
2. Wu, JH; Wheeldon; Guo, YQ; Lu, TL; Du, YN; Wang, B; He, JK; Hu, YQ; Khademhosseini, A, A sandwiched microarray platform for benchtop cell-based high throughput screening. Biomaterials, 2011. 32(3): p. 841-848.
3. Bibette, J., Gaining confidence in high-throughput screening. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(3): p. 649-650.
4. Lenhert S; Sun, P; Wang, YH; Fuchs, H; Mirkin, CA, Massively parallel dip-pen nanolithography of heterogeneous supported phospholipid multilayer patterns. Small, 2007. 3(1): p. 71-75.
5. Nafday, O.A., T.W. Lowry, and S. Lenhert, Multifunctional Lipid Multilayer Stamping. Small, 2012. 8(7): p. 1021-1028.
6. Lowry T. W., Kusi-Appiah A., Guan J., Van Winkle D. H, DavidsonM. W., Lenhert S, Materials Integration by Nanointaglio. Advanced Materials Interfaces, 2014: p. n/a-n/a.
7. Kusi-Appiah, AE ; Vafai, N ; Cranfill, PJ; Davidson, MW; Lenhert, S, Lipid multilayer microarrays for in vitro liposomal drug delivery and screening. Biomaterials, 2012. 33(16): p. 4187-4194.
8. Nafday, O.A. and S. Lenhert, High-throughput optical quality control of lipid multilayers fabricated by dip-pen nanolithography. Nanotechnology, 2011. 22(22).
H5: Nanomanipulation and Assembly
Session Chairs
Tuesday AM, December 02, 2014
Sheraton, 2nd Floor, Back Bay A
9:00 AM - *H5.01
Stimuli Responsive Micro and Nanotools for Drug Delivery and Surgery
David H. Gracias 1
1Johns Hopkins University Baltimore USA
Show AbstractAn important goal of modern medicine is the development of miniaturized devices with moving parts that can access hard to reach places in the human body to enable diagnostic and therapeutic procedures in a less invasive manner and more effective manner. In this talk, I will discuss the challenges associated with the development and deployment of untethered micro and nanosized mimics of macroscale surgical tools. I will describe the development of biocompatible devices that harness mechanical energy from stimuli responsive changes in thin film mechanical properties such as the release of residual stress or differential swelling. I will outline strategies that can be used to engineer devices that respond to their biological environment and can consequently operate in an autonomous manner. When composed of hydrogels or polymers and loaded with drugs, they enable a chemomechanical approach for sustained release in the GI tract. I will detail an important result in the use of dust-sized grippers to biopsy tissue in the gastrointestinal tract of live pigs. I will also discuss recent developments which show that these devices could be made small enough to capture even single cells and composed of bioresorbable materials which can enhance safety for in vivo procedures.
Relevant Publications: (a) Leong et al, Tetherless thermobiochemically actuated microgrippers, PNAS 2009; (b) Fernandes et al, Toward a miniaturized mechanical surgeon, Mater. Today 2009; (c) Bassik et al, Enzymatically Triggered Actuation of Miniaturized Tools, JACS 2010; (d) Solovev et al, Self-Propelled Nanotools, ACS Nano 2012; (e) Gultepe et al, Biopsy with thermally-responsive untethered microtools, Adv. Mater. 2013; (f) Solovev et al, Rolled-up magnetic microdrillers: Towards remotely controlled minimally invasive surgery, Nanoscale 2013; (g) Gultepe et al, Biologic tissue sampling with untethered microgrippers, Gastroenterology 2013; (h) Malachowski et al, Stimuli responsive theragrippers for chemomechanical controlled release, Angew. Chemie 2014; (i) Malachowski et al, Self-folding single cell grippers, Nano Lett. 2014.
9:30 AM - H5.02
Applications for Micro- and Nanomanipulators in Biomedical Research
Andrew Jonathan Smith 1 Andreas Rummel 1 Klaus Schock 1 Stephan Kleindiek 1
1Kleindiek Nanotechnik Reutlingen Germany
Show AbstractThe default application for micromanipulators in biomedical and life science research is patch clamping, where an electrolyte-filled glass pipette fitted with a electrochemically sensitized metal wire is used to measure potentials across cell membranes. This technique benefits from stable micromanipulators that do not drift. At the same time, ease of use as well as accessibility and portability are important features in day to day lab work.
Going beyond patch clamping there is a wide array of applications and research goals that rely on highly precise and easy to operate micromanipulators. A few examples are briefly discussed in this work.
Investigations on small vesicles can be aided by injecting a fluorescent die into the sample. Using pipettes similar to the ones described above, this injection process an easily be achieved.
Another application for micromanipulators is the dissection of chromosomes. Using a sharp needle in combination with a micro blade, sections of a single chromosome can be extracted for further analysis.
Micromanipulators can also be used to stimulate cells. Work performed at the University Ulm involved stretching and compressing in order activate specific strain-induced biochemical responses. Some of the tests were performed using a micro-spatula to put strain on the sample, other experiments relied on a micro gripper to gently squeeze a cell culture in cyclic manner.
Finally, a method for measuring adhesion forces between cells and their substrates using a image recognition-based approach is discussed. This approach utilizes the microscope's magnifying power to produce both deflection and force data by employing a cantilevered sample mount with a well-defined spring constant. This approach eliminates the need for expensive and overly sensitive measurement electronics in favour of a robust measurement platform combined with intelligent software analysis.
9:45 AM - H5.03
Biocompatible Mg-Based Micromotors
Chuanrui Chen 1 Fangzhi Mou 1 Lei Kong 1 Huiru Ma 2 Jianguo Guan 1
1Wuhan University of Technology Wuhan China2Wuhan University of Technology Whuan China
Show AbstractSelf-propelled micromotor has been attractive in the last decade for their diverse potential applications ranging from environment to biomedical engineering.1 Unfortunately, most of self-propelled micromotors developed so far only works in harsh surroundings of hydrogen peroxide, acidic and alkaline medium etc.. Thus, by taking account of the excellent biocompatibility and biodegradability of metal Mg, we firstly have developed a highly hemocompatible Mg/Pt Janus micromotor, which is propelled by hydrogen bubbles generated from Mg-H2O reaction with the assistance of HCO3-.2 Secondly, to the directly harvest energy from human blood plasma, we then developed a biologically-friendly Mg/Pt-Poly(N-isopropylacrylamide) (PNIPAM) Janus micromotors which can directly driven by simulated body fluids (SBF) or human blood plasma.3 The pit corrosion of chloride anions and buffering effect of SBF or blood plasma play major roles for accelerating Mg-H2O reaction to produce hydrogen bubble propulsion for the micromotors. The Mg/Pt-PNIPAM Janus micromotors also can effectively uptake, transport and temperature-control-release drug molecules by taking advantages of the partial surface-attached thermoresponsive PNIPAM hydrogel layers. Thirdly, to eliminate the residual micro-nano/motors component(such as metallic Pt, Ti or Polymers), which are harmful to living organisms, a new Mg baesd micromotor with fully biodegradability in different fuel and in human plasma is presented.4 This Janus micromotor consists of a biodegradable PLGA [poly (D,L-lacltide-co-glycolide)] or Zinc as shielding layer and metal Mg as main body, also shows self-propulsion rely on the Mg-H2O reaction. It has been found that the power conversion efficiency of the micromotor is highly dependent on the bubble ejection behaviors. The as-developed Mg based micromotors with excellent biocompatibility hold promises for diverse biomedical applications, including drug delivery, protein and cell separation, microsurgeries etc. if their configuration is optimized by design in the future.
REFERENCES
1 Alberto Credi, Angew. Chem. Int. Ed. 126 (17), 4360 (2014).
2 Fangzhi Mou, Chuanrui Chen, Huiru Ma, Yixia Yin, Qingzhi Wu, and Jianguo Guan, Angew. Chem. Int. Ed. 125 (28), 7349 (2013).
3 Fangzhi Mou, Chuanrui Chen, Qiang Zhong, Yixia Yin, Huiru Ma, and Jianguo Guan, ACS Appl. Mater. Interf. (2014). doi: 10.1021/am502729y.
4 Chuanrui Chen, Fangzhi Mou, Shun Jiang, Lei Kong, Yixia Yin, Huiru Ma, and Jianguo Guan, In Preparatioin.
10:00 AM - H5.04
Self-Folding Biocompatible Devices for Single Cell Capture and Analysis
Qianru Jin 1 David H Gracias 1
1Johns Hopkins University Baltimore USA
Show AbstractThe analysis of the biomolecular milieu within and around individual cells in a high-throughput manner and the ability to monitor biochemical changes of a specific cell and during cell division over time provide a means to understand progressive or dynamical events that might otherwise be averaged away in a population wide analysis. Hence, a long standing challenge in engineering has been the identification, capture and analysis of single cells with high sensitivity and selectivity. Previously in our group we have developed self-folding SiO2/SiO single cell grippers in a large array, which can capture individual live fibroblast cell and red blood cell [1]. Here, we leverage the optical transparency of the devices to analyze captured cells in situ through optical modalities. We will discuss surface patterning of the devices with optical molecular probes and studies on biomolecular detection and analysis of intra and extracellular biomolecules.
[1] K. Malachowski, M. Jamal, Q. Jin, B. Polat, C. Morris, and D. H. Gracias, “Self-folding single cell grippers,” Nano Letters, 2014, DOI: 10.1021/nl500136a.
10:15 AM - H5.05
Rotation Dynamics in Long-Time Operating Nanomotors
Jianhe Guo 1 Kwanoh Kim 2 Donglei Fan 1 2
1University of Texas at Austin Austin USA2University of Texas at Austin Austin USA
Show AbstractRecently, we reported an innovative type of nanomotors consisting of nanowires as rotors and patterned nanomagnets as bearings, the dimensions of which are less than 1 µm and can continuously rotate for 15 hrs. In this work, we rotated the nanomotors up to 44 hours and systematically investigated their rotation dynamics during the long-term operations. Using analytically modeling, we determined the time-dependent nanoscale torques and forces involved in the system, which showed high consistency with the extent of wear of nanomotors from the experimental characterization. We also revealed the linear dependence of the nanoscale loading and frictional forces in the submicron devices, proving previous theoretical results. This work is the first attempt in a newly reported system, which could inspire the future research of NEMS tribology and guide the system design for long operation lifetime.
10:30 AM - H5.06
Nanopropellers in Biological Media
Debora Walker 1 Tian Qiu 1 John Gibbs 1 Andrew Mark 1 Peer Fischer 1
1Max-Planck-Institute for Intelligent Systems Stuttgart Germany
Show AbstractNano- and microparticles have great potential for biomedical applications, including drug transport and delivery, localized sensing, and chemical/mechanical stimulation of individual cells, or even individual cell components. Active propulsion permits efficient, targeted delivery of such particles to their intended destination. However, transport in biological media is complicated by the presence of polymers, such as glycoproteins and polysaccharides, which form an interconnected network that results in gel-like properties. Biological gels effectively prevent transport of micrometer-sized particles, but smaller particles and molecules, necessary for nutrient supply, can pass through easily. The challenge is thus to develop artificial “probes” that are small enough to pass through biological gels, but remain propulsive and steerable.
Here, we report the smallest magnetic nanopropellers that have been realized to date.1-3 They are actuated by a gradient-free external magnetic field and their shape couples rotation into translational. We show that they can be actively and efficiently moved through biological media with micron-level precision.1 The nanopropellers have a filament diameter well below 100 nm and can easily be navigated through hyaluronan (HA) gels. In contrast, larger microparticles and propellers cannot move even at low HA concentrations. Interestingly, the nanopropellers are so small that they cannot be controlled with a gradient magnetic field (tweezer) setup, and experience very strong Brownian motion in water, but are very efficient in biological media.
We discuss the factors that affect the propulsion, including the growth method, the geometry of the screw-propellers, their passivation with appropriate surface coatings and the incorporation of enzymes that can influence the environment of the nanopropellers. We show that nanopropellers may even experience significantly enhanced propulsion efficiencies in viscoelastic gels compared to micropropellers, which paves the way for actively propelled “nanorobots” inside biological media and living organisms, and ultimately, within cells.
References:
(1) Schamel, D.; Mark, A. G.; Gibbs, J. G.; Miksch, C.; Morozov, K. I.; Leshansky, A. M.; Fischer, P.: Nano-Propellers and their Actuation in Complex Viscoelastic Media. ACS Nano2014, DOI: 10.1021/nn502360t.
(2) Gibbs, J.; Mark, A. G.; Lee, T.-C.; Eslami, S.; Schamel, D.; Fischer, P.: Nanohelices by Shadow Growth. Nanoscale2014,DOI: 10.1039/C4NR00403E.
(3) Mark, A. G.; Gibbs, J. G.; Lee, T.-C.; Fischer, P.: Hybrid Nanocolloids with Programmed Three-dimensional Shape and Material Composition. Nat. Mater.2013, 12, 802-807.
H6: Nanoelectric Biotechnology
Session Chairs
Tuesday AM, December 02, 2014
Sheraton, 2nd Floor, Back Bay A
11:15 AM - H6.01
Development of Vertically Aligned Nanoelectrode Array System for Photosynthetic Electron Extraction from Living Algal Cells
Lo Hyun Kim 3 Hyeonaug Hong 3 Dasom Yang 3 Yong Jae Kim 3 Myungjin Han 2 Gu Yoo 1 Youngcheol Chae 2 Jae-Chul Pyun 1 WonHyoung Ryu 3
1Yonsei University Seoul Korea (the Republic of)2Yonsei University Seoul Korea (the Republic of)3Yonsei University Seoul Korea (the Republic of)
Show AbstractNatural photosynthesis is one of the most efficient energy conversion processes. Solar energy can be converted into biochemical energy with nearly 100% quantum efficiency. However, most of photosynthetic energy is used for cell growth and small fragment of the converted energy has been utilized in a form of biomass or bioethanol. More efficient harvesting of photosynthetic energy can be conducted in non-cyclic electron transfer that occurs in thylakoid membranes before NADPH reduction. Recently, generation of bioelectricity using extracted photosystem II (PSII) or fragmented thylakoid membranes has been demonstrated with modified electrode systems. However, instability of extracted PSII or thylakoid membranes limits continuous harvesting of photosynthetic electrons from the extracts. In this study, we developed electrochemically-active nanoelectrode arrays that can be inserted into single algal cells and extract photosynthetic electrons directly from living algal cells. The nanoelectrode arrays were fabricated using template-based nanosphere lithography (NSL) and multiple steps of photolithography. Highly doped SOI wafer was used for electrical connection, while 1mu;m silica nanoparticles and polyimide films were used as etch mask. Using dry etching process, arrays of high A/R nanoelectrodes were fabricated. Subsequently, insulation layer was deposited followed by PR coating. Exposed nanoelectrode tip was metallized for enhanced redox interaction with carriers of photosynthetic electrons within chloroplasts of the algal cells. Using a custom-built microscope system, algal cells (Chlamydomonas reinhardtii) were captured by a glass micropipette and inserted by nanoelectrode using a micro manipulator. Then, using a chronoamperometry system, we measured light-responsive electron signals from the inserted algal cells. The maximum photosynthetic current from a single algal cell was 1.1pA with light intensity 99mu;mol#903;m-2s-1 and potential 0.4V (versus Ag/AgCl). The current signals disappeared when the light was off and it increased when the light intensity was increased. Since more photons make more excited electrons at high intensity, current and light intensity showed a proportional relation. The photosynthetic currents also showed gradual increase and saturation upon the increase of voltage bias. Photosynthetic electrons were harvested with voltage bias from 0V to 0.4V because most of photosynthetic components such as plastoquinone, plastocyanin have midpoint potentials within the similar range. However, when the voltage bias higher than 0.4V, the number of photosynthetic components to oxidize became limited and photosynthetic current showed gradual saturation. After 2 hours, green cell color faded and the photo currents were also reduced to below 0.3pA due to the leakage of subcellular components.
11:30 AM - H6.02
Intracellular Electrical Stimulation of PC-12 Cells through Vertical Nanowire Electrode
Hyungsuk Kim 1 Ilsoo Kim 1 Hye-young Lee 2 Eungjang Lee 3 Seong Yi 2 Seung-Han Park 3 Heon-Jin Choi 1 Jeongmin Park 4
1Yonsei University Seoul Korea (the Republic of)2Yonsei University Seoul Korea (the Republic of)3Yonsei university Seoul Korea (the Republic of)4Yonsei University Seoul Korea (the Republic of)
Show AbstractIt is known that neurons can be modulated by electrical stimulation that elicits the cell activation energy and rehabilitates the function of neurons. In the neural stimulation, metal films including platinum and gold are typically used as electrodes in an extracellular mode. Meanwhile, intracellular electrical stimulation should be effective compare to extracellular one. Patch clamp, a representative intracellular stimulation method, has been studied, however, it critically damaging the cells and thus not suitable for practical manipulations. Si nanowires (SiNWs) have a very small diameter that can stimulate the cells in intracellular mode without damages to the cells. Furthermore, SiNWs can be assembled in a large scale that makes possible to stimulate many cells in the neural networks simultaneously. SiNWs are also bio-compatible that are suitable for electrical stimulation in a long term without toxicity.
Here, we demonstrate intracellular electrical stimulation of Nerve Growth Factor (NGF) treated PC-12 cells, which are representative model of neuronal differentiation, through vertical SiNW electrodes. Vertical SiNW electrodes were prepared by firstly growth of SiNWs on Si wafers by vapor-liquid-solid (VLS) mechanism and then fabricated as electrodes by using semiconductor fine processing. We then cultured PC-12 cells on Poly-L-Lysin (PLL) coated SiNW electrodes with 105/cm2 density for 4 days with intracellular electrical stimulation (constant 50mV) and 2 days of stabilizing period. The morphological differentiation of PC 12 cells were observed by scanning electron microscopy (SEM). It was found that more PC-12 cells were cultured on NWs (about 4 times more dense) than the flat-films. It was also found that intracellular electrical stimulation with SiNW electrodes induce neural outgrowth and are effective in differentiation (40 um longer in average neurite length) compare to extracellular stimulation with Au films. The PC-12 cells cultured on SiNW electrodes were further characterized by z-step fluorescent scanning in two-photon microscopy. It showed that the cells were pierced by NWs that clearly demonstrate the intracellular mode electrical stimulations of the cells, i.e., SiNWs penetrate a cell&’s membrane, and subsequently release electrical stimulation directly into the cell&’s cytosol, and allows highly efficient stimulation. Our results indicate that SiNW electrodes could be a new way of stimulation of neuron cell efficiently under living states for a long time.
11:45 AM - H6.03
Bioelectrical Impedance Measurements to Detect Changes in Tight Junction Expression at Cell Junction
Ramsey Kraya 1 Peter Searson 1
1Johns Hopkins University Baltimore USA
Show AbstractWe analyzed the effect of PTP inhibitors phenylarsine oxide (PAO) induced proteolysis of occludins on the overall cell monolayer impedance of Madin-Darby canine kidney (MDCK) cells as a function of time using impedance spectroscopy. Occludins are a tight junctional proteins that have been shown to significantly contribute to the regulation of the transport of ions, water and other molecules through cell monolayers. The cell monolayer is modeled as a parallel RC circuit, and the cell resistance and capacitance are recorded before and after the addition of the PAO containing media, providing information on the effect of occludin proteolysis to overall cell behavior in a real time dynamic measurement system. The frequency ranged from 1 Hz to 100 kHz. The model accounts for the double layer capacitance at the electrode-electrolyte interface. Our results clearly show the effect of occludin proteolysis on the overall resistance and capacitance of the monolayer.
12:00 PM - H6.04
Fabrication of Sub-20 nm Nanochannels by Conformal Film Deposition Process
Sung-Wook Nam 1
1IBM T.J. Watson Research Center Yorktown Heights USA
Show AbstractWe report a method to build sub-20 nm nanochannel structures by conformal film deposition process, such as atomic layer deposition (ALD). Highly conformal ALD film-layer enables fine-tuning of the size of nanochannels [1]. At first, we demonstrated nanochannel structure surrounded by dielectric materials. To build nanochannels, we used trench structure patterned on amorphous-silicon (a-Si)/silicon-oxide (SiO2) film-layers, in which an undercut geometry was produced by dilute-HF (DHF) wet-etching process. Upon the undercut structure, ALD dielectric film (Al2O3) was deposited until the open tube (trench) structure was sealed. Due to the presence of undercut geometry, the sealing process by highly conformal ALD film resulted in a void structure left in the center of trench, through pinch-off process. The void structures can be utilized as nanochannels with sub-20 nm dimension. In second, we extend this method to build nanochannel ion-transistor structures, in which nanofluidic channels are surrounded by gate-metal/gate-dielectric layers. To couple sub-20 nm nanochannels with metal/dielectric layers, we carried out sequential depositions of an ALD 'metal' layer which does not pinch off at the top of the tube, followed by an ALD 'dielectric' layer which does pinch off the top. Based on the nanochannels surrounded by metal/dielectric layers, we propose a nanochannel ion-transistor analogous to solid-state field-effect-transistor devices. Application of electrical gate-voltage bias may allow for the regulations of ionic transport, which eventually controls the motion of charged molecules. In conclusion, conformal film deposition process enables precise control over the nanochannel feature-size beyond lithographic limit, such as sub-20 nm dimension. Metal/dielectric surrounded nanofluidic channels offers an insight of a nanochannel ion-transistor device designed for electrical manipulations of ionic transport.
[1] S.W. Nam et al. Nano Letters 10, 3324-3329 (2010)
12:15 PM - H6.05
Development of a Single Molecular Tunnel-Current Identification Method for Electrical Genome Sequencing
Takahito Ohshiro 1 Makusu Tsutsui 1 Kazumichi Yokota 1 Tomoji Kawai 1 Masateru Taniguchi 1
1Osaka University Ibaraki Japan
Show AbstractSingle-molecule genome electrical sequencing by using Nanogap-devices are promising for personal genome generation in the context of personalized medication. We have been proposed a tunneling-current based identification as a single-molecule DNA/RNA sequencing by nano-gap electrode devices. This methodology is based on sequentially reading the tunneling-current across individual single-nucleotides in the sequence, resulting in a high-speed electrical discrimination of the individual nucleotides without chemical probes and PCR amplifications.
In this study, we measured the time-trace of the conductance values of DNA / RNA nucleotide molecules flowing through a nanogap-electrode. When the molecules passed between the nano-electrodes separated by a sub-nanometer gap, the tunneling-current through the molecules was increased, relative to that in the absence of molecules. The current intensity is closely related to the individual electronic conductance. The single-nucleotide-size gap were reproducibly formed by using nanofabricated, mechanically controllable break junction technique. When a gold-nanowire was thermally broken under 0.1V dc voltage apply by using a piezo-controller, a pair of gold nanoelectrodes were formed, the size of the gap was found to be comparable to a single-nucleotides. Using the nanogap-electrode, we measured DNA/RNA monomers in the phosphate buffered solution. The magnitude of the peak conductance of four nucleotides was found to be in the following order: dGMP > dAMP > dCMP > dTMP, and rGMP > rAMP > rCMP > rUMP. This conductance values is due to the individual molecular energy level. Calculations based on density functional theory indicated that the order based on the highest occupied molecular orbital (HOMO) energy was similar to our experimental results. Based on this determined electrical conductivity for single-nucleotides, we electrically identify the base-type in oligonucleotides, and read the fragment of sample nucleotide passing through the sensing electrode. On the basis of a reconstruction of the read fragment sequences, we performed a resequencing and identify a specific let-7 RNA from other RNA samples. In addition, we found that the read fragment increased in length when nanofluidics partes were placed in the center of the nanogap-electrode devices. This method could be a promising small RNA profiling strategy.
12:30 PM - H6.06
Control and Manipulation of a Charged Macromolecule in a Nanosized Gap between Two Fluid Reservoirs: Theory and Experimental Results
Venkat S.K Balagurusamy 2 Stas Polonsky 1
1IBM Watson Research Center Yorktown Heights USA2IBM T.J.Watson Research Center Yorktown Heights USA
Show AbstractBiomolecules and therefore living systems indispensably depend on the water environment for their survival and carrying out many important biological functions. The ability of some biological assemblies to move along information-carrying polymers of nucleic acids with single nucleotide precision in water is vital for the machinery of life. DNA or RNA polymerase, for example, has to translocate along the nucleic acid molecule with single nucleotide accuracy in order to replicate it correctly. Controlling the position of a macromolecule like DNA at a single monomer accuracy is of great technological importance as well for developing high-speed electronic DNA sequencing devices, e.g., by electron tunneling through nanogap junctions. Non-uniformity of charge distribution along the length of charged linear polymer molecules, with DNA being a notable example, opens the theoretical possibility of controlling the position of such molecules with a single monomer accuracy. We use a simple Born ion continuum model to analyze the effect of solvation energy change on the translocation of a charged linear polymer molecule through a nanosized vapor gap between two fluid reservoirs. As the effective radius of the discretely spaced charges increases, our model predicts a transition from trapping to diffusion of the molecule in the vapor gap [1]. We introduce a new experimental technique that uses an FPGA-driven nanopositioner to control the coupling of a nanopipette with the liquid surface of a fluidic cell and create a transient nanogap [2]. We present results on creating such a nanogap, triggered by the translocation of double-stranded DNA between a nanopipette and a fluidic cell, and measure the probability to find the molecule near the tip of the nanopipette after closing the gap. The developed platform will enable testing some of our recent theoretical predictions for the behavior of a charged macromolecule in a nanogap between two fluidic reservoirs.
[1] S.Polonsky and V.S.K.Balagurusamy, Europhysics Letters, 103 (2013), 68007.
[2] S.Polonsky, V.S.K.Balagurusamy and J.Ott, Review of Scientific Instruments, 2014, to appear
12:45 PM - H6.07
Flexible Fiber-Based Multifunctional Platforms for Neural Recording, Interrogation and Repair
Polina Anikeeva 1 2
1Massachusetts Institute of Technology Cambridge USA2Massachusetts Institute of Technology Cambridge USA
Show AbstractOur ability to treat debilitating neurological conditions such as Parkinson&’s disease, trauma-induced para/tetraplegia and post-traumatic stress disorder is limited by the tools available for functional long-term interfacing with neural circuits in the central and peripheral nervous systems.
We use fiber drawing methods to create multifunctional flexible devices based on polymers and their composites. These fiber-probes permit electronic, optical and pharmacological interactions with neurons in freely moving animals, while inducing minimal damage to the surrounding tissue even over prolonged implantation (3 months). The fiber probes can be applied to neural recording in the brain as well as spinal cord and have already enabled optical control and electrical monitoring of locomotor functions. Furthermore, fiber-inspired fabrication has recently allowed us to create polymer-based optoelectronic neural scaffolds (OPTELS) - an electronically active platform for investigation of mechanical, chemical and optical stimuli as tools for neural repair.
Symposium Organizers
Donglei (Emma) Fan, University of Texas at Austin
Jianping Fu, University of Michigan
Xingyu Jiang, National Center for Nanoscience and Technology
Matthias Lutolf, Ecole Polytechnique Federale de Lausanne
Symposium Support
Air Force Office of Scientific Research
Biomaterials Science
National Science Foundation
H11: Innovative Biosensing
Session Chairs
Wednesday PM, December 03, 2014
Sheraton, 2nd Floor, Back Bay A
2:30 AM - *H11.01
Novel Nano-Optical Tools for the Manipulation and Sensing of Biomolecules
Srdjan Acimovic 1 Maria-Alejandra Ortega 1 Johann Berthelot 1 Mark Kreuzer 1 Vanesa Sanz 1 Romain Quidant 1
1ICFO-The Institute of Photonic Sciences Barcelona Spain
Show AbstractNanoscale control of plasmonic fields in engineered metal nanostructures offers unique opportunities to boost the interaction of light with tiny amounts of matter, down to the molecular level. In this paper we discuss how such enhanced interaction can benefit the detection and non-invasive manipulation of biomolecules.
We first present an integrated analytical platform that combines the sensing capability of plasmonic antennas with advanced microfluidic technologies for the label-less detection of protein cancer markers in blood. Our platform offers parallel, real-time inspection of tens of sensing sites distributed across independent microfluidic channels with very high reproducibility. This enables us to test various sensing strategies for the detection of biomolecules. In particular we demonstrate the fast detection of relevant cancer biomarkers (AFP and PSA) down to concentrations of 500 pg/mL in human serum.
A second technology exploits the optical properties of gold nanostructures to optically trap and 3D-manipulate single specimens as small as 10nm. A nano-optical trap is built by engineering a bowtie plasmonic aperture at the extremity of a tapered metal-coated optical fibre. Both the trapping operation and monitoring are performed through the optical fibre, making these nanotweezers totally autonomous and free of bulky optical elements. The achieved trapping performances allow for the trapped specimen to be moved over tens of micrometres over a period of several minutes with very low in-trap intensities.
3:00 AM - *H11.02
Single-Molecule Analysis with Nanomechanical Systems
Michael L. Roukes 1
1California Institute of Technology Pasadena USA
Show AbstractNanoelectromechanical systems (NEMS) resonators can detect inertial mass with unprecedented sensitivity - now down to the near-atomic scale. We have used this attribute to realize a new method for single-molecule mass spectrometry in real time: as each arriving molecule adsorbs upon a NEMS sensor, its mass and position-of-adsorption are determined by continuously tracking two driven vibrational modes of the device. Our NEMS-enabled analyses of individual large-mass biomolecular complexes, one-by-one in real-time, have demonstrated the power of this new method.
Recently we discovered a way to greatly enhance the analytical capabilities of real-time, NEMS-based measurements of single molecules. This paradigm shift enables imaging the spatial distribution of the mass of individual analytes - in real time and with molecular-scale resolution - when they adsorb onto a nanomechanical resonator. Each single-molecule adsorption event induces discrete, time-correlated perturbations to all of the modal frequencies of a NEMS sensor. By continuously tracking a multiplicity of vibrational modes, the spatial moments of mass distribution can be deduced for individual analytes, one-by-one, in real time as they adsorb. The lowest moment of the measured distribution function provide the total analyte mass; higher moments reveal the analyte&’s center-of-mass position of adsorption, its average diameter, and its skew and kurtosis - the latter characterize its molecular shape. Together these acquired moments can be inverted to yield an “inertial image” of each analyte.
NEMS provide a unique and powerful new method for single-molecule analysis: they can resolve neutral species; can provide resolving power that increases markedly for very large masses, unlike existing approaches; are readily scalable to millions of detection channels to provide high sample throughput; and are readily producible en masse by methods of large-scale integration from the semiconductor industry.
4:30 AM - H11.03
Ultra-Sensitive Time-Resolved Infrared Spectroscopy of Biomolecule Interactions with Plasmonic Nanoantennas
Ronen Adato 1 Hatice Altug 2
1Boston University Boston USA2Ecole Polytechnique Federale de Lausanne Lausanne Switzerland
Show AbstractInfrared (IR) absorption spectroscopy directly probes the vibrational modes associate with the molecular bonds in a sample by measuring absorption in the mid-infrared spectral region, ~ 3 - 20 microns. IR spectroscopic measurements are thus intrinsically endowed with a level of chemical specificity and information content far exceeding that of most other optical measurement techniques.[1] Despite their potential, IR absorption measurements suffer not only from limited sensitivity, but are severely hindered by the strong, broad absorption of water that overlaps the bands of most organic compounds of interest. While recent surface enhanced infrared absorption (SEIRA) spectroscopy measurements have shown that IR resonant nanoantennas can be leveraged to dramatically increase sensitivity, [2-3] these have all been performed in dry environments and without time-resolution.
In this work we show that utilizing plasmonic nanoantennas not only to for intensity enhancement, but also to efficiently localize and redirect incident radiation enables a highly sensitive, versatile means to perform SEIRA measurements while discarding interfering water absorption.[4] This opens the door for highly sensitive measurements of proteins and other biological significant molecules and nano particles in their native environment.
These capabilities are ideally suited to time-resolved studies of biomolecules and other chemical species at the monolayer level. We demonstrate the performance of our approach by monitoring protein-binding interactions via their specific amide band absorption. Highlighting the exquisite chemical sensitivity of infrared spectroscopy, we also perform measurements on chemically distinct particles. Here we are able to chart the movement of various molecular groups down to the displacement of minute volumes of water.[4]
Our plasmonic technology thus overcomes the limitations of infrared spectroscopy in performing sensitive measurements on trace samples in aqueous environments. Finally, unlike traditional, bulky infrared sampling accessories based on traditional effects such as total internal reflection, our plasmonic approach is chip-based and represents a dramatic improvement in the compatibility of infrared absorption spectroscopy with modern sample preparation and handling technologies, as well as complementary optical measurement techniques.
References:
[1] Barth, A. Infrared spectroscopy of proteins. Biochim Biophys Acta. 1767, 1073-1101 (2007).
[2] Neubrech, F, et al. Resonant plasmonic and vibrational coupling in a tailored nanoantenna for infrared detection. Phys Rev Lett, 101, 157403 (2008)
[3] Adato, R, et al. Ultra-sensitive vibrational spectroscopy of protein monolayers with plasmonic nanoantenna arrays. Proc Natl Acad Sci USA, 106, 19227-32 (2009)
[4] Adato, R and Altug, H. In-situ ultra-sensitive infrared absorption spectorsocpy of biomolecule interactions in real time with plasmonic nanoantennas. Nat Commun. 4, 2154, (2013).
4:45 AM - *H11.04
Magnetic Sensing Technology - A New Biosensor Platform for Medical Diagnosis
Hakho Lee 1
1Massachusetts General Hospital Boston USA
Show Abstract
A major challenge in medicine is the rapid and accurate measurement of protein biomarkers, cells, and pathogens in biological samples. Biosensors based on magnetic detection emerge as a promising diagnostic platform. Due to the intrinsically negligible magnetic susceptibilities of biological targets, magnetic detection experiences little interference from native biological samples; even optically turbid samples will often appear transparent to magnetic fields. Biomolecules or cells of interests, when magnetically labeled, however, can attain a high contrast against complex biological background. This presentation will review such magnetic sensing technologies, specifically focusing on a general detection platform termed diagnostic magnetic resonance (DMR). Similar to clinical MRI, the DMR utilizes magnetic nanoparticles to modulate the spin-spin relaxation time of neighboring water molecules. Various assay configurations and nanoparticles have been designed to detect a wide range of targets including DNA, mRNA, proteins, enzymatic activity, metabolites, drugs, pathogens, exosomes and tumor cells. Recently, the capabilities of DMR technology have been considerably advanced with the development of a miniaturized, chip-based NMR detector system that is capable of performing highly sensitive measurements on microliter sample volumes and in a multiplexed format. With these and on-going advances in system design, the DMR technology holds great promise as a high-throughput, low-cost, and portable platform in clinical and point-of-care setting.
5:15 AM - H11.05
Ultrahigh-Speed Rotary Nanomotors for Tunable Biochemical Release
Kwanoh Kim 1 Xiaobin Xu 2 Jianhe Guo 2 Donglei Fan 1 2
1The University of Texas at Austin Austin USA2The University of Texas at Austin Austin USA
Show AbstractThe development of rotary nanomotors is crucial for advancing nanoelectromechanical system (NEMS) technology. In this work, we report design, assembly and rotation of ordered arrays of nanomotors. The nanomotors are bottom-up assembled from nanoscale building blocks with nanowires as rotors, patterned nanomagnets as bearings and quadrupole microelectrodes as stators. Arrays of nanomotors rotate with controlled angle, speed (over 18,000 r.p.m.) and chirality by electric fields. Using analytical modelling, we reveal the fundamental nanoscale electrical, mechanical and magnetic interactions in the nanomotor system, which excellently agrees with experimental results and provides critical understanding for designing metallic NEMS. The nanomotors can be continuously rotated for 15 h over 240,000 cycles. They are applied for controlled biochemical release and demonstrate releasing rate of biochemicals on nanoparticles can be precisely tuned by mechanical rotations. The innovations reported in this research, from concept, design and actuation to application, are relevant to NEMS, nanomedicine, microfluidics and lab-on-a-chip architectures.
H9: Micro/Nano Engineering for Cell Mechanics and Physical Oncology
Session Chairs
Wednesday AM, December 03, 2014
Sheraton, 2nd Floor, Back Bay A
9:00 AM - *H9.01
Enabling Plasmon Resonance Molecular and Cellular Imaging on Ubiquitous Laboratory Equipment by Colorimetric Nanoplasmonic Device
Gang Logan Liu 1
1University of Illinois at Urbana-Champaign Urbana USA
Show AbstractLabel-free detection based on optical techniques has revolutionized the ability to detect a broad range of biological samples such as protein-protein interaction, DNA hybridization and cell membrane dynamics simply based on the intrinsic dielectric permittivity of samples without the need for labeling. In particular, label-free optical techniques such as surface plasmon resonance, photonic crystal, and ring resonator have been integrated with microfluidics to measure the changes in the refractive index near the surface of the sensor. However, these techniques require complex instrumentation for illumination and detection such as high-resolution spectrophotometer, high-intensity monochromatic light source, and prism coupling. In contrast, optical sensors consisted of nanohole array on a noble-metal film, which exhibit extraordinary optical transmission (EOT), can be used with similar sensitivity to detect the changes in the refractive index near the surface of the sensor, with the advantage of simple collinear broadband illumination and portable spectrophotometer. Previously, we have reported colorimetric surface plasmon resonance imaging using a nanohole array device called nanoLycurgus Cup Array (nanoLCA), in which we demonstrated high spectral sensitivity of the nanoLCA to the changes in the refractive index due to the presence of different refractive index solutions and to the surface binding of biological-relevant molecules such as protein-protein interaction and DNA hybridization. Due to the unique transmission/reflection peak wavelengths of nanoLCA in the visible wavelength range, we were able to demonstrate visible colorimetric changes simply due to the changes in the refractive index on and near the surface of the sensor upon the changes of the presence or the concentration of the sample of interest. In this work, we extended the capability of nanoLCA to on-chip applications by integrating relevant microfluidic and micro-well plate designs, such as parallel flow channels, droplet generator, and by demonstrating colorimetric visualization of static and transient dynamics of optically-transparent solutions, cell membrane dynamics and molecular binding events which previously could not be visualized in a colorimetric manner using ubiquitous laboratory equipment such as bright field microscope and plate reader.
H12: Poster Session II: Organic Biodevices and Biophysics
Session Chairs
Wednesday PM, December 03, 2014
Hynes, Level 1, Hall B
9:00 AM - H12.01
Organic Conversion Reactions with High Selectivity and Yield Using Photocatalytic Microreactor
Keisuke Yoshida 1 Akari Nakamura 1 Tomoka Ishiwata 1 Shota Kuwahara 1 Kenji Katayama 1
1Chuo university Tokyo Japan
Show AbstractIn general, the photocatalytic reactions are known as decomposition of organic molecules due to direct redox reactions with photoexcited electrons or holes or by active oxygen species generated from H2O and O2. In recent years, not only such decomposition reactions, but also organic syntheses have been proposed, but they typically need long reaction times like hours. Then, their selectivities and yields are still low in many cases due to the side reactions and the consecutive reactions followed by the main reactions. On the other hand, we have developed a photocatalytic microreactor in which photocatalytic materials are coated in the inside the quartz capillary,. Due to the high surface/volume ratio, this reactor is advantageous for photocatalytic reactions. Furthermore, we have made a device of the measurement of an absorption spectrum inside the photocatalytic microreactor, which could monitor the progress of the photocatalytic reactions inside it in real time. [1] In addition, we have developed a quantitative and qualitative analysis method for a small amount of reactants and products. By using the device and the analysis methods, we successfully established organic reactions with high selectivity and yield. [2], [3] At present, to increase the throughput of each reaction, we are developing a photocatalytic microchip using a commercially available glass slide with cannels sanded with a diamond drill which has a larger volume than that of the capillary. Here, we report on some examples of photocatalytic organic reactions we have developed.
[1] N. Tsuchiya, K. Kuwabara, A. Hidaka, K. Oda, K. Katayama, Phys. Chem. Chem. Phys. 14, 4734 (2012).
[2] K. Katayama, Y. Takeda, K. Shimaoka, K. Yoshida, R. Shimizu, T. Ishiwata, A. Nakamura, S. Kuwahara, A. Mase, T. Sugita, M. Mori, Analyst 139, 1953 (2014)
[3] K. Shimaoka, S. Kuwahara, M. Yamashita K. Katayama, Anal. Sci. 30, 619 (2014)
9:00 AM - H12.02
Engineering Remotely Triggered Liposomes to Target Triple-Negative Breast Cancer
Alexandra Sneider 1 Fulya Ekiz Kanik 2 Adeyinka C. Adejumo 2 Ikjot Singh Sohal 3 Prakash Rai 1 2 3
1University of Massachusetts Lowell Concord USA2University of Massachusetts Lowell Lowell USA3University of Massachusetts Lowell Lowell USA
Show AbstractRemote triggering of theranostic nanoconstructs (TNC) through photodynamic therapy (PDT) containing verteporfin or Benzoporphyrin derivative monoacid (BPD) may prove an effective method for targeting triple-negative breast cancer (TNBC) cells. TNBC has been shown to respond to chemotherapy, but has continued to have the worst prognosis among breast cancer patients. Many of the chemotherapy drugs are poorly water-soluble and are administered unmediated to the body, resulting in limited effectiveness and increased toxicity. The addition of surfactants to improve the effectiveness of direct drug delivery can create severe dose-limiting toxicity. Liposomes, an engineered biological TNC that can encapsulate both hydrophobic and hydrophilic drugs, are a practical solution to these solubility issues and are already clinically approved for the treatment of cancer. Drug loaded liposomes modified with ligands to specifically target cancerous cell receptors may reduce damage to healthy tissue. In this study, free BPD, passive PEGylated BPD-loaded liposomes, and folate targeted PEGylated BPD-loaded liposomes are administered to breast cancer cell line (MDA-MB-231) in vitro. Polyethylene glycol (PEG), covalently bonded to the liposomes, aids in the solubility and delivery of the nanomedicine by potentially lowering the immune reaction caused by the liposomes (in vivo). BPD-loaded liposomes are synthesized and characterized in terms of size, zeta potential, and drug encapsulation efficiency. PDT remotely irradiates light at a specific wavelength to trigger a non-toxic photosensitizer, BPD, to initiate the formation of toxic molecular species that destroy tumor cells. PDT is an effective remote trigger, and further adds to the capability of this approach to selectively target cancer. This study will observe the toxicity of BPD to the cell line before and after PDT in both monolayer and 3D culture. Results from this study will be presented. Similar procedures for encapsulation may be applied to other cancer drugs, like Curcumin that can kill cancer cells in the presence and absence of light, to treat other forms of cancer. Future studies can compare the effectiveness and toxicity of TNC loaded with multiple drugs.
9:00 AM - H12.03
Micropillar Array Embedded System for Single Cell Encapsulation in Hydrogel
Kyun Joo Park 1 Kyoung G. Lee 2 Seunghwan Seok 1 Bong Gill Choi 3 2 Seok Jae Lee 2 Do Hyun Kim 1
1KAIST Daejon Korea (the Republic of)2National Nanofab Center Daejon Korea (the Republic of)3Kangwon National University Samcheok Korea (the Republic of)
Show AbstractIntegration of single-microbial cell encapsulation with hydrogel becomes important for the discovery of valuable biological species for various applications. To be practical, screening the target cells from their heterogeneous cell population is essential. Traditional method required complicated processes with numerous bench-top equipments including external filters and centrifuges for single-cell analysis and sorting. Recently, the combination of microfluidic system, hydrogels, and fluorescence-activated cell sorting system (FACS) has opened a new door to overcome technical limit in the handling and isolation of cells.
With microfluidic system, previous single-cell researches mainly focused on spherical cells with low cell concentration. However, most of valuable biopharmaceuticals and biomaterials are obtained from non-spherical shaped microbial cells. Additionally, polymer-based hydrogel is rarely associated with single-cell isolation due to its property to induce cellular aggregation although hydrogel provided bio-friendly environment for cell culturing and drug screening platform. New breakthrough to overcome this weakness is desirable to utilize the benefits from both the microfluidic system and the hydrogel to handle non-spherical entities such as rod-shaped bacterial cells.
In this research, micropillar arrays were employed to disperse the cell aggregations effectively. Installed micropillar arrays played as filter to break E. coli cluster into single-cell level with the aid of flow around pillars. The clusters were collided sequentially with three different sizes of pillars and split into smaller size of clusters. Stepwise break up of cluster was achieved by the combined effect of cluster collision with pillar, hydrodynamic drag and elongation force from surrounding fluid. Thus, the microstructure incorporation and their unique features can be a key factor to enhance cell dispersion and encapsulation efficiency.
Herein, we developed a strategy to enhance the dispersion of cylindrical-shaped microbial cells by embedding micropillar arrays in microfluidic device and to encapsulate an individual cell in hydrogel particles for potential applications. The integrated micropillar arrays had different dimensions to separate the different size of cell cluster which is induced from the presence of polymers. Those pillar arrays and flow induced hydrodynamic forces enable the isolation of cells from the clusters through continuous breakup and the increase of the single-cell encapsulation efficiency. This pillar aided approach demonstrated the applicability of the device in wide range of cell concentration for the isolation and encapsulation of a cell in hydrogel for potential application.
9:00 AM - H12.04
Single Cell DNA Damage/Repair Assessment with HaloChip
Yong Qiao 1 Ming Su 1 Liyuan Ma 1
1Northeastern University Boston USA
Show AbstractWe have developed a single cell array based DNA damage assay in which mammalian cells are attached on an array of micro-fabricated (micro-contact printing) patterns through electrostatic interactions. After treatments with drugs, cells are patterned on substrate, trapped in agarose gel and lysed with alkaline solution. Under such condition, damaged DNA fragments diffuse out of the nucleus and form a halo around each cell. The results can be observed fluorescently after labeling DNA with SYBR green I dye. In this approach, cells and halos can be analyzed without overlapping issue. The level of DNA damage can be quantified by determining sizes of halo and nucleus with nuclear diffusion factor using MATLAB software. This method has been used to assess DNA damage induced by anticancer drugs, ionizing radiation, metal ions, and nanomaterials. Compared to existing DNA damage assays, HaloChip assay is more sensitive, flexible and reliable, and takes less time. It can also be used on printing paper for point-of-care diagnosis.
9:00 AM - H12.05
Nanoceria Induce miR-146a Expression in Diabetic Wounds
Irina Kalashnikova 2 3 Junwang Xu 3 1 Carlos Zgheib 3 1 Soumen Das 2 Sudipta Seal 2 Kenneth W Liechty 1 Laurene Tetard 2
1Sanford Burnham Medical Research Institute Orlando USA2UCF Orlando USA3Nemours Childrenamp;#8217;s Hospital Orlando USA
Show AbstractBackground: Diabetic wounds have been shown to have an acute inflammation during the wound healing response. Previous data indicated that Cerium Oxide Nanoparticles (nanoceria) treatment can improve healing in skin wounds of mice. However, the mechanisms by nanoceria improve wound repair is not clear. MicroRNAs (miRNAs) regulate the translation of mRNAs at the post-transcriptional level. Specifically, miR-146a has been shown to be an inflammatory brake in diabetic wounds. We hypothesize that nanoceria can attenuate inflammation in diabetic wounds by altering miR-146a expression.
Methods: 8mm full-thickness wounds were created on the flank of 12 week-old diabetic (Db/Db) and non-diabetic (Db/+) mice with a dermal punch instrument, together with an intradermal injection of 50ul of 10uM nanoceria, or vehicle. The wounds were harvested 7days after injury for total cellular RNA. Gene expression was analyzed using Real-time PCR.
Results: MiR-146a was significantly down-regulated in diabetic wounds compared to non-diabetic wounds. Wounds treated with nanoceria noticeably up-regulated miR-146a expression in both diabetic and non-diabetic wounds. Furthermore, increased miR-146a levels closely correlated with decreased gene expression of pro-inflammatory factors such as IRAK1, TRAF6, NFkb, IL6, and MIP2 in the wounds.
Discussion: These findings explore the mechanisms of nanoceria on wound repair. It provides evidence that down-regulated expression of miR-146a, in diabetic wounds may be responsible, in part, for the increased inflammation in diabetic wounds. In addition, nanoceria treatment may improve diabetic wound healing by increasing expression of miR-146a.
9:00 AM - H12.06
Synthesis and In Vitro Characterization of Poly(1,2-glycerol carbonate)-graft-Succinic Acid-Paclitaxel Conjugate Nanoparticles
Iriny Ekladious 1 Heng Zhang 2 Mark W. Grinstaff 1 2
1Boston University Allston USA2Boston University Boston USA
Show AbstractPolymeric nanoparticle (NP) drug delivery systems possess several advantages over conventional small molecule chemotherapeutics. Among these is the ability to provide controlled and sustained drug release, specific drug targeting and delivery, and high loading of insoluble agents such as paclitaxel (Pax). Pax is one of the most widely used chemotherapeutic agents for a variety of solid organ malignancies (lung, ovarian, breast, head and neck cancers, and advanced forms of Kaposi&’s sarcoma). However, despite its widespread use, Pax suffers from poor solubility, rapid systemic clearance, limited tumor exposure, and low target tissue concentrations (~0.4% of the systemically administered dose). Due to its poor aqueous solubility, Pax is often delivered in a Cremophor EL (C/E) adjuvant; and C/E itself is known to cause adverse side-effects and hypersensitivity reactions. We have engineered a novel poly(1,2-glycerol carbonate)-graft-succinic-acid-paclitaxel (PGC-Pax) NP system in which Pax can be incorporated at high loadings (>60 wt%). Additionally, the polymer backbone is readily degradable and biocompatible, with glycerol, succinic acid, and carbon dioxide as the degradation byproducts. Reducing the carrier material and maximizing the drug content departs from current Pax drug carriers that typically contain <10 wt% drug loading. We herein demonstrate the synthesis and in vitro characterization of PGC-Pax NPs, including the dose-dependent cytotoxicity of these particles in MDA-MB-231 cells. We also show the internalization of rhodamine labeled PGC (PGC-Rho) NPs in these cells via flow cytometric analysis and confocal microscopy.
9:00 AM - H12.07
Heuristics Applied the Fermatrsquo;s Principle
Yessica Yazmin Calderon Segura 1 Gennadiy Burlak 1
1Centro de Investigaciamp;#243;n en Ingenieramp;#237;a y Ciencias Aplicadas Mexico Mexico
Show AbstractOur numerical simulations have shown the transition between energy levels governed by Fermat's principle for found the sample irradiated in a solid with a laser beam,We systematically study the results of a heuristic algorithm are presented for calculating emissions from a laser nano in within a fractal percolation cluster. Our numerical simulations have shown the transition between energy levels governed by Fermat's principle in the sample irradiated in a solid with a laser beam, energy of the electrons is observed to generate an electromagnetic field that defines the beam excited state and by applying the traveling salesman problem is the minimum path of nano radiated emission for each sample used.
9:00 AM - H12.08
2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) as a Novel Fusion Expression Partner to Improve Solubility of Aggregation-Prone Proteins
Yoonsik Kang 1 Jong-am Song 1 Jeewon Lee 1
1korea university Seoul Korea (the Republic of)
Show AbstractOne of the effective methods for the production of active recombinant proteins in Escherichia coli is fusion expression using solubility enhancer proteins. The development of a novel fusion expression partner that can be applied to various aggregation prone proteins is crucial importance.
In our previous study, two-dimensional electrophoresis (2-DE) was employed to systematically analyze the E. coli BL21(DE3) proteome profile in response to heat treatment, and KHG/KDPG aldolase (EDA) was identified as a heat-responsive (i.e. aggregation-resistant) protein. When used as fusion expression partner, EDA increased the solubility of seven aggregation-prone heterologous proteins in the E. coli cytoplasm. The efficacy of EDA as a fusion expression partner was evaluated through the analysis of secondary structure or bioactivity of several target proteins. EDA-fusion expression resulted in the synthesis of bioactive bacterial arginine deiminase and human ferritin light chain and the formation of correct secondary structure of human granulocyte colony stimulation factor.
When used as a fusion expression partner, EDA dramatically enhanced the cytoplasmic solubility of seven heterologous aggregation-prone protein. Bioactivity or correct secondary structure of several target proteins were also confirmed.
9:00 AM - H12.09
Enhanced Solubility of Aggregation Prone Heterologous Proteins by Fusion Expression Using Stress Induced Escherichia Coli Protein, CysQ
Jeong-Hyeok Kwon 1 Jeewon Lee 1 Ji Yun Lee 1 Jong-Hwan Lee 1
1Korea University Seoul Korea (the Republic of)
Show AbstractWhen used as an N-terminal fusion expression partner, the E. coli stress-responsive protein, CysQ dramatically increased the cytoplasmic solubility of various aggregation-prone heterologous proteins: Pseudomonas putida cutinase (CUT), arginine deiminase (ADI), human activation induced cytidine deaminase (AID), human granulocyte colony-stimulating factor (hG-CSF), human ferritin light chain (hFTN-L), human interleukin-2 (IL2), and deletion mutant of human glutamate decarboxylase (GAD448-585). This is likely due to the intrinsic ability of CysQ to form its native conformation, probably promoting the binding of molecular chaperones during the folding of CysQ-fusion protein. When used as a substrate, p-nitrophenyl butyrate (PNB) was successfully hydrolyzed to p-nitrophenol by CysQ-CUT fusion mutant. In the cases of hFTN-L and hG-CSF, solubility, bioactivity, and/or secondary structure were successfully maintained even after CysQ was removed. Conclusively, it seems that CysQ is a highly effective solubility enhancer and fusion expression partner for the production of a variety of bio-active recombinant proteins.
9:00 AM - H12.10
Comparison of Microfluidics Strategies in the Preparation of the Alginate Microparticles
Anna Pittermannova 1 2 Frantisek Stepanek 1 Jerome Bibette 2
1Institute of Chemical Technology Prague Prague Czech Republic2amp;#201;cole Supamp;#233;rieure de Physique et de Chimie Industrielles Paris France
Show AbstractRecent research in the drug delivery systems has resulted in the first templates of alginate composite microparticles, which are able to release active component encapsulated in the particles. These composite alginate microparticles were prepared by the Ink-jet method with a smallest achievable size around 40 µm. For further use of these particles in the medicine, their adhesion properties in the living tissues will be a necessary step to investigate. It is therefore desirable to decrease the particle size of the alginate microparticles to be not larger than size of the red blood cell, which is approximately 6 µm.
Microfluidic technique is one of the possible ways how to produce such small microparticles. At the beginning we count into two main microfluidic strategies to produce alginate microparticles. The first possibility is to synthesize particles right on the chip using flow focusing device where the continuous phase is 1-undecanol with Ca2+ ions and disperse phase is aqueous solution of alginate. Flow focusing regime is producing w/o emulsion and gelation of particles is started due to diffusion of Ca2+ ions from continuous phase to droplets. Second way is to synthesize particles in a two-step process. In the first step, w/o emulsion of droplets with required sized were prepared on membrane chip by step emulsification where the disperse phase is 1% of alginate solution and continuous phase is mineral oil with surfactant. Emulsion is collect and gelation is established outside of the chip by introducing calcium ions. These two methods were compared and evaluated.
9:00 AM - H12.11
Experimental and Theoretical Study on the Electrospinning Nanoporous Fibers Process
Lan Xu 1 Xiao Peng Tang 1 Hong Ying Liu 1
1Soochow University Suzhou China
Show AbstractPore structure and connectivity determine how porous materials perform in applications such as adsorption, separation, filtering, catalysis, fluid storage and transport, electrode materials or as reactors. Porous materials can be prepared by sol-gel method, hydrothermal synthesis method, electrospinning and other methods. Electrospinning has been recognized as a simple and efficient technique for the fabrication of polymer nanofibers. Recently, research shows electrospun porous materials can be obtained simply by adjusting electrospinning parameters or postprocessing surface treatments. The porous structure further enlarges the specific surface area and enhances the hydrophobic property of the electrospun nanofibers, which alters the performance of the electrospun nanofibers greatly.
In this paper, the electrospun nanoporous fibers process was investigated experimentally as well as theoretically. To research the formation mechanism of electrospun porous nanofiber, a simplifying gas-liquid two-phase flow model was established. Based on the model, the effects of various spinning parameters on quality of product, such as the number of nanopores and diameter, will be systematically carried out. With the increase of the flow rate or the decrease of the applied voltage and the collect distance, the diameter of nanofibers decreased. The theoretical analysis results were further verified according to the experimental data. In addition, Bernoulli equation was used to study the electrospinning “splaying” process. As the jet accelerates and thins in the electric field, radial charge repulsion results in splitting of the primary jet into two filaments in the electrospinning process, we call this phenomenon as splaying. We found the ratio of pore width to pore length is varied along with the variation of the internal pressure of the jet, and the internal pressure of the jet increases with the velocity of the charged jet decreases. When the radial charge repulsion becomes larger the primary jet splitting into two filaments, the ratio of pore width to pore length decreases, the pores of the fiber will collapse.
9:00 AM - H12.12
Fabrication of Arbitrarily Shaped Nanofibrous Micropatterns and Their Hybridization with Hydrogels for Cell Encapsulation
Wei Song 1 Duo An 1 Der-I Kao 2 Yen-Chun Lu 1 Shuibing Chen 2 Minglin Ma 1
1Cornell University Ithaca USA2Weill Medical College of Cornell University New York USA
Show AbstractElectrospun fibers have attracted intensive research interests in the past couple of decades and found broad applications. Generally, the electrospun fibers have random nonwoven structures, resulted from the whipping motion of the electrospinning jet. However, it is highly desirable to endow these materials with more spatially organized architectures in order to engineer more functional structures and devices. Tremendous efforts have been made to manipulate electrospun fibers in spatially organized ways. Despite of varying degrees of successes, limitations still exist in the robustness and versatility of reported methods.
In this study, we report a new, versatile, and robust approach to fabricating nanofibrous micropatterns, particularly microposts and microwells, with arbitrary geometries. The key is the use of an intrinsically conductive and ductile low-melting temperature metal alloy that can be micropatterned, through simple imprint lithography, into arbitrary shapes with a relatively high resolution. When used as the substrate to collect the nanofibers, the alloy allowed conformal deposition of fibers on its topographical features. To our knowledge, this is the first report that metal alloy was micropatterned and used as a template to make hierarchical nanofibrous structures. We demonstrated various mechanically robust, free-standing nanofibrous microposts and microwells. In contrast to the reported microwells with smooth surface, these nanofibrous microwells structurally resemble the extracellular matrix and hence represent a biomimetic platform for high throughput cell culture. Additionally, we demonstrated that these nanofibrous microposts and microwells could be used as structural frames to form robust hydrogel micropatterns that may otherwise be fragile on their own for cell encapsulation applications. We showed that either non-adherent or adherent cells could be readily encapsulated in these hybrid micropatterns. The nanofibrous frames not only enhance the integrity of the hydrogel but also facilitate the formation of hydrogel micropatterns which in turn increase the surface area for mass transfer and cell loading.
We believe that this approach will facilitate the fabrication of hierarchical nanofiber structures of any microtopography, and the nanofibers-framed hydrogel micropatterns will have great applications in cell encapsulation and regenerative medicine.
9:00 AM - H12.13
Bioinspired Silk Fibroin/Octacalcium Phosphate Composite Coatings Biomaterials with Highly Ordered Nano-Micro Multiscale Structure
Hui Wang 2 1 Feng-Yi Yan 2 Guo-Qiang Chen 2 Ke-Qin Zhang 2 1
1Soochow University Suzhou China2Soochow University Suzhou China
Show AbstractNatural bone exhibits fascinating multi-functionality, originating from its hierarchical structure and multiple chemical compositions, which mainly consist of calcium phosphate (CaP) and organic protein matrix (collagen).1 Inspired by natural bone, the fabrication of hierarchical structured composite materials has turned out to be one of the crucial steps in tissue engineering to provide the vital framework for the seeded cells to organize into a functioning tissue.2Bombyx mori (silkworm) silk has been attracting much attention in biomaterials fabrication, due to its biocompatibility, robust biomechanical features, and relatively slow biodegradation, as well as sufficient supply based on the established mature sericulture industry.3 Octacalcium phosphate (Ca8H2(PO4)6middot;5H2O, OCP) is a significant calcium phosphate regarded as a precursor of biological apatite, and eventually converts into hydroxyapatite.4 In this study, silk fibroin (SF) and OCP have been adopt to construct a highly ordered nano-micro multiscale composite coatings on a titanium (Ti) substrate under electric field induced effect. Findings indicate that SF plays an important role in controlling the OCP crystal growth behavior. More interestingly, the samples exhibit the gradual change of the surface wettability from superhydrophilic to hydrophobic regimes with the SF concentration increasing. In vitro cell culture test demonstrates that the the nano-micro multiscale porous structure within the OCP/SF coatings may promot the cells adhesion, spreading and proliferation. The coatings developed in this study may have considerable potential for bone tissue engineering applications.
[1] M. M. Stevens, Mater. Today, 2008, 11, 18
[2] H. Wang, X. Y. Liu, Y. J. Chuah, J. C. H. Goh, J. L. Li, H. Y. Xu, Chem. Commun.2013, 49, 1431.
[3] F. G. Omenetto and D. L. Kaplan, Science, 2010, 329, 528.
[4] M. Iijima, H. Kamemizu, N. Wakamatsu, T. Goto, Y. Doi, Y. Moriwaki. J. Cryst. Growth., 1997, 181, 70.
9:00 AM - H12.14
Preparation of a Nano-Patterned Polymer Replica for Reducing Catheter Associated Infections
Luting Liu 1 Batur Ercan 1 Linlin Sun 1 2 Thomas Webster 1
1Northeastern University Boston USA2Northeastern University Boston USA
Show AbstractIntroduction: Nowadays catheter associated infections are the most serious and costly of all healthcare-associated infections. It is the hypothesis of this study that polydimethylsiloxane (PDMS), a commonly used catheter material, can be formulated to mimic the nano-patterned topography of natural tissue and decrease infections while remaining non-toxic to mammalian cells. Here we present a simpe and cheap method to prepare a nano-pattened PDMS replica by using highly ordered nanotubular anodized titanium (ATi) as the template.
Materials and Methods: For anodization, an etched Ti sample was used as an anode, while a platinum (Pt) mesh served as a cathode. Both were immersed in an electrolyte solution consisting of 1.5% hydrofluoric acid and were connected to a DC power supply. Next, the PDMS slurry was cast onto the ATi template and then was placed into a vacuum chamber for 1h. The PDMS sample was cured at 60 °C for 2h followed by cooling and was gently peeled away from the Ti template.
Human fibroblasts (ATCC CCL-110) at population numbers less than seven were cultured in Eagle&’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Cells were seeded onto the substrates at a density of 5000 cells/cm2 and were allowed to adhere for 4 h in a 37°C, humidified 5% CO2 atmosphere. MTT assays were used in this study.
Staphylococcus aureus and Escherichia coli (ATCC 25923 and 25922, respectively) were cultured in fresh tryptic soy broth (30 mg/mL). Sterile samples were treated with the prepared bacterial solution (×107) and cultured for either 24h or 48h in an incubator (37°C, 5% CO2, humidified). Afterwards, the samples were placed into 15 mL tubes with 4 mL PBS. These tubes were shaken at 3000 rpm for 15 minutes on a vortex mixer to release the bacteria attached on the surface into the solution. Solutions with bacteria were spread onto agar plates and bacteria colonies were counted after 18h of incubation. All the tests were performed in triplicate and repeated three times.
Results and Discussion: As expected, the nano-sized tubes were distributed uniformly on the Ti surface after anodization and nano-patterned structures were fabricated successfully on the surface of PDMS by using ATi as the template. It was found that the nano-patterned PDMS templates increased fibroblast adhesion by 20% and, thus, were non-toxic to fibroblasts. Results also showed that nano-patterned PDMS inhibited S. aureus growth by 70.5% and 79.2% after 24h and 48h, respectively. Moreover, data suggested decreased E. coli by 50% after 48h.
Conclusions: Nano-patterned structures were successfully fabricated on the surface of PDMS through an ATi template method. Furthermore, the nano topography on PDMS could inhibit S. aureus and E. coli growth significantly without using antibiotics and while remaining non-toxic to fibroblasts, and thus should strongly be considered for use in catheters.
9:00 AM - H12.15
3D Biocompatible Microenvironments to Evaluate MCF-7 Cell Development
Oriana I A Salas 1 Adriano J G Otuka 1 Laura M de Freitas 2 Vinicius Tribuzi 1 Carla R Fontana 2 Cleber R Mendonca 1
1Instituto de Famp;#237;sica de Samp;#227;o Carlos - Universidade de Samp;#227;o Paulo Sao Carlos Brazil2Department of Clinical Analysis, School of Pharmaceutical Sciences - UNESP Araraquara Brazil
Show AbstractCell growth monitoring is fundamental for understanding a variety of physiological processes. In order to replicate natural cell environments, it is necessary to produce structures with accurately defined features, since those can influence the attachment, migration, and proliferation of cells. One method that has been recently explored to fabricate microenvironments for cell culture is the use two-photon polymerization (2PP). In this work, we used 2PP to fabricate microenvironments (microstructures arrangements) with distinct geometrical features, to evaluate its influence on the development of Michigan Cancer Foundation-7 (MCF-7) cells, labeled with green fluorescent protein (GFP). Cells were cultured into microenvironments composed of a matrix of structures with different shapes (square and circular cross-section) and spacing between them (12, 18, 24 and 30 µm). The resin used for the microfabrication is composed in equal proportions of two three-acrylic monomers: tris(2-hydroxyethyl) isocyanurate triacrylate and ethoxylated (6) trimethylolpropane triacrylate. While the first one provides hardness to the structure, the later reduces the shrinkage tensions upon polymerization, preventing deformations on the final structure. As the photoinitiator for the polymerization process we used ethyl-2,4,6-trimethylbenzoyl phenylphosphinate, commercially known as Lucirin TPO-L. The monomers are mixed to the photoinitiator for 1 h to obtain a homogeneous solution. We used a Ti:sapphire laser oscillator (790 nm, 100 fs pulses, 82 MHz) to produce the microstructures. A droplet of resin was put between glass coverslips and spacers. The laser beam is focused into the resin using a microscope objective with 0.85-NA (60×). The intensity of femtosecond pulses at the focal volume is high enough to induce two-photon absorption and locally initiate the radical polymerization. The laser is scanned in the x-y direction by a pair of movable mirrors, while the sample's axial (z) positioning is performed by a motorized stage. An illumination source and a CCD camera allow for real time monitoring of the polymerization. After polymerization, the sample is immersed in ethanol to wash away the uncured resin, leaving on the substrate only the fabricated microstructures. Since the fabricated microenvironments provided favorable conditions for cell development, the influence of microenvironments' geometrical features on cells growth was analyzed by transmission and fluorescence microscopies. Our results indicate a dependence of the cell growth on the spacing between the structures on the microenvironment, but not on its shape.
9:00 AM - H12.16
Micro-Molding of Vascularized Liver-Like Tissues with iPS-Derived Hepatic Endoderm Spheroids
Tatsuya Osaki 1 2 Takanori Takebe 3 Junji Fukuda 2
1University of Tsukuba Yokohama Japan2Yokohama national university Yokohama Japan3Yokohama City University Yokohama Japan
Show AbstractEngineering implantable liver tissues is still challenging mainly because of a lack of highly organized vascular networks for blood circulation. To tackle the problem, we propose an approach for rapidly engineering vasculatures using micro-molding technique and subsequently inducing differentiation of human iPS-derived hepatic endoderm cells to hepatocytes.
For the micro-molding of vasculatures, an oligopeptide was designed to spontaneously absorb and form a self-assemble monolayer on a gold surface and to be desorbed from the surface by applying a negative potential [1]. Cells adhering on a gold surface via the oligopeptide layer were detached within 5 min of potential application along with desorption of the layer. We applied this approach to cylindrical gold rods to transfer human umbilical vein endothelial cells (HUVECs) to the internal surface of microchannels (phi;500 um) in a hydrogel. Furthermore, we encapsulated HUVECs, mesenchymal stem cells, and iPS-derived hepatic endoderm cells in the hydrogel between the HUVEC-lined vascular-like structures. In 7 days of perfusion culture, HUVECs form complex vascular networks around iPS-HE spheroids in the hydrogel. Liver-specific functions, such as albumin secretion and ammonia removal, of iPS-HEs spheroids increased overtime and were superior to those in stationary culture. This simple fabrication technique could open the door to a new strategy for engineering vascularized 3D thick tissues and organs.
9:00 AM - H12.17
Fabrication of Collagen Gel Hollow Fibers for Bioengineering of Renal Tubules with Highly Expressed Functions
Chong Shen 1 Qin Meng 1
1Zhejiang University Hangzhou China
Show AbstractThe bioengineering of renal tubules for highly expressing renal functions remains a key challenge but is of great interest for construction of bioartificial kidney. This study, for the first time, proposed the formation of the renal tubules via culturing renal tubular cells on the inner surface of collagen gel hollow fibers (Col HF). The Col HF, whose mechanical strength was acceptable, was fabricated delicately by liquefying the cell entrapped Ca-alginate core and keeping the collagen shell of Ca-alginate/collagen core-shell fibers made by microfluidic device. Within Col HF, human kidney-2 (HK-2) cells could self-assemble into renal tubular structure with diameters of about 50 µm, an equivalent size to renal tubules. The intact hollow lumens of bioengineered renal tubules were evidenced to be tightly distributed by the polar renal cells with distinct apical/basement membranes under hematoxylin-eosin/F-actin staining and transmission electron microscope imaging. The renal tubules in Col HF culture exhibited at least 1-fold higher activity/gene expression on brush border enzymes (alkaline phosphatase and gamma-glutamyltransferase), multidrug resistance protein 2 and glucose uptake than each traditional Transwell culture or hollow fiber membrane culture. In this regard, the bioengineered renal tubules in Col HF that closely mimicked the in vivo structure and expressed high renal functions could sustain great potential in construction of bioartificial kidney for both clinical use and pharmaceutical/biological investigation of kidney.
9:00 AM - H12.18
Polymer Based Nanoelectrode Array for Photosynthetic Electron Extraction from Green Algae Cells
Dasom Yang 1 Hyeonaug Hong 1 Lo Hyun Kim 1 Myungjin Han 2 Young Wook Chang 3 Youngcheol Chae 2 Jae-Chul Pyun 3 WonHyoung Ryu 1
1Yonsei University Seoul Korea (the Republic of)2Yonsei University Seoul Korea (the Republic of)3Yonsei University Seoul Korea (the Republic of)
Show AbstractNature photosynthesis is one of the most efficient mechanisms to convert solar energy into chemical energy. To harvest photosynthetic energy stored in plants, its extraction in the form of biodiesel or bioethanol have been investigated. Recently, extraction of photosynthetic energy in a form of electrons, “bioelectricity”, has been demonstrated with extracted photosystems or fragments of thylakoid membranes that contains photosystem I & II. However, bioenergy harvesting from such extracted photosystems or thylakoid membranes does not last long due to their low stability. In this work, we report polymer based nanoelectrode arrays for direct photosynthetic electron extraction from living algal cells, Chlamydomonas reinhardtii. In order to fabricated high A/R nanoprobe arrays, we employed a thermal drawing technique combined with screen-printed polymer patternning.First, SU-8 2150 was patterned on a glass substrate by a screen printing method using a laser-machined polyimide mask. After soft baking, a heated micro pillar was contacted on the SU-8 patterns and L-shaped nanoprobes were fabricated by modulating drawing speeds, distance, directions, and temperatures. An Au layer was sputter-deposited on the polymer nanoprobes and insulated with paralyene by thermal chemical vapor deposition process. The tip ends of the nanoprobes were surface-cut and the Au layer was exposed by focused ion beam (FIB) Finally, submicrometer band electrode was fabricated. Electrochemical performance of the fabricated polymer nanoelectrodes was verified by cyclic voltammetry (CV) with commercial potentiostat. Using our custom-built single cell electrochemical analysis system, single algal cells were inserted at the tips of each polymer nanoelectrodes. Their photo-sensitive electric signals were measured and analyzed using the chrono amperometry method.
9:00 AM - H12.19
Utilizing Shape Memory Polymers to Generate Complex Wrinkles for Active Cell Culture
Shelby L Buffington 1 Derek Loh 1 James H Henderson 1 Patrick T Mather 1
1Syracuse University Syracuse USA
Show AbstractMany tissues undergo continuous remodeling of the extracellular matrix (ECM) in response to environmental stimuli. However, in vitro biomaterial systems used to probe cell responses have traditionally been static and unable to replicate dynamic environmental changes. To address this gap, we and others have developed shape-memory polymer (SMP) substrates capable of changing topography during cell culture. [1] Guevendiern, et al. demonstrated that cells will respond to biaxial or complex “double” wrinkle patterns on a silicone.[2] Our group demonstrated that wrinkles can be triggered to form by buckling of a gold coating upon an SMP undergoing contraction.[3] Here, we introduce the use of triple shape polymeric composites (TSPC) to allow two sequential recovery events that lead to distinct wrinkle patterns upon the TSPC surface. These patterns may impact cell morphology, migration, and stem cell differentiation.
TSPCs were made using previously reported methods wherein an electrospun poly(ε-caprolactone) (PCL) fiber mat was embedded in a glassy shape memory polymer epoxy.[4] The resulting material allows programming of two temporary shapes, one via PCL crystallization and one via epoxy vitrification, and features one permanent shape set at the point of epoxy crosslinking. To achieve complex wrinkles, two tensile strains were first independently programmed into film samples with variation in the relative strains and angle between the two strains. The samples where then sputter-coated with gold and recovered in a stepwise fashion: first above the epoxy Tg to recover one strain then and above PCL Tm to recover the other strain. During recovery, the gold coating buckled sequentially to form “mixed” wrinkle patterns that were characterized using atomic force microscopy and scanning electron microscopy.
Wrinkle formation was affected by the total amount of strain in each phase, the ratio of the strains between each phase, and the angle between the two programmed strains. We found that the first wrinkles impact the second wrinkles significantly when the angle between the two is large. We postulate that this is due to a geometric stiffening of the coating that impacts the second buckling event. Hypothesizing that cells will display unique responses to these dynamic ECM scale patterns, future experiments will focus on assessing changes in cell morphology, migration behavior, and phenotype differentiation. Compositional modifications will be needed to enable two active transitions under cell culture conditions, enabling future experiments on how cells respond to multiple nano-scale topography transitions.
Acknowledgements: Funding from NSF IGERT Program, DGE-1068780 is gratefully acknowledged.
[1] Davis, K.A.,Biomat. 2011. [2] Guvendiren M, Adv Healthcare Mater. 2013. [3] Yang, P, Soft Matter, 2013. [4] Luo, X, Adv Funct., 2010.
9:00 AM - H12.20
From Tissue Engineering to Growing Artificial Plants in 3D Nano-/Microfibre Scaffolds
Stoyan K. Smoukov 1 CJ Luo 1 Raymond Wightman 2 Elliot Meyer 3
1University of Cambridge Cambridge United Kingdom2Sainsbury Lab Cambridge United Kingdom3Cal Tech Pasadena USA
Show AbstractMan-made nano-/microfibres have a broad range of applications in electronics, ultra-filtration, acoustics, sensors, drug delivery and biomedical engineering. In particular, the culture and manipulation of mammalian cells within a true three-dimensional (3D) scaffold represent a suite of techniques currently the subject of intense development with particular applications in tissue engineering and regenerative medicine for human healthcare. While such fibre scaffolds are emerging as key tools in the study of mammalian biology, their suitability for fundamental plant research has not been explored. The culture of cells and tissues of plants is a field that spans nearly a century with important roles in cell biology and agro-biotech applications. To date, traditional
plant cell culture is limited to obtaining a suspension of cells in aqueous medium without any structural definition. Here we have made use of shear spun micro- and nano-fibre scaffolds to immobilise, or encapsulate, cells from liquid cultures from the laboratory model plant Arabidopsis thaliana. We present data that show, within 72 hours of seeding, cells interact with the fibres and these fibres strongly influence the growth patterns of the cells. We report new patterns of growth that are not normally visible in planta. This work defines a new suite of techniques for the study of plant cells in fibra.
9:00 AM - H12.21
Building Kingdoms: From Tissue Engineering to Culturing Plants in Micro/Nanofibre Scaffolds
C. J. Luo 1 Raymond Wightman 2 Eliot Meyerowitz 3 Stoyan Smoukov 1
1University of Cambridge Cambridge United Kingdom2University of Cambridge Cambridge United Kingdom3California Institute of Technology Pasadena USA
Show AbstractMan-made polymeric nano/microfibres have a broad range of applications in electronics, ultra-filtration, acoustics, sensors, drug delivery and biomedical engineering. In particular, the culture and manipulation of mammalian cells within a true three-dimensional (3D) scaffold represent a suite of techniques currently under intense study, with important applications in tissue engineering and regenerative medicine for human healthcare. The synthesis of suitable scaffolds has been refined over the last 20 years and has benefited from the construction of biomaterials that mimic the animal extracellular matrix (ECM). A scaffold should be biocompatible and provide micropores that permit cell penetration, nano-topography and mechanical properties that are tuneable depending upon the tissue type. These properties can be fulfilled by a scaffold containing both micro- and nanofibres.
While such fibre-scaffolds are emerging as key tools in the study of mammalian biology, their suitability for fundamental plant research has not been explored. We rely on plants not only for air, clean water, building materials, textiles, resins and food, but also for fuel and medicine. The plant and animal cell share a number of biological features. Both have an extracellular matrix, an organ-specific, microscopic environment supporting a diverse range of cell types and functions. At the tissue level, unlike animal cells that lack the cell wall, plant cells grow by turgor driven cell expansion and their shape is dependent upon local cell wall reinforcements and rearrangements.
The culture of cells and tissues of plants is a field that spans nearly a century with important roles in cell biology and agro-biotech applications. Using medium containing a defined combination of synthetic hormones and nutrients, sterile explants from tissues such as root tips and leaf segments form unorganised cell masses known as callus. Further modification of the medium can result in whole organs and whole individuals from this cell mass or can be propagated as isolates in liquid cell culture, that are amenable to subcellular live imaging. One limitation is that these cells are not part of any 3D higher ordered space-limited and immobile structure that properly defines a plant tissue.
Here we have made use of shear-spun micro- and nanofibre scaffolds to immobilise, or encapsulate, cells from liquid cultures from the laboratory model plant Arabidopsis thaliana. We present data that show, within 72 hours of seeding, cells interact with the fibres and these fibres strongly influence the growth dynamics of the cells. We report new patterns of growth that are not normally visible in planta. This work defines a new suite of techniques for the study of plant cells in fibra.
9:00 AM - H12.22
Biocompatible Polarized Polymeric Films for Cell Stimulation and Tissue Regeneration
Paula Maria Vilarinho 1 Marisa Maltez-da Costa 1 Alicia Alves 1 Ana Roque 2 Odete da Cruz e Silva 2 M Helena Fernandes 1
1University of Aveiro Aveiro Portugal2University of Aveiro Aveiro Portugal
Show AbstractType I collagen is the most abundant protein in the skin, tendon and bones. Therefore it has been used to prepare scaffolds for cell adhesion and growth in tissue regeneration studies. Cell interactions with synthetic surfaces are of increasing significance, as they are utilized to mimic in vivo conditions for a variety of healthcare applications, namely in regenerative medicine strategies.
As the vast complexity of tissue regeneration mechanisms is still poorly understood, it is particularly difficult to replicate the native biological mechanisms. As a result, the in vitro assembled collagen substrates evidence a weak osteogenic capacity when compared to that presented by the bone native tissues. [1]
It has been reported that collagen fibers comprised in bone matrix display piezoelectric properties that can stimulate bone regeneration. [2,3] Ensuing this finding we aim to produce biocompatible type I collagen-based biomaterials, modified by electric polarization which are expected to stimulate the growth of new bone tissue at a higher rate and with lower rejection, leading to a faster recover from bone injuries.
In this study, different samples of collagen type I (crystalline, semicrystalline, non-polarized, positively and negatively polarized) were prepared as micronsized films. Samples were characterized by Differential Scanning Calorimetry, Atomic Force Microscopy, Scanning and Transmission Electronic Microscopy, X-Ray Diffraction, Infrared and Wettability analysis. All these assays are useful in establishing and discussing the relations between crystallinity, surface morphology and polarization, for collagen type I substrates.
Viability and proliferation cell assays were then performed using a human osteosarcoma cell line MG-63 and also a human Osteoblast primary cell culture. The results evidence that both MG-63 and Osteoblast cells adhered preferably to the polarized surfaces. Moreover, analysis of cell morphology revealed differences on cell cytoskeleton between polarized and non-polarized surfaces. Cell differentiation and mineralization assay have also been conducted so as to evaluate the effect of polarization onto the cell cultures. We envisage elucidating the versatility and potentiality of these biomaterials to be used in future studies of in vivo bone regeneration.
[1] A.M. Ferreira, P.Gentile, V. Chiono, G. Ciardelli, Acta Biomaterialia, 8, 9 (2012)
[2] Fukada, E.; Yasuda, I. J. Phys. Soc. Jpn. (1957), 12, 1158-1162.
[3] A. Marino, R. O. Becker, Nature 253, 627 (1975).
9:00 AM - H12.24
How the Morphology of Osteocytes Contributes to Their Mechanotransduction near Microdamage
Elisa R Budyn 2 1 Morad Bensidhoum 3 Patrick Tauc 4 Herve Petite 3 Eric Deprez 4
1University of Illinois at Chicago Chicago USA2Ecole Normale Superieure de Cachan Cachan France3University Paris 7 Paris France4Ecole Normale Superieure de Cachan Cachan France
Show AbstractIntroduction: With increasing life expectancy, bone pathologies related to massive bone loss occur later in life and carry $5-$10 billion financial burden on the U.S. healthcare system. Successful techniques for massive tissue regeneration can be however difficult to produce and often require addition of functional materials. Human Haversian cortical bone is a complex hierarchical heterogeneous tissue resulting from continuous remodeling. Microdamage are therefore resorbed by osteoclasts cells before tubular lamellar structures called osteons are formed by osteoblast cells laying Type I collagen fibrils mineralized by hydroxyapatite nano platelet crystals glued together with non-collagen proteins and proteoglycans. Trapped osteoblasts further differentiate into mechano-sensitive osteocytes that are able to sense stimulation produced by microdamage. Osteocytes have the particularity to bear 40 to 60 cytoplasmic processes extending into canaliculi to create a syncytial network with the neighboring cells with which they can transmit signals in a fashion similar to the nervous system. Because osteocytes regulate healthy bone turnover, it is essential to quantify the relationship between in situ mechanical stimulation and the cell biological response.
Materials and Methods: Dual experimental and numerical top-down investigations were applied through micro bending tests conducted on human femoral fresh cadaver samples to produce and image the growth of controlled nascent sub-microscopic damage near live osteocytes. The multiscale local constitutive fracture mechanisms has been identified scale by scale after the balance of the energies at the global scale to evaluate the in situ stress field near bone cells. The finite element model is based on explicit bone tissue morphology and digital image correlation. Multi-modal imaging techniques using SEM, UV and fluorescent confocal microscopy coupled to a hierarchical multi-level numerical simulations contributed to enhance the measurements of the cell cytoskeleton rearrangement and chemicals produced.
Results and Discussion: The numerical model shows nascent diffuse damage within osteon lamellae appearing prior to visible microcracks as local stresses increase from yield values of 58/-77 MPa close to HAP (hydroxyapatite) strength to 108/-216 MPa when mineralized collagen fibers still bridge cracks. These results confirm brittle/ductile fracture behavior of bone. Multi-modal microcrospic observations within osteon lamellae showed microdamage patterns near osteonal lacunae and canaliculi. Neighboring bone ECM and osteocyte cytoskeleton displacements and secreted chemicals are identified by different fluorochromes.
Conclusions: Hybrid experimental and numerical investigations quatified osteocyte lacunae and canaliculi morphology alterations in bone diffuse damage areas that are correlated to nascent sub-microcracks.
Acknowledgements: with the support from NSF CMMI BMMB 1214816 and the Farman Institute.
9:00 AM - H12.25
Stimuli-Responsive Capsules for 3D Spatiotemporal Biomolecular Gradients
Maneesh Kumar Gupta 1 Yong Kong 1 Limei Tian 2 Srikanth Singamaneni 2 Michael C. McAlpine 2
1Princeton University Princeton USA2Washington University St. Louis USA
Show AbstractA major challenge with the in vitro control over growth and differentiation in human primary and stem cells (especially towards forming functional tissues with differentiated structures) is the inability to replicate the complex spatiotemporal chemical gradients that are present in vivo. Extensive research has been conducted to understand the role other environmental cues such as presence of absence of chemical and biological signals, mechanical properties of scaffold, topography of substrate, and spatial clustering of anchoring sites. Currently only a handful of examples have been developed where spatial and temporal chemical gradients are capable of directing cell growth, migration and differentiation. These efforts include microfluidic systems capable of pumping biomolecules into a scaffold and thereby establishing gradients or polymeric scaffold with photolabile functional groups capable of releasing signaling molecules in response to light exposure. While insightful, these examples prove cumbersome to implement as routine practices.
In order to overcome these challenges, we report on the development of stimuli-responsive capsules capable of releasing biomolecules in response to light exposure providing the ability to create 3-dimensional spatiotemporal gradients within a hydrogel matrix. The capsules are fabricated from poly(lactic-co-glycolic acid) PLGA using an additive manufacturing layer-by-layer approach allowing accurate positioning and patterning of the capsules within the matrix while maintaining highly efficient encapsulation of the active biomolecule. The capsules are functionalized with gold nanorods to allow for wavelength selective rupture of the PLGA shell. The use of gold nanorods is advantageous in this application because the longitudinal LSPR peak is length dependent allowing for multiplexed and selective release of biomolecules at different wavelength. We have demonstrated this multiplexed functionality by selective release of horseradish peroxidase and alkaline phosphatase enzymes to demonstrate that the laser excitation and rupture process do not degrade enzyme activity. Additionally, we have demonstrated selective release of small molecules to modulate protein expression in E. coli.
9:00 AM - H12.26
Softening Substrates and Photolithographic Devices toward Chronic Neural Bioelectronics
Walter E Voit 1 2 6 Joseph J Pancrazio 3 Jason Carmel 4 Robert Rennaker 2 Michael Kilgard 2 Kenneth Lee 5 Stuart Cogan 2 Mario Romero-Ortega 2 Orlando Auciello 2 1
1UT Dallas Richardson USA2UT Dallas Richardson USA3George Mason Fairfax USA4Burke-Cornell Medical Research Institute White Plains USA5UT Southwestern Medical Center Dallas USA6UT Dallas Richardson USA
Show AbstractWe demonstrate peripheral and cortical neural stimulators and recording devices using engineered low cure stress softening polymer substrates, photolithography with 2 micron minimum features sizes and microfabrication processes at temperatures up to 300°C. Polymers can be implanted at moduli of more than 1 GPa and soften to approx. 20 MPa. Encapsulation materials include combinations of zwitterions, hydrophobic thiol-based polymers, Parylene-C, Silicon Carbide, Silicon Nitride, Aluminum Oxide and /or Ultrananocrystalline Diamond (UNCD). Neural interfaces allow for delivery of a large amount of information transfer, but current microstimulators or microrecorders fail chronically or are poorly suited for interfacing with small biological structures, such as sensory peripheral nerves. We discuss chronic device failure through design of both materials and devices to overcome various failure mechanisms. Bioelectronics implant failure is defined by the inability to drive sensory perception due to either device breakdown (abiotic mechanisms) or biotic mechanisms. Device survivability is adversely affected by impedance drops due to F1) delamination of insulating materials, F2) diffusion of fluids into the device, and impedance spikes due to F3) electrode corrosion and F4) conductor cracking. These failure mechanisms can be attributed to the aggressive, dynamic mechanical and chemical environment in the body. The dominant biotic failure mechanisms lead to a decoupling of tissue from the device and, as such a loss in device specificity, and are due to F5) insertion damage and chronic damage mechanisms resulting from F6) device geometry, F7) device stiffness, F8) surface chemistry of the foreign material, and F9) cyclic electrical stimulation. Insertion damage results in localized bleeding, death of the tissue of interest and/or changes in local physiology that complicate or wholly eliminate the ability to stimulate the appropriate fascicles and nerve fibers. Chronic damage due to geometry, stiffness and surface chemistry of the foreign material results in scar tissue encapsulation which ultimately electrically insulates the electrodes from the neural activity of interest.
We demonstrate improved device specificity and device survivability by mitigating the nine failure mechanisms through materials design and processing, We demonstrate 350 µV peak-to-peak signals from softening devices in the cortex of lab rats on intracortical electrodes. We demonstrate the effects of self-coiling vagus nerve stimulators and self-wrapping cochlear implants. We demonstrate spinal stimulators that reduce inflammation and tissue response and behave in a manner similar to ball electrodes for use in understanding long-term muscle plasticity. We demonstrate softening peripheral neural interfaces for modulating sensory and motor input toward closed-loop feedback for prosthetics.
9:00 AM - H12.27
On-Chip Detection and Molecular Analysis of Central Nervous System (CNS) Lymphoma
Jun Song 1 2 KyungHeon Lee 1 Anna Turetsky 1 Randy J Giedt 1 Eunha Kim 1 Alexandra E. Kovach 3 Ephraim P Hochberg 4 Cesar M Castro 1 Ralph Weissleder 1 Hakho Lee 1
1Massachusetts General Hospital Boston USA2Harvard University Cambridge USA3Massachusetts General Hospital Boston USA4Massachusetts General Hospital Boston USA
Show AbstractDetecting central nervous system (CNS) lymphoma, especially in cerebrospinal fluids, is a challenging clinical task due to low cell density and limited sample volumes. We herein present a new microfluidic platform for high thoughtput analysis of CNS lymphomas. The platform is designed to captures individual lymphoid cells in sub-nanoliter traps. Subsequently, the captured cells can be stained on-chip for molecular analyses or treated with chemotherapeutic agents for drug screening. To profile lymphocyte cell populations, we established a three-step labeling strategy, namely, 1) the use of CD19 and/or CD20 to determine B cells; 2) the use of kappa or lambda light chains to identify different clonal populations of lymphomas and 3) additional phenotypic markers for subtyping and prognostic purposes. To facilitate such analyses, we also established an automated image processing algorithm for clonality assessment. The developed platform achieved capture efficiency of >90%, considerably minimizing cell-loss. Through on-chip staining, we imaged both intracellular and extracellular protein markers from individual lymphoma cells; the image processing algorithm further enabled fast quantitation of target markers at the single cell level. The entire assay was completed in less than one hour, demonstrating the potential for on-site disease diagnosis and characterization. By enhancing the accuracy and ease of CNS lymphoma diagnosis, this new platform is expected to aid rationalized, biomarker-based therapy as well as in treatment monitoring over time.
9:00 AM - H12.28
Monitoring Interstrain Microbial Interactions by Dielectrophoretic Tracking of the Characteristic Phenotype of Individual Cells
Nathan S Swami 1
1University of Virginia Charlottesville USA
Show AbstractMicro-organisms are present in heterogeneous samples that are spread over subpopulations with varying antibiotic persistence and strains with differing levels of toxicity. Herein, we present a single-cell image tracking technique based on the simultaneous measurement of dielectrophoretic velocities of microbials under a spatially non-uniform AC field of a wide bandwidth (10 kHz - 10 MHz) as a method to sensitively quantify fractional subpopulations based on their characteristic phenotype [1, 2]. Interactions across the microbiome, especially between different strain types within a species, can affect the colonization of nutrient environments and control the emergence of infections. For instance, Clostridium difficile (C.difficile) infection (CDI) is a toxin-mediated intestinal disease that is commonly attributed to exposure to pathogenic C.difficile strains following the elimination of healthy microflora in the gut by broad-spectra antibiotics [3]. Asymptomatic colonization with non-toxigenic C.difficile (NTCD) strains can reduce the incidence of CDI from toxigenic C.difficile (TCD) strains [4], leading to much interest in controlling the antagonistic interstrain interactions. However, due to the lack of specific antibodies or surface markers, there is no independent way to simultaneously monitor physiological alterations in each of these strains, especially during antibiotic and therapeutic interventions. Based on simultaneous dielectrophoretic tracking of microbials, towards or away from localized microfluidic constrictions, we demonstrate that well-defined differences in the surface layer (S-layer) proteins on the cell wall of each C.difficile strain, which determine gut colonization and host-pathogen adherence [5], can be identified by dielectrophoresis for fingerprinting each strain. Similarly, persistent subpopulations of Cryptosporidium parvum, a water-borne pathogen, can be identified and separated based on the dielectrophoretic fingerprint of their sporozoite structure. We envision the application of this microfluidic diagnostic platform for the development of therapies to identify and arrest the emergence of infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the microengineering of nutrient environments to control microbiomes.
[1] Y. H. Su et al. "Quantitative dielectrophoretic tracking for characterization and separation of persistent subpopulations of Cryptosporidium parvum," Analyst, vol. 139, pp. 66-73, 2014.
[2] A. Rohani, et al. “Electrical tweezer methodology for highly parallelized electro-rotation measurements over a wide frequency bandwidth”, Electrophoresis (2014), DOI: 10.1002/elps.201400021
[3] K. C. Carroll and J. G. Bartlett, Annu Rev Microbiol, 2011, 65, 501-521.
[4] M. Natarajan, S. T. Walk, V. B. Young and D. M. Aronoff, Anaerobe, 2013, 22, 1-5.
[5] R. P. Fagan and N. F. Fairweather, Nature reviews. Microbiology, 2014, 12, 211-222.
9:00 AM - H12.29
Scaffold-Free Three-Dimensional Cell Assembly for Bottom-Up Tissue Engineering
Pu Chen 1 2 Sinan Guven 1 2 Ljupcho Prodanov 3 4 Berk Usta 3 4 Utkan Demirci 1 2
1Stanford University School of Medicine Palo Alto USA2Stanford School of Medicine Palo Alto USA3Massachusetts General Hospital Boston USA4Shriners Hospital for Children in Boston Boston USA
Show AbstractScaffold-free assembly of cells into three-dimensional (3D) architectures is requested for engineering tissues with high cell packing density and low extracellular matrix (ECM), such as liver, brain and pancreas. For example, ECM in liver tissue only constitutes 0.5-3% of the liver wet weight. Although many approaches have been demonstrated for spatial organization of cells with cell-seeded microcarriers such as cell encapsulating hydrogels and cell-seeded microcarrier beads, these approaches can&’t achieve high cell packing density due to large ratio of cell scaffold in the cell-seeded microcarriers. Here, we demonstrate a novel bottom-up approach for scaffold-free 3D cell assembly, which enables generation of repeating and symmetric cellular structures with high cell packing density and good cell viability. Standing waves established at the air-liquid interface by hydrodynamic instability are used to pack cells without scaffold into 3D architecture at the bottom of a fluid carrier chamber. The pattern of 3D architecture is determined by the topography of standing waves and can be dynamically controlled by vibrational frequency and amplitude of standing waves. We demonstrated packing of a large amount (106) of hepatocytes into periodic concentric ring-square structures with a thickness of 200 µm in a few seconds. These 3D architecture was further immobilized by chemical cross-linking for tissue culture. Bile canaliculi staining and connexin staining indicated formation of bile canaliculi and gap junctions after 3-day tissue culture. Functionalization of engineered liver tissue was confirmed by measuring albumin secretion and urea synthesis. We expect this scaffold-free cell assembly will find broad applications in engineering ECM-less tissues.
9:00 AM - H12.30
Imposing and Analyzing Static Mechanical Strain on Cell Monolayers
Adam S Zeiger 1 Frances D Liu 1 Krystyn J Van Vliet 1
1Massachusetts Institute of Technology Cambridge USA
Show AbstractCells comprising blood vessels can both exert and receive mechanical strains, which can in turn affect cell functions. Although engineering devices are well developed to supply and analyze effects of fluid flow and dynamic (cyclic) loading on individual cells, cell monolayers, and intact vessels, the capacity to apply controlled static strain in these contexts are less explored. Such capabilities are of interest in exploring whether cell-generated strains, for example from contractile pericytes that surround microvessels, affect vessel stability or vascular endothelial cell behavior. Here we describe and demonstrate such an engineering approach, applied to explore how modest and time-invariant mechanical tension can elicit cell response. This device comprises optically transparent polymers for 2D and 3D culture of one cell type, while enabling automated mechanical strains of magnitudes and durations comparable to that exerted by a second cell type. Using this device, we show that such specific magnitudes and rates of mechanical strain can directly and rapidly modulate the morphology and phenotype of microvascular cell monolayers. More generally, this engineering approach allows for systematic analysis of how both extracellular mechanical and chemical stimuli can induce intracellular responses within minutes.
9:00 AM - H12.31
Quantitative Analysis of Signaling Molecule for Pollen Tube Guidance
Naoki Yanagisawa 2 1 Tetsuya Higashiyama 2 1 3 Hideyuki Arata 2 1
1JST-ERATO Higashiyama Live-Holonics Project Nagoya Japan2Nagoya University Nagoya Japan3Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University Nagoya Japan
Show AbstractPollination in flowering plants takes place by transferring pollen to a stigma. In order fertilization occurs, each pollen grain on a stigma germinates a pollen tube (PT), which carries sperm cells and travels toward the ovary [1]. During this journey, PTs are guided to the target embryo sac by sensing multiple signaling molecules so that they reach to their goals successfully. Recently, signaling molecules that attract PTs have been identified in Torenia Fournieri [2]. While it is considered that PTs are following concentration gradients of those attractants [3], quantitative information such as the amount of chemical species as well as the distribution of concentration gradients required for PT&’s attraction still remains unknown. One of the major reasons is that PTs grow randomly on a conventional platform (e.g., agarose gel dish), and it is very challenging to judge if the change in their growth direction is due to the response to signaling molecules. For the observation of the effect of attractants on PT&’s growth direction, the experimental condition will be more suitable if each PT elongates in structurally constrained conduits.
In this work, we developed a microfluidic platform that contained series of channels where a single PT could pass through. By placing a pollinated style onto a microfluidic reservoir, PTs grown in a style (in vivo environment) subsequently entered microfluidic channels (in vitro environment). A concentration gradient of an attractant was created in these channels by a diffusion-based method, and the PTs response to the chemical was observed under various attractant&’s concentrations.
[1] Kurihara, D.; Hamamura, Y.; Higashiyama, T. Develop. Growth Differ. 2013, 55, 462-473.
[2] Okuda, S. et al. Nature, 2009, 458, 357-362.
[3] Higashiyama, T.; Hamamura, Y. Sex. Plant Reprod. 2008, 21, 17-26.
9:00 AM - H12.32
Electroactive Scaffolds for Skeletal Muscle Tissue Engineering
Daniel Browe 1 Caresse Simmonds 1 Joseph Freeman 1
1Rutgers University Piscataway USA
Show AbstractNormal muscle function can be impeded by traumatic injury, disease, or tumor ablation. Although muscle autografts may be used in some cases, tissue engineering holds the most promise for regenerating large volume defects without the risk of donor site morbidity. Both mechanical and electrical stimuli have been investigated to enhance myoblast development in vitro. These two stimuli can be applied simultaneously through the use of ionic polymer gels (IPGs), which move in an electric field. Previously, our group has developed composite electrospun nanofibrous scaffolds with a hydrogel component serving as the IPG. These scaffolds have shown the ability to bend in an electric field. The objective of this study was to arrange the scaffold components to create scaffold contraction instead of bending and determine the configuration that would lead to optimal scaffold contraction in an electric field while enhancing myoblast groth in vitro.
Scaffolds were fabricated through electrospinning a 20% (w/v) polycaprolactone solution with multi-walled carbon nanotubes (MWCNT). After electrospinning, a 6% (w/v) poly(acrylic acid) and poly(vinyl alcohol) (PAA/PVA) (83/17) solution with 10% (w/w) ammonium persulfate was spin-coated onto the scaffolds at a density of 40 mu;L/cm2. Scaffolds were exposed to UV (365 nm) radiation for 20 min with various photo-masks applied to selectively block the UV radiation. Cross-linked PAA/PVA bands were perpendicular to the primary direction of the fibers. Actuation experiments were performed by submerging 6 cm x 1 cm scaffold sections in a 30% saline (NaCl) solution and applying 20 V via alligator clips attached to the sample and a Platinum electrode. Videos of the experiments were analyzed and contraction was calculated from the positions of the scaffold endpoints. Statistics were performed with Tukey's test (p<0.05).
Scaffold sections with a uniform distribution of cross-linked PAA/PVA bent under the electrical field leading to an overall decrease in length of 5.8% (±8.1%) while banding patterns led to overall contraction. The scaffolds with 1 cm bands and 0.5 cm bands of cross-linked PAA/PVA contracted 6.7% (±3.2%) and 11.0% (±6.7%) respectively (gaps between bands were equal to the size of the bands). Changes in the banding pattern appeared to alter the degree of contraction, although these differences were not significant. Ongoing experiments are investigating micro-scale photo-masks to create different micro-patterned crosslinking of PAA/PVA and measure the effect on actuation. In addition, ongoing in vitro experiments will determine how electrical stimulation can be used to promote cell differentiation on these scaffolds. Our preliminary in vitro studies have shown that the use of polypyrrole nanoparticles lead to increased cell proliferation over MWCNT while maintaining scaffold conductivity. Thus, our future experiments will use scaffolds with polypyrrole.
9:00 AM - H12.33
A Solid Tumor Model with Opportunities for Studying Immune Component Interactions
Ryan Cecchi 1 Kristie M Charoen 1 Mark W Grinstaff 1 2
1Boston University Boston USA2Boston University Boston USA
Show AbstractA tumor undergoes a variety of interactions with the surrounding stroma, with a large part of these interactions concerning both the immune system and the tissue of origin. As a result of this crosstalk, two major cell types recruited by tumors are cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs). This project focuses on the effect of TAMs within a model system, and if their function is location dependent, whether they are within the tumor or in the surrounding stroma. These two models mimic what is clinically observed.
In order to utilize these relationships, an in vitro model was created with its main component being a spheroid of human breast cancer cell line (MDA-MB-231). The spheroid is embedded in collagen so as to allow three-dimensional shape, movement, and to mimic the stromal environment. The immune cells, mouse macrophages (RAWs), are added into the system in two different ways in order to gain insight about differing activities based on location in reference to the tumor. The first method is to create a heterospheroid, a spheroid containing both MDA-MB-231 and RAW cells. The first method mimics the activity of TAMs that have infiltrated the tumor mass thus being exposed to hypoxia, and metabolic gradients. The second method diffusely seeds the macrophages in the collagen so that they surround the spheroid, thus modeling TAMs in the cancer stroma. Observation of the system will demonstrate how the incorporation of macrophages affects the model.
H9: Micro/Nano Engineering for Cell Mechanics and Physical Oncology
Session Chairs
Wednesday AM, December 03, 2014
Sheraton, 2nd Floor, Back Bay A
9:30 AM - H9.02
Biomechanical Phenotyping of Single Suspending Cancer Cells Using Deformability Microcytometer
Raymond H. W. Lam 1 Baoce Sun 1 Shuhuan Hu 1
1City University of Hong Kong Kowloon Hong Kong
Show AbstractBiomechanical properties of human cells can reflect the sub-cellular cytoskeletal reorganization caused by cancerogenesis. The genetic alterations can affect synthesis of intracellular components such as the viscoelastic actin-myosin networks. It has been proven that viscoelasticity of cancer cells correlates strongly with the progression from normal non-tumerigenic, to tumorigenic, and to metastatic and malignant cells. In bloodstreams of some cancer patients, metastatic circulating tumor cells (CTCs) share some common mechanical characteristics such as elasticity changes, typically quantified by measuring cell deformation (in microns/tens of microns) under an external force (in the nano-Newton scale). Over the past years, many microfluidic devices have been developed for quantitative measurements of suspending CTC deformability/elasticity. Cell elasticity can be considered as one of the diagnostic biomarkers, yet the measurement can be affected by other cell properties such as the cell size and viscosity. Here, we report a high-throughput microfluidic device to quantify for all the size, stiffness and viscosity of each cells flowing along the device, in order to achieve quantification of comprehensive viscoelastic properties of cells. Technically, we designed the device with multiple funnel-shaped microchannels to capture cells from the samples under known physical configurations. We selected normal human epithelial cells and cancer cell lines for the measurements by continuously flowing them along the confining channels, with their dynamic shape changes recorded by a high-speed camera. The cells first contacted with the channel side walls and squeezed further into the channels with larger deformations before departure. Further, we applied the Hertz&’s and Tatara&’s theories to derive a first-principle model effectively converted the measured shape dynamics to multiple physical cell properties (size, elasticity and viscosity) simultaneously. We validated that the measured values have shown reasonable agreements with the previously reported cell properties. Our results demonstrate that this strategy can recognize statistical significant differences in stiffnesses and viscosities between metastatic and non-metastatic cells using only a small number (~10) of cells. Such high measurement sensitivity is an important detection criterion for potential clinical applications, due to the limited number of metastatic CTCs existing in the patient&’s blood (<10 cells/ml). Altogether, we anticipate this biomechanical measurement method can examine suspending cancer cells for their viscoelastic properties, and provide implication for the metastatic potentials. With further development including system automation, it may achieve an effective metastasis diagnosis of clinical samples from cancer patients.
9:45 AM - H9.03
Development of a Novel 3D Bioprinted In Vitro Nano Bone Model for Breast Cancer Bone Metastasis Study
Benjamin Holmes 1 Wei Zhu 1 Lijie Grace Zhang 1 2
1The George Washington University Washington USA2The George Washington University Washington USA
Show AbstractBreast cancer (BrCa) is the second commonest cause of cancer-related deaths in women. The metastatic breast cancer exhibits a high affinity to bone, leading to debilitating skeletal complications associated with significant morbidity and poor prognosis. Traditional in vitro and in vivo BrCa bone metastasis models contain many inherent limitations with regards to controllability, reproducibility, and flexibility of design. Thus, the objective of this research is to use a 3D bioprinting system and nanomaterials to recreate a biomimetic and tunable bone modelsuitable for the effective simulation and study of metastatic BrCa invading and colonizing a bone environment. For this purpose, we designed and 3D printed a series of scaffolds, comprised of a bone microstructure and nano hydroxyapatites (nHA, inorganic nano components in bone). The size and geometry of the bone microstructure was varied with 250 and 150 µm pores, in repeating square and hexagon patterns, for a total of four different pore geometries. 3D bioprinted scaffolds were subsequently conjugated with nHA, using an acetylation chemical functionalization process and then characterized by scanning electron microscope (SEM). SEM imaging showed that our designed microfeatures were printable with the predesigned resolutions described above. Imaging further confirmed that acetylation effectively attached nHA to the surface of scaffolds and induced a nanoroughness. Metastatic BrCa cell 4 h adhesion and 1, 3 and 5 day proliferation were investigated in the bone model in vitro. The cell adhesion and proliferation results showed that all scaffolds are cytocompatible for BrCa cell growth; in particular the nHA scaffolds with small hexagonal pores had the highest cell density. Given this data, it can be stipulated that our 3D printed nHA scaffolds may make effective biomimetic environments for studying BrCa bone metastasis.
10:00 AM - H9.04
Nanomechanical Responses in Micro-Patterned Cardiomyocytes Using Combined Scanning Probe and Fluorescence Microscopy
Varun Vyas 1 Neerajha Nagarajan 2 Pinar Zorlutuna 2 Bryan D Huey 1
1Institute of Material Sciences Storrs USA2Department of Biomedical Engineering Storrs USA
Show AbstractCoupled optical and scanning probe systems provides unique opportunities to investigate not just the nanomechanical properties of living cells and tissue in-vitro, but also their living response to nanomechanical, chemical, optical, or other signals. As an example, the nanomechanical properties of isolated and micropatterned cardiomyocyte cultures are studied using a colloidal AFM probe in contact with the cells to monitor their natural beating processes, both asynchronous and synchronous. Essentially achieving nano-stethoscopy, the cells are found to exert displacements on the order of micrometers, and forces up to hundreds of nN, at frequencies typically of 0.5 to 5 Hz. Simultaneous optical micrsocopy, including fluorescence imaging to track Ca ions that are integral to beating events, reveals the spatial distribution of the beating process. Altogether, this provides novel insight into cell signaling pathways.
The nanomechanical behavior of the cells before, during, and after loading has also been investigated. Specifically, the beating rate and strength of contraction for myocytes are found to adjust upon nanomechanical perturbation. Contact forces are varied from tens to hundreds of nN, with load frequencies of 1-5Hz. The combined imaging and nanomechanical interrogation thereby allows the emergent behavior of collective cells to be uniquely investigated, with applications for nanobiology including the development of BioMEMS functionalities.
10:15 AM - *H9.05
Microtechnologies for Screening Cell Mechanobiological Responses
Craig A. Simmons 1
1University of Toronto Toronto Canada
Show AbstractCells reside in three-dimensional, soft extracellular matrices where they interact with other cells and, in the case of cardiovascular and musculoskeletal tissues, are subjected to dynamic mechanical loading. However, in traditional cell culture platforms (e.g., microtiter well plates), cells are grown on rigid, static two-dimensional surfaces. Thus, current platforms for studying cardiovascular and musculoskeletal cell biology poorly represent the in vivo environment, which limits the novelty and translatability of the biological information they generate.
In this talk, I will describe some of the microtechnologies that we are developing to address these limitations. These microfluidic platforms are designed to allow precise control over the cellular microenvironment, including matrix stiffness and proteins, soluble proteins, cell-cell interactions, and biophysical forces. Compared with standard culture platforms, these microsystems better mimic key components of the in vivo cellular mechanobiological microenvironment, offer more precise control over microenvironmental cues, and avoid some of the confounding factors associated with application of dynamic mechanical forces at the macroscale. They are also compatible with on-chip imaging and are highly parallelizable, and therefore can be used in high-content and high-throughput screening applications. Current screening applications include fundamental studies of cell-matrix and cell-cell interactions in mechanically active environments, screening of biomaterial properties for stem cell-based tissue regeneration, and drug screening in microfluidic vascularized tissue arrays.
H10: Micro/Nano Engineering for Cell Fate Control
Session Chairs
Wednesday AM, December 03, 2014
Sheraton, 2nd Floor, Back Bay A
11:00 AM - *H10.01
Single-Cell Cytokine Profiling of Myeloproliferative Malignancies
Rong Fan 1
1Yale University New Haven USA
Show AbstractIt is widely recognized that clonal evolution led to intratumor genetic heterogeneity, which can be characterized by single-cell sequencing. Non-genetic variability of cancer cells at the functional level has emerged as another layer of mechanism contributing to intratumor heterogeneity and pathogenesis. However, it remains a challenge to fully characterize functional heterogeneity at the single cell level and in a highly informative manner. In this work, we show a microchip technology for highly multiplexed (40+) profiling of effector function proteins secreted from single cells. Applying this technology to the study of myeloproliferative neoplasms (MPNs) delineated how a spectrum of inflammatory cytokines are produced and correlated to MPN pathogenesis in a JAK-mutation mouse model and myelofibrosis patients. Principal component analysis of cytokine spectra in all single hematopoietic cells reveals that the cytokine functions of “normal” non-mutant hematopoietic cells were significantly skewed and contributed substantially to MPN pathogenesis. Interestingly, mutation-specific deletion of STAT3, a master transcription factor controlling the secretion of a host of cytokines, has little effect on disease trajectory. JAK inhibitor treatment has to suppress both malignant and non-malignant hematopoietic cells to achieve therapeutic benefit. This work demonstrated the power of our single-cell technology to characterize functional cellular heterogeneity in disease lesion and revealed an unexpected crucial role of “normal” hematopoietic cells in MPN pathogenesis and therapeutic response.
11:30 AM - H10.02
Engineering Cell-Matrix Interactions on Biomaterials to Guide Cellular Programming and Reprogramming
Kristopher Alan Kilian 1
1University of Illinois at Urbana-Champaign Urbana USA
Show AbstractThe architecture of the cellular microenvironment provides physical, chemical and biological cues to guide cell fate and orchestrate normal and pathological processes. Substrates that are designed to emulate this architecture are useful tools for exploring the process of cell fate determination. In this presentation I will discuss our efforts in modifying both “hard” and “soft” biomaterials with patterned biomolecules to explore cellular programming and reprogramming. First, I will show how microarrays presenting combinations of bioactive peptides can serve to identify surfaces that promote cellular differentiation and de-differentiation. By combining short peptides derived from adhesion proteins, proteoglycan binding domains and growth factors, multiple pathways can be stimulated to modulate cell fate decisions. Next I will demonstrate how soft lithography can be used to pattern biomolecules in defined geometries—on substrates of varying mechanical properties—to explore how physical cues guide cellular form and function. Inspired by the geometry of tissue, regions of curvature at the perimeter of single cells and cell populations can be designed to foster variable regions of tension and compression for in vitro morphogenetic guidance. Finally, I will discuss how these tools can be translated to designer 3-D tissue-mimetic architectures for the establishment of model in vitro systems, and to aid the development of clinically viable hydrogel materials. Taken together, these studies reveal how a well-defined presentation of biophysical and biochemical signals at the cell-biomaterial interface is critical for directing cell fate decisions.
11:45 AM - H10.03
Effect of Tension on Myelination
Anna E Jagielska 1
1MIT Cambridge USA
Show AbstractOligodendrocytes are cells that insulate neurons via synthesis and wrapping of a myelin sheath around axons. These key cells in the central nervous system differentiate from oligodendrocyte precursor cells (OPCs), but the mechanisms required to stimulate OPC migration and differentiation are incompletely known. Specifically, it remains unclear whether mechanical strains exerted on OPCs by their microenvironment - due either to axon growth or to traumatic impact and swelling - may affect these cell functions. Here, we engineer devices that can modulate mechanical strain applied to OPCs over weeks in vitro, concurrent with optical imaging that facilitates tracking of cell differentiation. We show that both static and dynamic tensile strains decreased OPC proliferation and promoted differentiation toward myelin-producing oligodendrocytes. We correlate this mechanically accelerated differentiation with in situ analysis of chromatin dynamics, as well as changes in gene expression. This engineering approach enables in vitro analysis of which in vivo mechanical stimuli can stimulate oligodendrocyte differentiation, and shows that strains associated with axon growth can promote this cell transition to perform its healthy function of myelination.
12:00 PM - H10.04
Roughness of Nanostructured Zirconia Surface Modulates Neuronal Cell Fate by Alteration of the Cellrsquo;s Mechanic Properties
Paolo Milani 1 Carsten Schulte 1
1University of Milan, Italy Milan Italy
Show AbstractNeurodegenerative diseases are disorders that eventually lead to the death of cells of the nervous system. Due to the limited intrinsic regeneration capacity of the nervous system, cell replacement strategies are a promising approach in the search for therapies for these diseases. The strategies aim to recapitulate the processes in neurogenesis in vitro with the objective to regenerate damages in the nervous system. Cellular mechanosensing and mechanotransduction play an important role in neuronal differentiation. Cells sense the condition of their environment and react to it very specifically; in particular the topographical and mechanical characteristics have a strong impact on the cell&’s fate. In nanobiotechnology scientists attempt to exploit this by engineering biomaterials with nanostructured surfaces permitting cell/nanostructure interaction that eventually enable the manipulation of cell behavior. Our approach for biomaterial production is based on the deposition of zirconia nanoparticles by a supersonic cluster beam (SCBD). This technique creates a film with a nanostructure composed of randomly distributed nanoparticles featured with reproducible roughness parameters. We found that a specific nanostructure roughness induced neurite outgrowth in PC12 cells (a model cell line for neuronal differentiation) even in the absence of a canonical stimulus (i.e. NGF). Atomic force microscopy measurements revealed that the cell/nanostructure interaction changed the cell&’s biomechanic properties in a way that they display decreased membrane/cytoskeletal tension. A compensation of this effect by a hypoosmotic gradient led to a gradual inhibition of the nanostructure-induced events. Further cell biological experiments showed the involvement of mechanosensitive components (i.e. β1 integrins, calcium channels) and corresponding signaling cascades which eventually alter transcription factor localisation and protein expression. In addition to the results obtained for the cell line PC12 we extended our research to a primary neuronal cell system. The nanostructure had an impact on the differentiation of primary neurons derived from rat neonatal hippocampus. The maturation of functional synapses was significantly accelerated so that they showed action potentials and neuronal network activity earlier than the control cells. A profound understanding of the molecular mechanism underlying biomaterial-induced cell biological processes is crucial for the intelligent development of devices with biomedical applications but is, to date, only superficially realized. Our data emphasise the potential of the nanostructured surfaces produced by SCBD in inducing neuronal differentiation and proposes a mechanism based on mechanotransduction. The objective is to design a cell culture device favoring neurogenesis in adequate (stem) cell systems. Such a device would have significant relevance for cell replacement strategies in neurodegenerative diseases.
12:15 PM - *H10.05
Orthogonally Engineering Matrix Topography and Rigidity to Regulate Multicellular Morphology
Xiaodong Chen 1
1Nanyang Technological University Singapore Singapore
Show AbstractThe formation and regulation of multicellular morphology, during which cells can actively respond to their environment and collectively build into the entire functional organization, is vital in synthetic tissue biology. Here, we reported a platform to control the multicellular morphology by orthogonally modulating the mechanical and topological cues of extracellular matrix based on the biocompatibility of polymer materials and patterning flexibility of micro-contact printing. It was found that morphogenetic ring formation process is the result of a mechanical equilibrium established and maintained by multicellular cooperation with the extracellular matrix. This demonstrates that the extracellular matrix parameters are comparable to biochemical cues from growth factors in profoundly influencing cell behavior. Weakened cell-matrix anchorage may lead to the smoothening of multicellular ring borders and enlargement of ring size, leading to unclosed rings even with long-term cell proliferation. The formation of rings can increase the permeability of the cell sheet and thus allow better delivery of nutrients or therapeutics, while enhanced cell-matrix anchorage would promote the formation of confluent monolayer, serving as a protective barrier which can resist pressure in vivo. Consequently, this protective function determined by the efficiency of tumor cells in maintaining its continuity and integrity can be switched on or off by modifying their environment, thereby suggesting ways in developing implantable materials to specify the assembly of biological structures and contributing to a better understanding of synthetic tissue biology.