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1 Gold nanoparticles have shown great potential applications in biomedical diagnosis and therapeutic treatments due to their shape and size-dependent optical properties. However, densely packed CTAB double layer, a surfactant used to prevent nanoparticle aggregation, on the nanorod surfaces presents challenges for effective biofunctionalization in setting up a nanoparticle-based platform with high detection sensitivity. In this study, we systematically studied the functionalization of gold nanorods with various bioconjuate linkers in pursuit of developing nanorod-based immunosensors with maximal sensitivity and specificity. First, gold nanorod chips were fabricated by chemically binding nanorods onto a silanized glass substrate; the gold nanorods were then functionalized with carboxylic acid or an amine group via thiol linkers for antibody conjugation. The effects of thiol linkers with different lengths such as 11-mercaptoundecanic acid (MUDA), 4-aminothiophenol (4-ATP), 2-mercaptopropionic acid (MPA), and 6-mercaptohexanoic acid (MHA) were investigated. The optimized functionalization of the gold nanorod chip was applied to develop a nano-biosensor to detect human serum IgG as a model system. Since the localized surface plasmon resonance is highly sensitive to the biological binding on the nanorod surface, this mechanism provides a label-free biodetection, eliminating signaling labels such as fluorophore and radioactive agents.
EFFECT OF UREA ON THE SURFACE ACTIVITY OF AMYLOID BETA PEPTIDEMelissa Hernandez, Eva Chi.
University of New Mexico, Albuquerque, Albuquerque, NM.
The aggregation of amyloid beta (Aβ40) peptide into insoluble fibrils rich in beta-sheets is a major pathological hallmark of Alzheimer’s disease. Previously, we showed that Aβ40 is highly surface active and that its binding to model lipid membranes seeds fibril formation. In an effort to better understand the aggregation of Aβ40 in vivo, we studied the surface activity of Aβ40 in the presence of the naturally-occurring osmolyte, urea. Urea is known to preferentially bind to and increase the solubility of proteins in solution. We hypothesized that urea will similarly increase the solubility of Aβ40 in solution and thereby decrease the tendency of Aβ40 to adsorb to the air-water interface. To test our hypothesis, we assessed the surface activity of Aβ40 by measuring its adsorption isotherms (surface pressure versus time) in a Langmuir trough with increasing urea concentration in the subphase. Our results showed that urea lowered the equilibrium adsorption pressure of Aβ40, indicating lower surface activity. However, the rate of adsorption increased sharply, and the lag time of adsorption was eliminated in the presence of urea. To better understand our results, we are investigating the roles of some secondary effects, such as pH and ionic strength, on Aβ40 surface activity. Understanding how Aβ40 behaves in cell-mimicking environments will provide insight as to why and how Aβ40 aggregates into insoluble fibrils in the brain, thereby creating a foundation to begin treating and preventing Alzheimer’s disease.
ENGINEERING HYALURONIC ACID BASED HYDROGELS FOR 3D STEM CELL CULTUREVivian Le, Sylvia Natividad-Diaz, Amit Jha, Kevin Healy.
University of California, Santa Barbara, Santa Barbara, CA, 2University of California, Berkeley, Berkeley, CA.
1 Heart failure arising from myocardial infarction (MI), initially caused by the blockage of coronary arteries, is one of the leading causes of death around the world. Currently, the only available treatment to restore cardiac function after MI is heart transplantation, which is risky and limited by the availability of donor organs. One potential alternative treatment is the use of induced pluripotent stem (iPS) cells to regenerate cardiac tissue that has been damaged by MI. Preliminary experiments with magnetic activated cell sorting (MACS) of mesoderm-differentiated human iPS cells
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have demonstrated a cell subpopulation that is positive for CD105, a cell surface marker for proangiogenic endothelial cells. These cells have the potential to form vascularized networks within an appropriate 3D matrix, which we plan to engineer using hyaluronic acid (HyA) hydrogels. HyA is a natural substance found in the extracellular matrix of human tissue, and it can be used in the synthesis of scaffolds for wound healing applications. In addition, hyaluronic acid is biocompatible, biodegradable, and has readily accessible functional groups for chemical modification. The sorted cells can be encapsulated into HyA-based hydrogels to improve their viability in cardiac tissue post-MI. During this project we will determine whether HyA hydrogels will promote the sustained incorporation of the CD105+ cells with native cardiac tissue.
FABRICATION OF ACOUSTIC FOCUSING FLOW CELLS FOR HIGH THROUGHPUT FLOW CYTOMETRY
ANALYSIS OF PLANKTON
Biomedical Engineering, University of New Mexico, Albuquerque, NM.
Microbes account for most of the primary productivity and biomass in the ocean, and the structure of the microbial community determines in large part that of higher trophic levels. A fundamental understanding of the factors that regulate community structure requires detailed and sustained observations of the plankton. A fully submergible hydrodynamic flow focusing bases flow cytometer has been developed to analyze large phytoplankton and microzooplankton in the ocean. Ocean phytoplankton are continuously monitored for about 6 months via fluorescence, scattering, and high resolution images of phytoplankton acquired from the flow cytometer. However, the main drawback of the current setup is its limited throughput. Signal and image quality are diminished with increases in flow rates of sheath fluid and sample fluid in order to enhance throughput. In addition, the maximum flow rate for hydrodynamic focusing is limited to 125 mL/min. Thus, it is important to investigate alternative flow focusing techniques with enhanced throughput capabilities. The purpose of current research is to develop a flow focusing cell that uses standing acoustic waves and investigate its capability to focus and concentrate plankton at very high flow rates prior to analysis. Here, we report design and fabrication of acoustic flow cells and our initial finding on focusing of phytoplankton.
PEEL STRESS ASSESSMENT OF COMMERCIAL WOUND-TREATMENT POLYMER FILMSRita Thornton, Vitaly Kheyfets, Ender Finol.
University of Texas at San Antonio, San Antonio, TX.
There are different types of polymer films used as temporary substitutes on damaged skin to aid in wound treatment.
It is important to understand the tackiness of these films to investigate whether encounters with environmental materials such as stainless steel surfaces, will cause them to be torn from a wound. The objective of this study is to conduct a peel test to determine the tackiness of four commercially available isotropic and nonlinearly elastic wound care films: New Skin, Smith & Nephew, Skin Shield, and Silesse. We designed an apparatus using a 20 mm stainless steel dolly that compressed (≈4 N) porcine skin for 3 hours at 37 °C. The dolly was attached to an Electroforce 3200 tensile testing instrument, which peeled it off the skin surface at a rate of 100 cm/s and collected force measurements at 20 Hz. A MATLAB code was used to calculate the peel stress, i.e., the maximum force per surface area of the dolly at the time of detachment from the skin for each polymer. We found that Silesse [0.5 ± 0.5 kPa] did not adhere, while Smith & Nephew [13.8 ± 4.8 kPa], New Skin [12.1 ± 5.4 kPa], and Skin Shield [10.1 ± 4.3 kPa] demonstrated much greater adherence. These results showed that the polymer Silesse is less likely to adhere to household items and may offer reduced chance of damage or reinjury due to contact with environmental surfaces. (This work was partially supported by UTSA MARC-U*STAR GM007717.) FRI-264
DETERMINING FORCE PRODUCED BY GROUPED VS. SINGLE MOTOR PROTEINS THROUGH DNA ORIGAMISCAFFOLDS Enrique Daza, Jing Xu.
University of California Merced, Merced, CA.
Neurodegenerative diseases (NDD) pose a great problem in elderly populations and, less commonly, in adolescents.
One of the widely known causes of NDD is the inability for motor proteins to function within the brain’s neuronal
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axons. These proteins are essential in delivering information, such as neurotransmitters, across the brain in compact vesicles. In order to better understand the capabilities of these motor proteins along the axons on microtubule tracks, a rigid, programmable DNA origami scaffold is created as a synthetic vesicle to mimic in vivo movement. We predict that a grouped kinesin 1 motor protein ensemble will perform with an increased pico-newton force when compared to a single kinesin 1 motor protein. Kymograph imaging will be used to measure the force produced from 1, 2, 3, 4, and 7 kinesin 1 motor proteins by attaching them to programmed DNA origami synthetic cargo. The images provide detail on motor-protein velocity, distance. Run time and force produced is measured with the known mass of the scaffold. The labeled DNA origami scaffold has been designed with oligo specific nucleotides for protein his-tag cross linkage.
FABRICATION OF CYTOKINE-LOADED CANCER TRAPSLinda Dao, Liping Tang.
University of Texas at Arlington, Arlington, TX.
Metastatic cancer, which unfortunately often leads to more death than primary cancer, must be better understood in order to treat and control this almost ubiquitous illness. Recent studies have shown that inflammation caused by biomaterial implantation can attract metastatic cancer. It is believed that inflammatory cytokines might be responsible for this. We hypothesize that implanting biomaterials loaded with specific inflammatory cytokines might attract metastatic cancer cells. The role of specific cytokines in cancer metastasis will be examined. Current research in the cytokine and cancer area is inconsistent, possibly due to the high variability of in vitro experiments. Hence, cell culture conditions like trypsinization time (that can affect cell phenotype) will be optimized to generate reproducible outcomes. Thereafter, a Boyden Chamber seeded with 50,000 B16F10 melanoma cells on the top and different cytokines at the bottom were used to determine the most effective cytokines. Subsequently, these cytokines will be loaded into a cancer trap comprising of a 3D multilayered PLGA, poly(lactic-co-glycolic acid), scaffold fabricated using a novel protein microbubble-based technique. Using B16F10 cells as a model, our optimization experiments show that trypsinization for 2 minutes proved to be the optimum time for future experiments. Thus far, erythropoietin appears to be a melanoma-cell attracting cytokine. The scaffold’s pore size ranges from 75 to 150 µm, with approximately 90% porosity. Currently, drug release from the traps is being studied. Our studies show good potential for the development of cytokine-loaded cancer traps that could localize metastatic cancer cells, which could then be eradicated by localized treatments.
BIOCOMPATIBLE SCAFFOLDS IN ENGINEERING CARTILAGE IN THE TREATMENT FOR MICROTIALesley Eldridge1, Jeremy Meier2, Patrick Tresco2, Jonathan Curtis3.
University of New Mexico, Albuquerque, NM, 2University of Utah, Salt Lake City, UT, 3School of Medicine, University 1 of Utah, Salt Lake City, UT.
Microtia is a congenital malformation of the external ear, affecting 1 in 6,000 children. Current treatment options for microtia include complex reconstructive surgery using rib cartilage, an external prosthesis, or artificial implant. A novel approach using biocompatible extracellular matrix (ECM) as a scaffold to develop cartilage has been developed in our lab. We hypothesize that this technique can become a new treatment option for microtia. Preliminary investigations to understand the time for differentiation of mesenchymal stem cells (MSCs) to chondrocytes are currently being done. We used rat GFP-labeled mesechymal stem cells grown in DMEM over a period of 10 days of incubation until confluence. MSCs were subjected to three conditions: DMEM, sedo minus (S-), and chondrocyte differentiation media (CDM). In MSCs subjected to CDM, we observed differentiation of the MSCs into cells morphologically resembling chondrocytes with lacunae formation after 9 days. Significant confluence of the differentiated cells was noted at day
21. Results from this study indicate the ability to successfully differentiate MSCs into chondrocytes. Our next step is to seed and differentiate the MSCs in an extracellular matrix scaffold. We will then implant the engineered cartilage in an animal model with the ultimate goal of using this technique to treat children with microtia.
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NOVEL METHODOLOGIES TO INDUCE OSTEODIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS
FOR TISSUE ENGINEERING APPLICATIONSMarissa Wechsler, Senthilnath Laksmanachetty, Vanessa Wechsler, Rena Bizios.
University of Texas at San Antonio, San Antonio, TX.