Symposium Poster Abstracts - University Of Cincinnati Regional... · 2018-08-17 · Symposium...

2011 Regional Symposium on Bioapplications of Membrane Science and Technology

Symposium Poster Abstracts

Degradation of Potent Cyanobacteria Toxin Microcystin-LR Using Photocatalytic Cellulosic Electrospun Fibers

Nicholas M. Bedford, Miguel Pelaez, Changseok Han, Dionysios D Dionysiou, Andrew J. Steckl

University of Cincinnati, Cincinnati, OH

Non-woven, high surface area photocatalytic cellulosic electrospun fibers were created for solar light driven water treatment purposes and tested for photocatalytic decomposition of the potent cyanobacteria toxin microcystin-lr (MC-LR). Electrospun fibers of cellulose acetate were converted to succinylated cellulose and then loaded with titania nanoparticles using a simple solution based technique. It was found that the type of titania nanoparticle (visible light activated or UV light activated), the surface area of the fiber mat, and solution loading pH all have an effect on the distribution of titania along the fibers. The titania coverage was found to correlated well with the degree of MC-LR degradation under both visible and solar irradiation. These photocatalytic electrospun fibers could be advantageously used for waste water and drinking water treatment applications using solar light as a renewable source of energy.

Scaffold Composition Affects Mechanical Properties of Tendon Tissue-Engineered Constructs in vitro

Andrew P. Breidenbach1, David L. Butler1, and Karl E. Kadler2

1University of Cincinnati, Cincinnati, OH

2Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK Introduction. Tendon and ligament injuries account for nearly half of all musculoskeletal injuries at a cost of $30 billion annually in the United States[1]. Tissue engineering strategies seek to improve upon current standards of medical repair by implanting cell-seeded tissue-engineered constructs (TECs) to expedite the repair process and to restore normal mechanical function to the damaged tissue. Understanding how cells interface with the TEC material and remodel the extracellular environment is integral in identifying methods that lead to improved biological and mechanical function of tissueengineered repairs. Collagen and fibrin biopolymers are attractive for these applications, as they are bioresorbable materials that naturally occur in the body, allow for flexibility in construct design, and can be readily recognized and remodeled


by cells. This study sought to compare TEC mechanical properties, TEC structure and gene expression of cells seeded in fibrin and type I collagen gels over time in culture. We hypothesized that the collagen TECs would exhibit greater linear modulus (LM) than the fibrin TECs at all time points. Additionally, we hypothesized that cells in the collagen TECs would have increased fibrillogenic gene expression (type I collagen, fibromodulin) compared to cells in the fibrin TECs. Methods. Experimental Design. TEC mechanical properties (n=9), gene expression (n=3) and scaffold structure (n=2) were examined on the day that the gel contracted into a linear construct (T0) and three days later (T3). TEC Preparation. TECs were prepared using day 13 embryonic chick metatarsal tendon fibroblasts (passage 6-7) as previously described[2]. Fibrin and collagen gels were at initial concentrations of 3.4mg/ml and 2.6mg/ml, respectively, in order to maintain similar porosity[3]. Mechanics. TECs were failed in tension at a 10%/s strain rate and material properties were determined as previously described[4]. Gene Expression (qPCR). RNA was isolated, converted to cDNA, and amplified in triplicate using primers and SYBR Green detection for Col1a1, Col3a1, Fmod, Dcn, FN, Scx, integrin α11, integrin β1, and 18S. Scaffold Structure. TECs were prepared for transmission electron microscopy (TEM) as previously described[4]. Fibril diameter (FD) distributions and fibril volume fraction (FVF) were determined using ImageJ on three randomly selected images from each TEC. Statistics. Response measures were compared either via two-way ANOVA (mechanics and scaffold structure) or MANOVA (gene expression) analysis with time and material as fixed factors (p<0.05). Results. Mechanics. Time increased linear moduli with collagen TECs increasing from 0.37±0.09 MPa (mean±SD) at T0 to 0.99±0.26 MPa at T3 (p < 0.01), while fibrin TECs increased from 0.32±0.12 MPa at T0 to 2.39±0.67 MPa at T3 (p < 0.01). Material did not affect linear modulus at T0, however fibrin TECs showed higher LM than collagen TECs at T3 (Fig. 1). Gene Expression. Time did not have an affect on gene expression (p > 0.05). Fibrin TECs exhibited greater expression of Col3a1, integrin α11 and integrin β1 than collagen TECs at T3 (Fig. 2). Material did not affect the remaining genes, and time did not affect expression of any genes investigated. Scaffold Structure. Data to be obtained by presentation. Discussion. The purpose of this study was to investigate how TEC material affects mechanical properties, cellular gene expression, and structural organization. Both TEC materials exhibited similar LM at T0 and showed significant increases in LM from T0 to T3. However, fibrin TECs experienced much greater increases in LM (2.4 times higher) than collagen TECs at T3. Tendon-related gene expression was

Figure 1: Comparing TEC material properties over time and across materials. (*significantly different with respect to time; #significantly different with respect to material; p < 0.01)

Figure 2: Gene expression data showing increased expression of Col3α1 and integrins α11 and β1 in fibrin compared to collagen at T3. (*significantly different with respect to material; p < 0.05)


quantified to elucidate how cells respond biologically to differing extracellular environments. Fibrin TECs exhibited increased expression of type 3 collagen relative to collagen TECs at T3. Fibrin TECs also exhibited increased expression of α11 and β1 integrin subunits, which can combine at the cell surface and bind to collagen. TEM data is expected to show that there is an increased number of fibrils aligned with the longitudinal axis in the fibrin TECs, which may explain the increased mechanical strength. While type I collagen is the primary structural component that gives tendon its tensile strength, the cells did not appear to efficiently remodel the type I collagen gel to resist tensile forces during the time period of this study. Future efforts will focus on identifying how cell-material interactions affect mechanical and structural properties at later time points and how this translates to repair response in an in vivo tendon injury model. Acknowledgement. Helen-Muir Fund, NIH grant 56943-02, and student support by NSF IGERT 0333377. References. 1. Praemer et al, AAOS, 1999; 2. Kapacee et al, Matrix Biol, 2008; 3. Pedersen and Swartz, Ann Biomed Eng, 2005; 4. Kalson et al, Matrix Biol, 2010.

Computational modeling of SCMTR: A synthetic chloride channel

Jonathon Burkhardt and Joel Fried

University of Cincinnati, Cincinnati, Ohio

Transmembrane Chloride ion transport is a critical function within cells of living organisms. Synthetic Anion Transporters are much simpler than Chloride specific proteins but allow for transmembrane ion transport. Their basic make up consists of a heptapeptide group that interacts with the membrane surface along with hydrophobic groups on either end to interact with the tail groups of the lipid bilayer (having the general for R’-GGGPGGG-R’’). Recent experimental studies of these compounds find that many derivatives of this general form create active Chloride channels and form dimers/trimers to function. Computational modeling set out to investigate the self-assembly and pore dynamics of these compounds at an atomistic level and to shed light on possible improvements. Current findings suggest that hydrogen bonding plays a role in how these compounds function. Additionally, water pocket formation is seen in the presence of this compound, which would stabilize a chloride ion within the lipid bilayer. How these compounds functions is not yet fully understood. However, further atomistic and coarse grain simulations are being done to better understand how these compounds self-assemble, and how they function as a channel.


Pro-angiogenic Microenvironment Restores Angiogenic Potential of Diabetic Endothelial

Cells

H. Cho1, S. Balaji1, A.Q. Sheikh1, M. Bloomer1, B. King1, K. Nolan1,F.Y. Lim2, T.M. Crombleholme2, D.A. Narmoneva1

1University of Cincinnati, Cincinnati, Ohio 2Cincinnati Children’s Hospital Medical Center

Purpose: Altered phenotype of endothelial cells (ECs) exposed to chronic hyperglycemia may contribute to insufficient neovascularization and impaired wound healing in diabetic ulcers, however, this is not completely understood. We hypothesized that 1) diabetes impairs angiogenic potential of endothelial cells and 2) this deficiency can be improved by a pro-angiogenic extracellular microenvironment. Methods: Microvascular ECs were isolated from lungs of diabetic (db/db) and C57BL/6 (WT) mice. To quantify the difference in angiogenic potential between diabetic and WT ECs, migration towards VEGF (20ng/ml) (modified Boyden chamber assay), proliferation (ki67), network formation assay on matrigel, and VEGF expression in cell lysates (ELISA) were compared. To determine the effect of microenvironment on diabetic and WT ECs, angiogenic peptide nanoscaffold was used as an in vitro 3D-system to quantify EC angiogenic response, and as an in vivo treatment for excisional wounds in db/db mice to quantify neovascularization (CD31), proliferation (ki67) and VEGF expression (ELISA) in the wounds. Results: In vitro: Diabetic ECs demonstrated significant impairment in chemotactic migration (p<0.001), proliferation (p<0.01), VEGF expression (p<0.01) and network formation (p<0.05), as compared to WT cells. However, in the angiogenic microenvironment, there were no significant differences (p>0.2) in network formation and VEGF expression between diabetic and WT ECs. In vivo: Db/db mice wounds treated with nanoscaffold had enhanced overall cell proliferation, wound neovascularization and VEGF protein levels, as compared to controls (PBS and hyaluronic-acid) (p<0.05). Discussion: Our results show that the angiogenic potential of ECs is impaired in diabetic condition and that it can be restored to that of WT ECs in angiogenic microenvironment. These results are consistent with our previous in vivo results of improved healing in nanoscaffold-treated db/db mice wounds. Overall, these results suggest that modifying wound microenvironment is a promising strategy to improve neovascularization and diabetic wound healing.


Enthalpic measurements of the hydrophobic interactions governing lysozyme adsorption onto mesoporous alkyl-functionalizsed silica

Rebecca J. Desch, JungSeung Kim, Stephen W. Thiel


Many protein separation schemes include a reverse phase process step using a

stationary phase modified with nonpolar alkyl chains. Hydrophobic interactions between the analyte and the stationary phase determine the degree of adsorption. Modifying the mobile phase composition and ionic strength impacts the interactions between the analyte and the stationary phase. Flow microcalorimetry was used to quantitatively measure the enthalpy of lysozyme adsorption onto a C18 mesocellular foam silica stationary phase in the presence of 0.01 M acetate buffer pH 5.2 mixed with varying ethanol and sodium sulfate concentrations. In all cases, a tailing exothermic peak was followed by a small desorption endothermic peak. The enthalpy of lysozyme adsorption was observed to have a maximum at 10% EtOH, and was attenuated in the presence of high EtOH or sodium sulfate. Improved understanding of the adsorption thermodynamic fundamentals can be used to create more accurate system models and ultimately design more efficient separations.

THE RELATIONSHIPS AMONG SPATIOTEMPORAL COLLAGEN GENE EXPRESSION, HISTOLOGY, AND BIOMECHANICS FOLLOWING FULL-LENGTH INJURY IN THE MURINE

PATELLAR TENDON

Nathaniel Dyment1; Nami Kazemi1; Lindsey Aschbacher-Smith2; Nicolas Barthelery2; Keith Kenter1; Cynthia Gooch1; Jason Shearn1; Christopher Wylie2; David Butler1

1University of Cincinnati, Cincinnati, OH

2Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH INTRODUCTION. Tendon and ligament injuries present a considerable socioeconomic impact as close to 50% of the 32 million musculoskeletal that occur in the US each year [1]. Improving repair of these injuries dictates that we better understand the natural healing process that leads to a non-functional scar. Type-I and type-II collagens are important structural proteins within the midsubstance and insertion, respectively. The spatiotemporal expression of these genes has not been studied extensively during healing. Therefore, we developed pOBCol3.6GFPtpz (Col1) and pCol2ECFP (Col2) double transgenic (DT) reporter mice to monitor expression on a cell-by-cell basis. Thus, the objectives of this study were to monitor changes in: 1) spatiotemporal Col1 and Col2 gene expression patterns, 2) tissue morphology, and 3) healing biomechanics following a full-length, central PT injury in Col1/Col2 DT mice and to compare these natural healing results to contralateral surgical shams and normal PT in age-matched controls. METHODS. Experimental Design. Histology and biomechanics were investigated at 6 time points post-surgery in sixty-four (64) 20-wk old (19.5±0.2 weeks; mean±SD) Col1/Col2 DT mice. Natural healing of a central PT defect was compared with healing of a contralateral sham for both histology at 1, 2, 3, 4, and 5 weeks (n=3 each) and biomechanics at 1, 2, 5, and 8 weeks (n= 12-13 each). Comparisons were also made to normal 20-wk old mice (n=18). Surgical


Procedure. The PT was accessed via longitudinal incisions along its borders. A jewelers forceps was slid under the tendon and a full-length, full-thickness, central PT defect was created. The sham procedure was performed by slipping forceps under the PT without creating the defect. Histology. Limbs were fixed in 4% PFA, decalcified in 0.5M EDTA and sectioned sagittally. Sections were imaged for Col1 (GFPtpz) and Col2 (ECFP) fluorescence or stained with H&E and imaged. Biomechanics. The native medial and lateral struts were removed and the limb was dissected down to a patella-PT-tibia unit. The tibia and patella were gripped, preloaded (0.02N), preconditioned (25 cycles, 0-1% strain, 3m/sec), and failed in uniaxial tension (3m/sec) in saline at 37°C. Statistics. Comparisons against normal and comparisons among defect groups over time (9 comparisons total) for structural properties were made via independent student t-tests with bonferroni correction for multiple comparisons (p < 0.006). RESULTS. The success rate of the surgeries was 89.3% with only 8 animals presenting lameness or ruptured tendons. The defect width measured 42% of the total tendon width (0.63 ± 0.13mm vs 1.50 ± 0.29mm, respectively). Histology. The defect region consisted of loose granulation tissue at 1 week with inflammatory cells and very little Col1 expression (Fig. 1). At 2 weeks, the defect was filled with a swollen disorganized matrix and Col1 was expressed throughout the defect length. From 3 to 5 weeks, the matrix became more aligned and Col1 expression diminished to normal levels. The sham tendons exhibited similar morphology to

normal PT with elevated Col1 expression at 1 week diminished to normal levels by 3 weeks. No Col2 expression was seen at any time point. Fig. 1: H&E (A-F) and Col1-GFPtpz/Col2-ECFP (a-f) micrographs from serial sagittal sections from normal PT (A,a) and the defect region at 1 (B,b), 2 (C,c), 3 (D,d), 4 (E,e) and 5 (F,f) weeks of healing. Fig. 2: The healing tissue showed significantly decreased structural properties at 2-, 5-, and 8-weeks compared to normal PT (p < 0.05). Horizontal lines indicate measured peak IVFs in the rabbit (21%) and goat (40%) [2-3]

Biomechanics. Ultimate loads of the 2-, 5-, and 8-week natural healing tissues were only

37%, 50%, and 48% of normal PT values, respectively (p < 0.006; Fig. 2). Similarly, stiffnesses for the natural healing tissues were 48%, 62%, and 63% of normal PT (p < 0.006). The healing tissue at 1 week was too compliant to dissect and was frequently damaged; therefore, it is not


reported. The surgical shams were not significantly different than normal at any time point (p > 0.05). DISCUSSION. The natural healing tissue did not recreate normal morphology by 5 weeks or normal mechanics by 8 weeks. The morphology exhibited characteristics of inflammation (1 week), repair (2 weeks) and remodeling (>3 weeks). There was a marked increase in Col1 expression at 2 weeks, which correlates to the repair stage of healing where matrix laydown occurs. While the limited size of the mouse prevents us from recording in vivo forces (IVFs), previously recorded IVFs from the rabbit and goat models [2-3] were translated to this model to provide a better understanding of functional healing. The defect tissue, while matching normal PT displacements at the 21% level (p > 0.05), required at least an additional 4% strain to reach the 40% level (p < 0.006) suggesting that the healing tissue was non-functional. Given its genetic power, the murine full-length PT defect injury serves as a potent tool for biological and biomechanical study of natural tendon healing. Future studies will compare natural healing in the adult to normal development in the embryo to elucidate potential treatment modalities for regeneration in the adult. REFERENCES. 1. Praemer A, 1999; 2. Juncosa N, 2003; 3. Korvick D, 1996. ACKNOWLEDGMENTS. Study supported by NIH AR46574-10, NIH AR56943-02, and NSF IGERT 0333377.

The Effects of CD151 on Prostate Cancer Cell Adhesion to the Vascular Endothelium

Jennifer L. Fischer1, Adrianne L. Shearer1, Xinyu Deng2, Xiuwei Yang2, Richard E. Eitel1, Kimberly W. Anderson1

1Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 2Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY

Cancer is the second leading cause of death in the United States, with metastatic lesions

resulting from circulating tumor cells (CTCs) most often causing the mortality. The basic steps of the metastatic cascade, from initial tumor formation to growth at a secondary site, can be conceptually sequenced as a series of events that are poorly understood. Modeling the physical properties of metastasis in order to unlock the mechanism of cancer cell adhesion to the endothelial cells (dissemination) is the focus of this research. There are two main hypotheses by which dissemination is likely to occur. Honn and Tang proposed a “docking and locking” mechanism in which CTCs roll along the endothelium breaking and forming weak bonds until velocity is slowed to a stop. Alternatively, CTCs may become lodged in vessels of smaller diameter and firmly adhere. Neither of these hypotheses has been proven definitively more influential. The objective of this study was to quantify the significance of CD151, a gene that encodes for a cell membrane glycoprotein, in the dissemination of CTCs. CD151 is suspected to enhance cancer cell motility and invasion. The prostate cancer cell line, PC3, and a PC3 line in which expression of CD151 was knocked down using shRNA were introduced into a parallel plate flow chamber and assays were performed to assess differences in relative adhesion rates and strengths. An initial attachment assay was conducted to determine the influence of CD151 under the “docking and locking” hypothesis. Tumor cells were introduced


into the flow system at various shears and the number of firmly adherent cells over time was tabulated to assess the role of CD151 in adhesion under flow conditions. A detachment assay was also employed to simulate the lodging mechanism. In this assay, tumor cells were introduced at a low shear and allowed to settle statically for 30 minutes prior to subjection to a light wash followed by shear. The ratio of cells present after the wash to those present initially was calculated to assess the role of CD151 in static attachment. The fractional adhesion of those firmly adherent tumor cells present after shear was also calculated to assess relative adhesive strengths between the PC3 lines. Preliminary results indicate that CD151 is influential initial attachment under both flow and static conditions, but the presence of CD151 does not appear to affect adhesive strength.

One-step fabrication of nanofibrous membranes for dual drug delivery using multi-axial electrospinning technique

Daewoo Han and Andrew Steckl


We have demonstrated dual drug/protein release behavior from core/sheath nanofiber

membranes produced by in a single step by coaxial electrospinning. Membranes have been characterized by measuring releasing kinetics as a function of core/sheath drug concentration and flow rate ratio effects. Higher drug/proteins loading and higher flow rate ratio results in stronger drug/proteins release. Enabling two different profiles of dual drug/proteins release will be greatly beneficial for many biomedical applications by providing both a quick release from the outer sheath layer for short-term treatment and a sustained release from the core fiber for long-term treatment. However, sustained release from coaxial fibers consisting of hygroscopic sheath layer will be a challenging issue because drugs from core will diffuse very quickly through wetted hygroscopic sheath layer. Therefore, we have suggested tri-axial electrospinning, in which an intermediate layer between the inner core and the outer sheath will act as a buffer region preventing leaching from the core. Therefore, sustained release from core will be maintained over many conditions and time periods, which will be beneficial for in-vivo wet application.

Self-Assembling Peptide Nanoscaffold for MMP Delivery and Cardiac Regeneration in the Diabetic Heart

Jennifer Hurley, AQ Sheikh, DA Narmoneva


In the diabetic heart, increased collagen accumulation, stiffness and cardiac dysfunction may be linked to the reduced expression and activity of matrix metalloproteinase 2 (MMP-2),


suggesting that diabetes-associated cardiac fibrosis may be attenuated through stimulation of native MMP-2 expression or delivery of exogenous MMP-2. Peptide nanofibers were investigated as a microenvironment for endogenous MMP-2 stimulation or exogenous MMP-2 delivery to promote the matrix remodeling response by wild type and diabetic cardiac fibroblasts. Cells were isolated from wild type or diabetic rat hearts and embedded in nanofibers, nanofibers with exogenous MMP-2, and Matrigel controls for 1, 6 and 14 days. Responses associated with matrix remodeling were assessed, including cell survival, native MMP-2 expression, ECM deposition and construct stiffness. The results demonstrate that nanofiber scaffolds provide an effective protein delivery vehicle with gradual MMP-2 release, while supporting long term survival and temporal matrix remodeling response by cardiac fibroblasts. At day 1, increased native MMP-2 expression and ECM deposition was observed in nanofiber scaffolds, whereas a shift towards active matrix remodeling was seen at day 14, with decreased MMP-2 and ECM levels and decreased stiffness. No significant differences were observed between wild type and diabetic fibroblast matrix remodeling response. No significant effects in fibroblasts matrix remodeling response was seen with incorporation of exogenous MMP-2 into the nanofibers. The results of this study suggest that peptide nanofibers may be a uniquely suited to increase local MMP-2 concentration in the diabetic heart and may be promising for applications focused on therapeutic matrix remodeling and cardiac regeneration.

Maladaptive Matrix Remodeling and Regional Biomechanical Dysfunction in a Mouse Model of Aortic Valve Disease

Varun K. Krishnamurthy1,2, Amy M. Opoka1, Christine B. Kern3, Farshid Guilak4, Daria A.

Narmoneva2, Robert B. Hinton1

1Division of Cardiology, the Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

2University of Cincinnati, Cincinnati, Ohio 3Department of Regenerative Medicine and Cell Biology, Medical University of South

Carolina, Charleston, South Carolina 4Departments of Orthopaedic Surgery and Biomedical Engineering, Duke University

Medical Center, Durham, North Carolina Aortic valve disease (AVD) occurs in 2.5% of people and remains a surgical problem.

Aortic valve malformation (AVM) underlies the majority of cases, suggesting a developmental etiology. Elastin haploinsufficiency results in complex cardiovascular abnormalities, and 20-45% of patients have AVM and/or AVD. Eln+/- mice demonstrate early AVM and latent AVD. The mechanism of pathologic ECM remodeling and its impact on the mechanical microenvironment are unknown. Aortic valve tissue from juvenile, adult, and aged Eln+/- mice was studied. Immunohistochemistry was performed for MMP-2 and 9, ADAMTS-5 and 9 (remodeling), Sox-9 and Ctrl-1 (cartilage), and intact and cleaved Versican (proteoglycan processing). Gelatin zymography determined MMP-2 and 9 enzyme activity. Regional (annulus and cusp) valve tissue mechanical properties were tested using micropipette aspiration. Cartilage-like nodules were identified within the Eln+/- valve annulus at all stages, consistent with increased Sox-9 and Ctrl-1 expression. Juvenile Eln+/- mice demonstrated AVM without AVD and were characterized


by increased valve MMP-2 and 9 expression and activity. These changes markedly increased with AVD manifestation in aged Eln+/- mice (p<0.0001). ADAMTS-5 but not ADAMTS-9 expression was increased in aged Eln+/- valve annulus, consistent with increased annular cleaved versican. Valve tissue biomechanical properties were significantly altered in aged Eln+/- cusp and annulus regions, as shown by lower Young’s modulus values (p<0.0001). These findings demonstrate that maladaptive ECM remodeling occurs early in the context of AVM and progresses, ultimately resulting in abnormal biomechanics and latent AVD. These mechanistic insights may inform the search for durable valve bioprostheses and new therapeutic targets.

In vitro characterization of nickel and chromium transport and polar pathway parameters through human stratum corneum

TD La count, MA Miller and GB Kasting

University of Cincinnati, Cincinnati, Ohio USA

The molar conductance skin of excised human skin immersed in electrolyte solutions

comprised of four cationic (Na+, K+, Ni2+, Cr3+) and five anionic (Cl, NO3, SO4

2, CrO42,

Cr2O72) species was determined as a function of concentration in Franz diffusion cells. Parallel

experiments were conducted in solutions alone to establish the validity of the method and verify the values of soln. Molar conductance decreased with increasing concentration, following the Kohlrausch Law over a 5- to 6-fold concentration range. Molar conductance values at infinite dilution were extrapolated from these data and used to estimate ionic conductances at infinite dilution. These values were used to calculate ion mobilities, transport numbers and diffusivities in solution and skin. Results for solutions were in fair agreement with published values. Results for skin showed the expected increase in cation permselectivity for monovalent cations and a 40- to 110-fold reduction in effective diffusivities with respect to those in solution. However, Ni2+ and Cr3+ were relatively less mobile in skin than in solution. Salt diffusivities calculated from ionic mobilities in skin provided a partial explanation for the difference in allergenic potency of NiCl2 and NiSO4 and also for Cr3+ and Cr6+ salts.

Identifying Novel Cellular Components Necessary for Shiga Toxin Toxicity in Primary Human Renal Cells

K. A. MacMaster1, C. A. Fuller1, D. E. Taylor1,2, T. J. Lamkin1,2 and A. A. Weiss1

1University of Cincinnati, Cincinnati, OH 2 Air Force Research Laboratory, 711th HPW/ RHPCB, WPAFB, OH

Shiga toxin (Stx) is a ribosome-inactivating protein that irreversibly removes an adenine

from the eukaryotic 28S rRNA leading to the inhibition of protein synthesis. Stx is the main virulence factor of Shiga toxin producing Escherichia coli (STEC), of which O157:H7 is a


predominate serotype. Stx is an AB5 toxin, composed of a single enzymatically-active A-subunit and five identical B subunits which make up the binding portion of the toxin. Conditions for a genome wide siRNA screen using primary human cells have been determined to identify novel cellular components important for the function and toxicity of Stx. Primary human renal proximal tubule epithelial cells (RPTECs) from a 7 year old male were used in this study. Efficient transfection of RPTECs was achieved in a 384-well format with 2000 cells/well using the transfection reagent INTERFERinTM. An initial screen utilizing a kinase siRNA sub-library was conducted. Transcriptional profiling was also performed to determine off-target effects caused by the siRNA delivery agent. As determined by an alamarBlue® metabolic assay, effective silencing was observed at 5nM using a death siRNA. Initial screen from a kinase sub-library identified potential hits, casein kinase 1, gamma 2 (CSNK1G2) and EPH receptor B4 (EPHB4). Additionally, transfection reagent INTERFERinTM did not alter transcriptional profiles, suggesting that RPTEC gene expression is unaffected by the agent. A genome-wide siRNA screen in primary cells known to be targets of toxin during clinical disease will allow for the identification of previously unknown cellular components that are important for the mechanism of action of Stx. A better understanding of Stx toxicity on the whole cell level may offer potential therapeutic targets in the future.

Biocompatibility Evaluation of HUVECs Directly onto LTCC Materials for Microfluidic Applications

William L. Mercke, Thomas Dziubla, Richard E. Eitel, and Kimberly Anderson

Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY

The expansion of Low-Temperature Co-fired Ceramic (LTCC) materials into microfluidic

systems technology has many beneficial applications. LTCC systems have the ability to quickly and inexpensively combine complex three dimensional structures with optical, fluidic, electrical functions. There have been a variety of LTCC-based microfluidic systems generated and some of them with biological applications in mind. Evaluations of the biocompatibility of these LTCC materials are vital in the expansion of LTCC microfluidics into biomedical research. Few biocompatibility studies have been conducted on LTCCs and these show negative cellular response to the thick film pastes used in the generation of electronic circuitry patterns in LTCC systems. The goal of this research is to develop an LTCC microfluidic device that monitors the permeability of an endothelial cell monolayer using “real-time” measurements. In this study, the biocompatibility of Human Umbilical Vein Endothelial Cells (HUVECs) was examined on Heraeus’s LTCC tape and two of their conductive pastes. The biocompatibility of LTCC materials was assessed by monitoring cellular attachment and viability (Live/Dead assay) at one and three days. Because of a recent study that suggests the possibility of harmful leachates within the LTCC system, this study also examines the possibility of leachates being detrimental to cells on these LTCC materials. Results indicate difficulty in the initial attachment of HUVECs to sintered LTCC tapes. However, there was no hindrance of cellular attachment and growth of HUVECs on the two conductive pastes. This study also demonstrates that possible harmful leachates from LTCC materials don’t thwart cellular attachment and growth for up to three days of cell culturing. These results provide a basis for biological devices using LTCC materials.


Construction, Optimization and Testing of a Coherent Anti-Stokes Raman Scattering Microscope

Minette Ocampo and Heather Allen

The Ohio State University, Columbus, Ohio

Coherent anti-Stokes Raman scattering (CARS) microscopy is a nonlinear vibrational

microscopy technique that generates an anti-Stokes signal that is used to detect the presence of chemical species based on their vibrational signature. CARS microscopy involves the combination of spectroscopy and microscopy that allows noninvasive characterization and imaging of chemical species without preparation or labeling. Three-dimensional sectioning with high sensitivity and resolution can be obtained from the contrast mechanism generated from the intrinsic molecular vibrational properties of the specimen.

A broadband epi-CARS microscopy system was built in the Allen laboratory and is currently being optimized. Broadband CARS microscopy allows fast data acquisition and recording of the entire CARS spectrum without tuning the Stokes frequency point by point and without changing alignment. A femtosecond Ti:Sapphire laser is used as the laser source and is divided into two arms to form the pump and the Stokes beam. A photonic crystal fiber is used to generate the broad supercontinuum of light for the Stokes beam that will interact with the narrowband pump beam to generate a CARS signal at the sample focus. To effectively suppress the background signal from the bulk medium, CARS signal is detected in the backward detection using an inverted microscope. Research is focused on use in cancer margin assessment to assist in complete resection of tumor tissues.

Shiga Toxin A and B Subunit Assembly Into Functional Toxin

Christine A. Pellino, Cynthia A. Fuller and Alison A. Weiss

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati,

Cincinnati, OH

Shiga toxin (Stx) producing E. coli (STEC) are responsible for a significant amount of food borne illness each year. The primary virulence factor, Stx, is responsible for disease symptoms. Stx is comprised of a receptor binding B-pentamer and an enzymatically active A-subunit that inhibits protein synthesis. There are two major antigenic forms, Stx1 and Stx2. While they share significant amino acid identity, Stx2 is associated with more severe disease. The molecular basis for this difference is not understood. We have identified a novel property of Stx, in which dissociated forms of the A- and B-subunits cooperate in vivo and in vitro to mediate toxicity. This property may play a role in explaining the differing potencies of the toxins.

Subunit assembly was first assessed in vitro by determining the toxicity of combined purified StxA and StxB in a protein synthesis assay. The ability of toxin assembly to occur in


vivo was then tested through bilateral IP injections of purified StxA and StxB subunits into mice. In vitro, while neither the A- nor the B- subunits alone produced any effect on the cells, when added together (A+B), they were capable of inhibiting protein synthesis similar to equivalent amounts of Stx1 and Stx2 holotoxins. Mutagenesis studies reveal that disruption of the A and B subunit interface or loss of the A subunit C-terminal residues results in a considerable reduction in mixed subunit protein synthesis inhibition. In vivo, mice that received purified Stx 1A, 1B, 2A or 2B showed no signs of illness or death. In contrast, groups receiving either doses of 1A+1B or 2A+2B exhibited signs of illness, as exhibited by extreme weight loss, and were lethal in many cases. Taken together these data reveal a means by which Stx A- and B-subunits are able to retain activity if introduced in a dissociated form, both in vitro and vivo. This newly characterized ability of Stx subunit re-association may play a role in potency differences between Stx1 and Stx2. Our long-term goal is to further characterize the interactions that mediate subunit assembly and determine their effects on potency. We plan to exploit this property to develop small molecule inhibitors of subunit association thus preventing toxicity in vivo.

Fate of Emerging Contaminants in Biomass Concentrating Reactors

William E. Platten, III and Makram T. Suidan

University of Cincinnati, Cincinnati, Ohio Chemicals of concern (COCs), which include endocrine disrupting compounds (EDCs), pesticides, and personal care products (PPCPs), have been found widely distributed in water and wastewater systems. Their potential to have a significant impact on human health and natural ecosystems makes it desirable to remove them wherever possible. Conventional wastewater treatment plants do not provide an effective barrier against the release of these COCs to receiving waters, while membrane bioreactors (MBRs) have shown better performance. A new, innovative MBR, called a biomass concentrating reactor (BCR), is being tested for removing ten COCs; ethinylestradiol (EE2), progesterone, testosterone, nonylphenol, nonylphenol-ethoxylate, nonylphenol-diethoxylate, atrazine (ATR), caffeine, carbamazepine (CMP), and triclosan. Two BCR systems were operated to test both aerobic conditions and hybrid aerobic/anoxic conditions. Both systems were operated at 6, 15, and 30-day solids retention time (SRT), while the hybrid system was also operated at different recycle ratios for returning the mixed liquor from the anoxic zone to the aerobic zone. The initial results show almost complete removal (>90%) of most the COCs at both the 6-day and 15-day SRTs. ATR (50-60% removal), CMP (40-50% removal), and EE2 (70-90% removal) were only partially removed to varying degrees. Further work is continuing on the 30-day SRT as well as the recycle ratio changes for the hybrid BCR.


Biosensing Using Voltage Control of Droplet-Interface-Bilayers

S. Punnamaraju


Droplet-interface-bilayer (DIB) lipid membrane is an artificial (model) lipid bilayer that is formed at the interface of phospholipid monolayer-coated aqueous droplets. Lipid-monolayer coated droplets are obtained by injecting aqueous droplets in dodecane-lipid oil. The DIB approach is versatile and has several attractive characteristics: choice in selecting type and volume of aqueous droplet volume (ranging from picoliters to microliters); ease of experimenting with different lipids (even assorted mixtures); mechanical overlap control of DIB dimensions; formation of various types of DIB networks; easy reformation. We report on the use of voltage enabled control over the anchor-free DIB dimensions. This electrofluidic approach, involving simultaneous optical and electrical recordings of DIB, allows active control and sensing of nanopore insertions in the DIB membrane. Both AC and DC voltages, applied across DIB, are shown to effectively modulate the DIB area and the current flow across DIB. Voltage-induced changes in interfacial tension modulated the DIB-oil contact angle and the membrane contact length, which provided control of membrane dimensions. Experimental results indicate substantial similarities between the DIB overlap control mechanism and that of electrowetting on dielectric. The process is reversible, robust and reproducible.

DIB is a good platform for ion channel studies. Biological nanopores (alpha-hemolysin ‘HL’ and Nystatin) insertions are studied using DIB. Single HL insertions are recorded at different voltages. Experiments are conducted as a function of voltage and nanopore concentration in the droplets. A smaller (larger) DIB region results in the insertion of fewer (more) ion channels and, in turn, results in lower (higher) currents being measured. The voltage control of the DIB dimensions enables an active control on the number of HL ion channels available in the lipid bilayer membrane and, thus, ion channel studies can be carried out with greater flexibility using anchor-free DIBs.

Triggered Release of Contents across Droplet Interface Bilayer (DIB) Lipid Membrane using Photopolymerizable Lipids

S. Punnamaraju


An asymmetric mixture of non-polymerizable phospholipids (DPPC or DPhPC) and cross-linking photopolymerizable phospholipids (23:2 Diyne PC) is incorporated in an unsupported artificial liid bilayer that was formed using droplet interface bilayer (DIB) approach. In DIB approach, lipids are mixed in dodecane and minute quantities of aqueous drops (typically


nanoliters) are then injected into the dodecane-lipid mixture. Lipid-monolayer is formed on the surface of aqueous drops. When two lipid-monolayer coated aqueous drops are contacted against each other, a lipid bilayer is formed at their interface. Photopolymerization of 23:2 Diyne PC is characterized from spectrophotometer analysis and color changes of 'dodecane-DPPC-23:2 Diyne PC' mixture. In DIB, domains of 23:2 Diyne PC cross-linked upon exposure to UVC (254 nm) radiation and resulted in the increase of DIB porosity. Current-voltage recordings and release of encapsulated fluorescent molecules across DIB after exposure to UVC radiation confirm the increase of DIB porosity. DIB is a versatile approach for artificial lipid bilayer formation and hence, these results suggest the potential use of DIB for studying radiation based drug delivery systems in-vitro.

Ultrasound-Enhanced Transfollicular Delivery of Polystyrene Nanoparticles into Ex Vivo

Human Skin

Kyle T. Rich,1 Steffan Nye,2 Marna Ericson,2 Robert Hoerr,3 T. Douglas Mast1

1University of Cincinnati, Cincinnati, Ohio

2Cutaneous Imaging Center, Department of Dermatology, University of Minnesota, Minneapolis, MN

3Nanocopoeia, Inc., St. Paul, MN

Topical administration of therapeutics to the skin potentially offers noninvasive local and systemic drug delivery. However, the skin provides a highly ordered, multilayered barrier that prevents the penetration of most nanoscaled therapeutics to the viable layers. This study investigated the use of ultrasound as a noninvasive approach to increase nanoparticle penetration into ex vivo, full thickness human skin. Fluorescent 840 nm polystyrene nanoparticles, modeling a nanoscaled therapeutic agent, were topically applied to skin samples. Ultrasound (center frequency 0.41 or 2.0 MHz, 10% duty cycle, dermal heating 5° C) was then applied for 30 min, followed by 90 min passive penetration. Passive (no ultrasound) controls were also conducted with 120 min duration. At each frequency, two ultrasound intensities were employed, one optimized to induce microbubble oscillations (stable cavitation) and the other optimized to additionally induce bubble collapse (inertial cavitation). Acoustic emissions from microbubble cavitation were monitored throughout insonation by a passive cavitation detector. Nanoparticle penetration and distribution in skin samples were visualized using confocal laser scanning microscopy. Confocal microscopy images revealed that follicular transport was the preferential and most significant pathway for nanoparticle penetration. Nanoparticles penetrated the dermal tissue via the follicular pathway in all skin samples treated with ultrasound at either frequency. In samples treated with 2.0 MHz ultrasound, nanoparticles significantly penetrated the dermal tissue and accumulated around the dermal vasculature. There was no clear nanoparticle penetration of either the follicle or dermis in passive (no ultrasound) controls. These results show that ultrasound increases the transfollicular penetration and dermal distribution of nanoparticles, and demonstrate the feasibility of ultrasound-enhanced delivery of nanoscaled drugs into human skin.


Biocatalytic Membranes for Carbon Dioxide Capture

Shada M Salem


Carbon dioxide is commonly considered to be the major greenhouse gas that is causing global climate change. The average temperature at the Earth's surface could increase 1.8-4.0K above the 1990 levels by the end of this century. Such warming is anticipated to cause sea level rise, increased intensity and frequency of extreme weather events, ice shelf disruption, and changes in rainfall patterns. As a result, reducing CO2 emissions from anthropogenic sources is a high priority.

The rate of hydration of CO2 can be improved by carbonic anhydrase (CA) enzyme. This enzyme is highly adapted to the hydration of CO2 in the sense that the reaction rates are almost diffusion limited. CA catalyzes the hydration of CO2 at rates of more than 1 million molecules of CO2 per second .This means that less than 1 kilogram of carbonic anhydrase will be necessary to sequester the approximately 290 tons of CO2 produced per hour from a typical 300-MW coal fired power plant.

The proposed research focuses on the development of a biocatalytic membrane for carbon dioxide capture from combustion flue gas.The membrane consists of an inert matrix in which carbonic anhydrase (CA) is immobilized. The pore volume of the membrane is filled with liquid in which CO2 and water are both soluble, allowing formation of bicarbonate and carbonate species that can diffuse through the liquid to a receiving fluid. The receiving fluid can be either a liquid or a gas with a low carbon dioxide partial pressure. If the receiving fluid is a liquid, it can contain a species such as an amine that can capture carbonate, so that a bicarbonate or carbonate concentration gradient is established to drive total carbonate transport through the membrane. The receiving fluid can be contacted with a solid adsorbent to remove captured carbonate species, carbonates can be removed by precipitation, or carbon dioxide can be removed by stripping or distillation. If the receiving fluid is a gas, CA can catalyze the formation of carbon dioxide from bicarbonate in the pore fluid. Carbonic anhydrase can be either used as an independent carbon dioxide extraction catalyst or it can be combined with conventional CO2 extraction technologies such as chemical absorption using amine-based solvents.

Development of Molecularly Templated Silica Materials for the Adsorption of Soluble Sugars and Protein Capture

Daniel Schlipf, Barbara L. Knutson and Stephen Rankin

Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY


Mesoporous silica materials (MSM’s) synthesized via surfactant templating are envisioned as a versatile materials platform for the separation of proteins and glycosylated proteins. MSM’s have been templated with ionic surfactants and block copolymers to obtain ordered porous networks with precisely controlled pore diameters. Adsorbing nearly three times their own weight, we have recently demonstrated that MSM’s templated with CTAB, a surfactant, have exceptional capacities as adsorbents for sugars. Utilizing MSM’s high sugar capacities, large pore SBA-15 materials will provide size selective capturing of proteins and glycosylated proteins. Using the ionic surfactant cetyl-trimethyl ammonium bromide (CTAB), MSM’s were template via a silica precursor condensation method. After removal of CTAB, the templated materials had a hexagonal pore arrangement with a pore size of approximately 3 nm. Hydrothermally aging of SBA-15 materials, templated with a block copolymer, at different temperatures provides a large range of meso and macro-porous pore sizes (approximately 6 and 10 nm). The effect of pore size on the adsorption of sugars was analyzed via sugar depletion experiments using high pressure liquid chromatography. Fourier Transform IR was used to determine complete surfactant or copolymer removal. Using nitrogen adsorption, the pore sizes of the materials were determined to be between 3 and 10 nanometers. The surface areas were also reported to be as great as 800 m^2/gram and decreased to 630m^2/gram with increasing pore size. TEM images and XRD confirm the presence of hexagonally arranged nanopores. In future studies, MSM’s large affinity for sugar will be utilized by creating a stabilizing environment for glycosylated proteins. The pore diameters achieved by using SBA-15 materials will provide large enough pore sizes allowing for the capture of proteins and larger bio-molecules.

Electromagnetic field mediates capillary-like network formation via mapk/erk signaling cascade

Abdul Q. Sheikh

University of Cincinnati, Cincinnati, Ohio Tissue stimulation using electromagnetic fields (EMFs) is a novel therapy to enhance

angiogenesis and treat chronic wounds. EMFs act as directional cues in cellular migration and activation of angiogenic signaling cascades, although the underlying mechanisms remain unclear. This study aimed to elucidate these mechanisms and to determine whether EMF-mediated angiogenesis 1) depends on EMF frequency, and 2) is regulated via the MAPK/ERK pathway. Mouse endothelial cells were seeded (105 cells/cm2) on the 3D peptide nanoscaffold and exposed to high frequency (7.5GHz) or low frequency (60Hz) EMF using a custom-built EMF setup, with or without MAPK/ERK inhibitor (n=4/group). Capillary-like networks were quantified using images of DAPI stained cell nuclei. High frequency EMF exposure resulted in significantly larger capillary networks as compared to non-EMF controls (30±4um vs. 18±3um, respectively, mean±SD, p<0.05), while no difference was observed between controls and low frequency EMF (20±4um). Interestingly, high frequency EMF exposure in the presence of


MAPK/ERK inhibitor resulted in reduced network size (10±4um). Western blot analyses showed higher MEK and ERK phosphorylation levels in high frequency EMF group as compared to 60 Hz EMF and non-EMF controls. These findings suggest that high frequency, but not low EMF may enhance the binding affinity between MEK1/2 kinase and its binding partner (c-Raf kinase or MAPK/ERK inhibitor). In summary, our results show that pro-angiogenic effect of EMF on endothelial cells is frequency-dependent and is regulated via MAPK signaling pathway.

Silicone Adhesive Membrane for Improving Oral Delivery of Ionizable Drugs

Gaurav Tolia and S.K. Li

College of Pharmacy, University of Cincinnati, Cincinnati, Ohio Purpose: Oral controlled release tablets are exposed to a wide range of pH conditions and mechanical agitation along the gastrointestinal tract. This leads to undesirable fluctuations in release rates of ionizable drugs and variable drug plasma concentrations in vivo. The purpose of the study was to a) determine the release rates of weakly basic model drug verapamil hydrochloride from matrix tablets prepared using cohesive, flexible and water insoluble silicone pressure sensitive adhesive under different pH conditions, b) compare the results of silicone adhesive tablets with those of the tablets prepared using non-cohesive and rigid ethyl cellulose, and c) study the mechanism of drug release from silicone adhesive tablets. Methods: Silicone pressure sensitive adhesive Bio-PSA® 7-4202 (Dow Corning Corporation) was used in this study. Ethyl cellulose (48% ethoxy content and 2.5 degree of substitution) was used for direct comparison. Matrix tablets containing 20% w/w polymer and 80% w/w verapamil hydrochloride were tested using USP dissolution apparatus 1. The effect of dissolution medium pH on verapamil hydrochloride release was studied using either simulated gastric fluid pH 1.2 or simulated intestinal fluid pH 6.8. Polymer membrane properties were studied using electrical resistance measurements. Tablet morphological changes during drug release were studied using a pH-indicator dye methyl red. Results: Silicone adhesive tablets decreased in size during drug release, showed Hindependent release and demonstrated high resistivity to ion influx. In comparison, ethyl cellulose tablets were highly porous, showed low resistivity to ion influx, and failed to provide pH-independent release of verapamil hydrochloride. Conclusion: Silicone adhesive can be useful for the construction of controlled release matrices of ionizable drugs in order to reduce the variability in drug release due to the changing dissolution conditions encountered in vivo.

Symposium Poster Abstracts - University Of Cincinnati Regional... · 2018-08-17 · Symposium...

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