2016 S r 2 Year Talks

2016 S u m m e r r e S e a rc h r e v i e w
2nd Year Talks
Wednesday, June 1, 2016
Schedule and Abstracts
DEPARTMENT of CHEMICAL
& BIOMOLECULAR ENGINEERING
U N I V E R S I T Y of D E L AWA R E
Summer Research Review-Second Year Talks
Colburn Laboratory/Spencer Laboratory
Colburn 102/Spencer 114
June 1, 2016
8:50-9:00
Welcome (Colburn 102)
Amber Hilderbrand, Colburn Club President
Session 1
(Colburn 102)
9:00-9:20
Andrew S. Gaynor, Advisor: Wilfred Chen
“Traceless Shielding-Mediated Rescue of Yeast Cytosine Deaminase for the Targeted
Treatment of Cancer”
9:20-9:40
Julia Rohlhill, Advisor: Eleftherios T. Papoutsakis
“Engineering a Native E. coli Formaldehyde-inducible Promoter for the Development of
Synthetic Methylotrophy”
9:40-10:00
Brian McConnell, Advisor: Maciek R. Antoniewicz
“Symbiotic Growth of the Photoautotrophic Microalga Chlorella vulgaris with Heterotrophic Microbes”
10:00-10:20
Camil A. C. Diaz, Advisor: Maciek R. Antoniewicz
“Powering Nitrogen Fixation: The Metabolism of the Aerobic Diazotroph, Azotobacter vinelandii, as
Revealed by 13C-metabolic Flux Analysis”
10:20-10:40
Break
Session 2
(Colburn 102)
10:40-11:00
John Ruano-Salguero, Advisor: Kelvin H. Lee
“Development of a Dynamic, 3D Blood-brain Barrier Model to Study Drug Transport”
11:00-11:20
Andrew Swartz, Advisor: Wilfred Chen
“Enhanced Affinity Precipitation of Antibodies using Functionalized Protein Nanoscaffolds”
11:20-11:40
Glenn Ferreira, Advisor: Christopher J. Roberts
“Predictive Approaches to Biophysical Property Assessment for Protein Pharmaceutical
Development”
11:40-12:00
Stijn Koshari, Advisors: Norman J. Wagner and Abraham M. Lenhoff
“Understanding Microstructure and Stability in Solid-State Biopharmaceutical Formulations”
12:00-1:15
Lunch (Colburn 102, Colburn 104, Colburn 109)
Summer Research Review-Second Year Talks
Colburn Laboratory/Spencer Laboratory
Colburn 102/Spencer 114
June 1, 2016
Session 3
(Colburn 102)
1:20-1:40
Jannat Nayem, Advisors: Norman J. Wagner and Yun Liu
“Effects of Surfactant Degradation on Solution Physiochemical Properties Relevant for
Protein Formulations used in Cancer Treatment”
1:40-2:00
Rashida E. N. Ruddock, Advisors: Millicent O. Sullivan and Thomas H. Epps, III
“Design of RNA-responsive Polymeric Nanocapsules for Personalized Therapeutic Delivery”
2:00-2:20
Katherine L. Wiley, Advisor: April M. Kloxin
“Development of Dynamic Hydrogels to Understand Cell Response to Matrix Remodeling”
2:20-2:40
Thomas E. Gartner, III, Advisors: Thomas H. Epps, III and Arthi Jayaraman
“Utilizing Gibbs Ensemble Molecular Dynamics and Hybrid Monte Carlo/Molecular
Dynamics Simulations for Efficient Study of Polymer-Solvent Phase Equilibria”
2:40-3:00
Hao Wang, Advisor: Yushan Yan
“Highly Stable Anion-Exchange Membranes for High-Voltage Redox-Flow Batteries”
3:00
END
Summer Research Review-Second Year Talks
Colburn Laboratory/Spencer Laboratory
Colburn 102/Spencer 114
June 1, 2016
8:50-9:00
Welcome (Colburn 102)
Amber Hilderbrand, Colburn Club President
Session 1
(Spencer 114)
9:00-9:20
Chen-Yu Chou, Advisor: Raul F. Lobo
“Reverse Water-Gas Shift Iron Catalyst Derived from Fe 3 O 4 ”
9:20-9:40
Garam Lee, Advisor: Dionisios G. Vlachos
“Homologous Series Study in Small Alkane Total Oxidation and Subsequent Kinetic and X-Ray
Absorption Spectroscopy Study on Concentration Hysteresis in Methane Total Oxidation over
Pt/Al 2 O 3 , Pd/Al 2 O 3 , and 1:3 Ag-Pd/Al 2 O 3 ”
9:40-10:00
Ziwei Cheng, Advisor: Dionisios G. Vlachos
“Structural Characterization of Fructose-derived Humins”
10:00-10:20
Pratyush Agarwal, Advisor: Michael T. Klein
“Software Tool Development for a Molecular-Level Vacuum Gas Oil Hydroprocessing
Kinetic Model”
10:20-10:40
Break
Session 2
(Spencer 114)
10:40-11:00
Wesley W. Luc, Advisor: Feng Jiao
“Catalyst Development for an Advanced Oxygen Recovery System”
11:00-11:20
Nicholas Gould, Advisor: Bingjun Xu
“Effect of Liquid Water on Acid Sites of NaY: An in-situ Liquid Phase Spectroscopic Study”
11:20-11:40
Jeffrey Heyes, Advisor: Bingjun Xu
“CO 2 Reduction on Cu at Low Overpotentials with Surface Enhanced In-Situ Spectroscopy”
11:40-12:00
Jared Nash, Advisors: Bingjun Xu and Yushan Yan
“Effect of pH on the Activity of Hydrogen Oxidation Reaction/Hydrogen Evolution Reaction
over PtRu Bimetallic Catalysts”
12:00
END OF TALKS IN SPENCER 114
12:00-1:15
Lunch (Colburn 102, Colburn 104, Colburn 109)
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Software Tool Development for a Molecular-Level Vacuum Gas Oil Hydroprocessing
Kinetic Model
Pratyush Agarwal
Advisor: Michael T. Klein
Committee Members: Antony N. Beris, Prasad S. Dhurjati
With increasing regulations and demands of information at the molecular level coupled with the
ability of modern analytical chemistry to provide detailed molecular classifications of feedstocks,
molecular-level kinetic modeling has become increasingly relevant for profit maximization and
product quality control in industry. An I/O (input/output) converter application has been
developed to study the impacts of reactor conditions and feedstocks on the industrially relevant
reactor outlets of a vacuum gas oil hydroprocessing unit. The application builds a vacuum gas oil
composition model by representing the feedstock molecules as a set of user-defined probability
density functions with adjustable parameters that are tuned to experimental data. The output of
the composition model, mole fractions of the individual species, along with a reaction network
mapping the change from reactants to products are used as the inputs of a kinetic model. The
kinetic model is solved as a fixed bed reactor under plug-flow with LHHW rate laws for different
process conditions, which are typically around 350˚C and 60 bar for hydrotreating. Additionally,
model solution time reduction was studied with the use of statistics and regression for model
parameter minimization in empirical models. These empirical models are based on fundamental
kinetic models in a limited working range but have their main benefits in being able to produce
answers quickly and with good accuracy as compared to the fundamental models.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Structural Characterization of Fructose-derived Humins
Ziwei Cheng
Advisor: Dionisios G. Vlachos
Committee Members: Dionisios G. Vlachos, Raul F. Lobo and Bingjun Xu
Humins are carbonaceous, polymeric by-products that are almost inevitably formed during acidcatalyzed, hydrothermal processing of sugars to bio-based platform molecules, such as 5hydroxymethylfurfural (HMF). 1 It is generally believed that they form as a result of
uncontrolled cross-polymerization reactions of HMF and other reaction intermediates. 1
Currently, humins are a low-value product only used for combustion in biorefineries.2 Any effort
of valorizing humins, however, is hampered by limited understanding of their structure. In
previous studies, only infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies have
been used to characterize humins.2–5 These techniques revealed little information beyond the
functional groups present in humins. Here, we aim at further understanding the structure of
humins by combining spectroscopic techniques with liquid chromatography coupled with mass
spectrometry (LC-MS). Different classes of solvents were used to test the solubility of humins.
The humin species solubilized in select solvents were separated using LC before entering the MS
for molecular weight determination. The LC-MS showed that different solvents solubilize
different species. These findings can be explained by a secondary structure present in humins,
which has not been proposed in previous work. Studies are underway to separate and purify the
small humin molecules for structural characterization.
(1)
(2)
(3)
(4)
(5)
van Zandvoort, I.; Koers, E. J.; Weingarth, M.; Bruijnincx, P. C. A.; Baldus, M.;
Weckhuysen, B. M. Green Chem. 2015, 17, 4383–4392.
Hoang, T. M. C.; Lefferts, L.; Seshan, K. ChemSusChem 2013, 6 (9), 1651–1658.
Patil, S. K. R.; Heltzel, J.; Lund, C. R. F. Energy and Fuels 2012, 26 (8), 5281–5293.
Sumerskii, I. V; Krutov, S. M.; Zarubin, M. Y. Russ. J. Appl. Chem. 2010, 83 (2), 320–
327.
Van Zandvoort, I.; Wang, Y.; Rasrendra, C. B.; Van Eck, E. R. H.; Bruijnincx, P. C. A.;
Heeres, H. J.; Weckhuysen, B. M. ChemSusChem 2013, 6 (9), 1745–1758.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Reverse Water-Gas Shift Iron Catalyst Derived from Fe3O4
Chen-Yu Chou
Advisor: Raul F. Lobo
Committee Members: Michael T. Klein, Dionisios G. Vlachos, Bingjun Xu
Unsupported iron oxides, specifically magnetite (Fe3O4), were used as a catalyst for the
reverse water-gas shift (RWGS) reaction at temperatures between 723 K and 773 K. This
catalyst exhibited fast catalytic CO formation rate (35.1 mmol h-1 g-1), high turnover frequency
(2.33×106 s-1), high CO selectivity (>99%), and high stability. Reaction rates over Fe3O4 catalyst
displayed a strong dependence on H2 partial pressure (reaction order of 0.79) and a weaker
dependence on CO2 partial pressure (reaction order of 0.33) under equimolar flow of both
reactants. X-ray powder diffraction patterns and XPS spectra reveal that the bulk structure of the
post-reaction sample was mostly metallic Fe and Fe3C while the surface contained Fe2+, Fe3+,
metallic Fe, and Fe3C. The activity test on pure Fe3C (iron carbide) suggests that Fe3C may not
be the major active sites. Gas-switching experiments (CO2 or H2) indicated that a redox
mechanism is the predominant reaction pathway, but the inverse kinetic isotope effect suggested
a secondary pathway for CO formation, which is probably the so-called associative mechanism.
Potential reaction mechanisms for this reaction will be analyzed.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Powering nitrogen fixation: The metabolism of the aerobic diazotroph,
Azotobacter vinelandii, as revealed by 13C-metabolic flux analysis
Camil A. C. Diaz
Advisor: Maciek R. Antoniewicz
Committee Members: Wilfred Chen, Kelvin H. Lee
Diazotrophs, or organisms capable of the enzymatic conversion of atmospheric nitrogen
into ammonia, present a metabolic paradox. On the one hand, nitrogen fixation is an energyintensive process, requiring 16 moles of ATP and 8 moles of reducing equivalents per mole of
N 2 fixed. On the other hand, the nitrogenase enzyme is highly oxygen-labile, readily inactivated
by oxidation of its metal cluster. Therefore, while an anaerobic lifestyle would circumvent the
issue of the oxygen sensitivity of the nitrogenase, low ATP yields from fermentation present a
significant limitation to the net ammonium output of anaerobic diazotrophs. Meanwhile,
diazotrophs that are obligate aerobes (e.g. Azotobacter vinelandii, Azorhizobium caulinodans)
are thought to cope instead by employing high respiration rates and protective membranes that
limit the diffusion of oxygen. One of the most well studied diazotrophs, A. vinelandii, is an
aerobic, fast-respiring bacterium found in soils globally. Although the biochemistry of this
organism has been extensively documented, the features of its core metabolism that allow it to
fix nitrogen and thrive in an aerobic environment have yet to be fully elucidated.
In this work, we have constructed a metabolic network model that captures the central
carbon metabolism and amino acid biosynthesis pathways of A. vinelandii. The model was
thoroughly validated by performing complementary parallel labeling experiments using all singly
labeled glucose tracers, that is, [1-13C]glucose through [6-13C]glucose and measuring the isotopic
labeling of biomass by GC-MS. Subsequent 13C-metabolic flux analysis (13C-MFA) confirmed
three key aspects of the metabolism in this organism, namely that A. vinelandii:
(1) Exclusively employs the Entner-Doudoroff pathway for glucose catabolism.
(2) Produces alanine from cysteine instead of pyruvate as a byproduct during nitrogenase
biosynthesis.
(3) Diverts a significant portion of its carbon flux towards respiration rather than cell growth.
Additionally, for the first time, we present conclusive evidence that this diazotroph exhibits high
flux through the TCA cycle, surprisingly during both nitrogen-starved and nitrogen-replete
conditions.
Finally, to probe the ability of this diazotroph to support the nitrogen needs of another
organism, an engineered strain of A. vinelandii that overproduces ammonium was grown in coculture with a panel of microbes known to secrete carbon sources utilizable by A. vinelandii. By
adaptively evolving these organisms in co-culture and identifying mutations that promote
improved symbiosis, we seek to gain additional insights into the metabolism that supports
nitrogen fixation. The knowledge gained from studying the core metabolism that enables
microbial nitrogen fixation in A. vinelandii offer valuable insight for future efforts to engineer
diazotrophs as more environmentally and economically favorable alternatives to man-made
sources of fixed nitrogen (e.g. the Haber-Bosch process) for industrial fermentation and
agricultural applications.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Predictive Approaches to Biophysical Property Assessment for Protein Pharmaceutical
Development
Glenn Ferreira
Advisor: Christopher J. Roberts
Committee Members: Abraham M. Lenhoff and Arthi Jayaraman
Protein therapeutics are the fastest growing sector within the pharmaceutical industry with sales
exceeding sixty billion USD/year and greater than 10% year-over-year growth [1]. Protein
therapeutics treat a plethora of chronic and/or life-threatening diseases. Monoclonal antibodies
(mAbs) are the largest subset of protein therapeutics [1] and are excellent drug candidates
because of their specific and tight bind to drug targets.
Non-native protein aggregation is a ubiquitous concern when bringing a new drug or formulation
to market. Aggregation can proceed through a variety of mechanisms, resulting in many different
aggregated morphologies [2]. At best, the resulting aggregates adversely affect product potency
and must be closely monitored and controlled to meet regulatory requirements [3]. At worst,
protein aggregates pose a safety and immunogenicity concern [4], [5]. Rapidly creating a stable
formulation that minimizes aggregation is an on-going challenge within the pharmaceutical
industry. This project seeks to utilize a combination of experimental and computational
techniques to predict mAbs long-term stability with various formulation conditions.
The present work focuses on mapping aggregation rates and temperature dependences of IgG1
and IgG4 mAbs at range of pH values and NaCl concentrations that include commonly selected
processing and formulation conditions. The aggregation behaviors were related to thermal
scanning calorimetry measurements, diffusion interaction parameters (k D ), and second osmotic
virial coefficients (B 22 ) that are practical to obtain from dynamic and static light scattering,
respectfully. The objective of this initial work is to provide a starting point for assessing how
biophysical properties and protein-protein interactions relate with the aggregation kinetics for
each mAb. Future efforts will seek to expand this understanding with additional biophysical
properties, both experimental and computational in nature. The ultimate objective is prediction of
medium- and long-term aggregation rates and behavior from a sample-sparing set of biophysical
properties and short-term aggregation behaviors, combined with structural details for a given
protein and solvent environment.
(1)
(2)
(3)
(4)
(5)
S. Aggarwal, Nat. Biotechnol. 32, 32–39 (2014).
C. J. Roberts, Curr. Opin. Biotechnol. 30C, 211–217 (2014).
W. F. Weiss, T. M. Young, C. J. Roberts, J. Pharm. …. 98, 1246–1277 (2009).
W. Wang, C. J. Roberts, Aggregation of Therapeutic Proteins (John Wiley & Sons,
Hoboken, NJ, ed. 1st, 2010).
A. M. Fathallah et al., J. Pharm. Sci., in press, doi:10.1002/jps.24592.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Utilizing Gibbs ensemble molecular dynamics and hybrid Monte Carlo/molecular
dynamics simulations for efficient study of polymer-solvent phase equilibria
Thomas E. Gartner, III
Advisors: Thomas H. Epps, III and Arthi Jayaraman
Committee Members: Eric Furst, Michael Mackay
Polymer thin films have seen increasing use in multiple technological applications such as
organic electronics, nanopatterning, battery and fuel cell membranes, and others.1-3 A primary
driver of a polymer film’s properties (and thus its technological usefulness) is its morphology, or
spatial arrangement of film components. Common polymer solution casting methods such as spin
coating or flow coating often feature rapid and uncontrolled solvent evaporation that kinetically
traps the polymer film into undesirable non-equilibrium and/or disorganized morphologies.3
Understanding the mechanisms underlying morphological rearrangement during solution
processing is key to producing reliable and useful polymer films, however tracking the spatial and
time evolution of solvent content and film morphology though in-situ experimental methods is a
major challenge.3 Therefore, we are motivated to apply simulation and theory to the
solvent/polymer film system to provide further mechanistic insight. Herein, we describe an
extension of the Gibbs ensemble molecular dynamics (GEMD) simulation method,4 which can
capture the phase equilibrium between polymer film and solvent vapor while probing important
dynamic properties relevant to the solvent processing of polymer films. Our modifications to
GEMD allow for direct control over particle transfer between phases and improve the method’s
numerical stability. The performance of this modified GEMD method is compared to a hybrid
Monte Carlo (MC)/molecular dynamics (MD) Gibbs ensemble simulation scheme in the context
of single and multi-component Lennard-Jones fluids and polymer/solvent mixtures relevant to
polymer film solution processing. We found that GEMD has significant advantages in
computational efficiency relative to hybrid MC/MD and achieves similar phase equilibrium results
across much of the phase window. However, GEMD is less stable than the hybrid method for
conditions close to the critical point. We propose that the computationally efficient GEMD
simulations be used to map out the majority of the phase window, with hybrid MC/MD used as a
follow up for conditions where GEMD may be unstable. In this way, we can capitalize on the
contrasting strengths of these two methods to facilitate the computational study of solvent effects
in polymer film processing.
1. Luo, M.; Epps, T. H., III, Macromolecules 2013, 46 (19), 7567-7579.
2. Hallinan, D. T., Jr.; Balsara, N. P., Annual Review of Materials Research, Vol 43 2013, 43,
503-525.
3. Sinturel, C.; Vayer, M.; Morris, M.; Hillmyer, M. A., Macromolecules 2013, 46 (14), 53995415.
4. Kotelyanskii, M. J.; Hentschke, R., Physical Review E 1995, 51 (5), 5116-5119.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Traceless Shielding-Mediated Rescue of Yeast Cytosine Deaminase for the Targeted
Treatment of Cancer
Andrew S. Gaynor
Advisor: Wilfred Chen
Committee Members: April Kloxin and Millicent Sullivan
While chemotherapeutics remain the gold standard for treating cancer in the clinic, they
frequently exhibit off-target effects that greatly diminish patients’ quality of life. Targeted
chemotherapies would alleviate this issue while maintaining high efficacy. One means of
distinguishing cancer cells from healthy cells is by exploiting the drastic proteome differences
between the two cell types. We envision regulating the activity of an enzyme, yeast cytosine
deaminase (yCD), by utilizing a conditional degradation tag, which flags a protein of interest for
degradation by default unless a specific trigger is presented to stabilized and preserve it. Once a
cancer cell is identified, (yCD) can be used to convert the non-toxic prodrug, 5-fluorocytosine,
into the toxic chemotherapeutic, 5-fluorouracil, in that cell. Here, we demonstrated that
Traceless Shielding (TShld) is an attractive conditional degradation tag system that leads to
enhanced yCD-mediated HeLa cell death in the presence of its trigger. Furthermore, we used
orthogonal fluorescent proteins to provide real-time information about the status of the system.
Since TShld represents one conditional degradation system out of several that have been
previously characterized, we hope to integrate a combination of systems to build complex protein
circuit architectures featuring multiple levels of control. This powerful technology should
minimize background yCD activity in healthy cells and the development of smarter targeted
chemotherapies.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Effect of Liquid Water on Acid Sites of NaY: An in-situ Liquid Phase Spectroscopic Study
Nicholas Gould
Advisor: Bingjun Xu
Committee Members: Raul Lobo, Dionisios Vlachos
Liquid phase catalyst characterization is experimentally challenging, but it deserves
consideration because solvent choice can affect catalyst structure and acidity. Thus, in vacuo
spectroscopy of adsorbed probe molecules like pyridine, while valuable, may provide incomplete
information on available catalytic sites in liquid phase reactions. To this end, an Attenuated Total
Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) cell was used to detect pyridine
adsorbed on sodium faujasite (NaY) in liquid water. The slow accumulation of pyridinium on NaY
in water was observed in the ATR cell, suggesting water promotes Brønsted acidity on NaY.
Transmission FTIR and ATR-FTIR experiments revealed pyridinium concentration increased with
increasing water concentration, making the liquid phase technique more apt than the established
vacuum technique for observing the Brønsted site. The accumulation of protonated pyridine in
NaY pores led to the question of whether NaY can support Brønsted acid catalyzed pathways in
protic solvent.
The Brønsted catalyzed ring opening of 2,5-dimethylfuran (DMF) to 2,5-hexanedione is
not active on NaY in water. However, this is attributed to the lack of basicity of DMF. A weaker
base than pyridine, deuterated acetonitrile, demonstrated no Brønsted character when adsorbed on
NaY in water, while stronger bases such as 2,6-dimethylpyridine, were protonated. The possibility
of probe molecule induced latent Brønsted acidity will be discussed. Thus, although NaY is
capable of forming pyridinium in water, NaY may not be active with respect to Brønsted catalyzed
pathways unless a reactant (or probe molecule) is sufficiently basic. This also suggests that
although probe molecule adsorption can detect acid sites, these sites may be induced by the probe
molecule of choice, and detecting acid sites says little about site activity.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
CO2 Reduction on Cu at Low Overpotentials with Surface Enhanced In-Situ Spectroscopy
Jeffrey Heyes
Advisor: Bingjun Xu
Committee Members: Feng Jiao, Yushan Yan
In-situ surface enhanced spectroscopic and reactivity investigations on the
electrochemical reduction of CO2 at low overpotentials (<0.7 V) was conducted on Cu surfaces.
Vibrational bands corresponding to adsorbed hydrogen (Had) and CO (COad) on Cu have been
identified at 2090 cm-1 and 2060 cm-1, respectively. Spectroscopic investigations show that Had is
capable of partially displacing COad, however, COad is unable to displace Had to any detectable
level. The preferential adsorption of H over CO on Cu is consistent with the high selectivity
towards the hydrogen evolution reaction at potential > -0.8 V vs. RHE.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Understanding Microstructure and Stability in Solid-State Biopharmaceutical
Formulations
Stijn Koshari
Advisor: Dr. Norman J. Wagner and Dr. Abraham M. Lenhoff
Committee Members: Dr. Eric M. Furst, Dr. Yun Liu, and Dr. Christopher J. Roberts
Although most macromolecular biotherapeutics are administered as aqueous solutions through
subcutaneous injection or intravenous infusion, sometimes there is a need to develop a solid-state
formulation. Solid-state formulations help mitigate major protein stability challenges including
chemical and physical degradation mechanisms and enable long-term storage and transport. The
optimal drying processes and conditions and the resulting long-term stability of the
biopharmaceutics are major topics in current research. In contrast, the physical characteristics of
the dried formulations themselves have been of lesser importance, as the powders are typically
reconstituted before administration. This is unfortunate, as the solid-state particle morphology and
particularly the protein distribution within contain important information that could potentially
impact long-term stability. This research project, in collaboration with Genentech, focuses on
using novel experimental techniques to investigate the morphology and microstructure of solidstate biopharmaceuticals, with particular interest in protein aggregation and protein-excipient
microheterogeneity. The results of these techniques are used not only to better understand the
effect of microstructure on protein stability, but also to model some processes fundamental to
solid-state biopharmaceutics.
We have pioneered the use of two experimental techniques to investigate the behavior of proteins
and excipients in solid-state pharmaceuticals: (1) confocal fluorescent microscopy (CFM) and (2)
small-angle neutron scattering in a vapor cell (VC-SANS). By studying industrially relevant
monoclonal antibody and antibody fragment formulations, we have shown that while freeze-dried
(or lyophilized) formulations lead to a uniform protein-excipient distribution and spray-dried
formulations have the tendency to lead to protein-excipient microheterogeneity, the resulting nanostructure seems to be similar for both processes. In addition, we are using these results to (1) create
better models for spray-drying processes and (2) better understand D2O diffusion and proteinexcipient interactions in solid-state hydrogen-deuterium exchange with mass-spectroscopy
(ssHDX-MS) experiments, an upcoming and promising technique in the biotech industry to predict
long-term protein stability in solid formulations.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Homologous series study in small alkane total oxidation and subsequent kinetic and X-ray
absorption spectroscopy study on concentration hysteresis in methane total oxidation over
Pt/Al2O3, Pd/Al2O3, and 1:3 Ag-Pd/Al2O3
Garam Lee
Advisor: Dionisios G. Vlachos
Committee Members: Raul F. Lobo and Bingjun Xu
The catalytic activity of seven monometallic (Pt, Pd, Rh, Ag, Ni, Cu, and Co/Al2O3) and
three nominal compositions (1:3, 3:1, and 1:10) of Ag-Pd/Al2O3 bimetallic catalysts are
evaluated for the total oxidation of small alkanes (methane, ethane, propane, and isobutane) in
the 280-400 ˚C temperature range. Volcano-type dependences of the methane turnover frequency
(TOF) on the C and O binding energy are observed for all small alkanes tested. Promising 1:3
Ag-Pd bimetallic exhibits superior activity than the existing most active single-metal Pt/Al2O3
catalyst in the oxidation of alkane which has carbon number 2 or higher while Pt shows superior
activity in methane oxidation. The kinetics of methane oxidation over Pt/Al2O3, Pd/Al2O3, and
Ag-Pd/Al2O3 are then investigated as a function of O2/CH4 ratio at 340 ˚C. We show that there
are three distinct kinetic regimes in which the TOF is (1) first-order, (2) negative-order, and (3)
zero order with respect to oxygen concentration. Also, we found that at low to intermediate
oxygen concentration (O2/CH4 ratio < 3), Pd-based catalysts (both Pd and Ag-Pd) exhibit
superior catalytic activity, while at high oxygen concentration, Pt shows superior activity.
Moreover, it is found that methane oxidation over Pt, Pd, and Ag-Pd can demonstrate two
distinct activity regimes at identical compositions of the reaction mixture: a high activity regime
and a low activity regime. Oxygen concentration change and its direction of change can switch
between two activity regimes: a high activity upon increasing oxygen concentration and a low
activity upon decreasing oxygen concentration. For Pt, Pd, and Ag-Pd catalyst, the activity
increased linearly with increasing oxygen partial pressure. After reaching the maximum, the
activity decreases with increasing oxygen concentration. At high O2/CH4 ratio, the activity
reaches plateau, completely switched to low activity regime. Upon decreasing oxygen
concentration, reactivation of Pt, Pd, and Ag-Pd catalyst take place at low O2/CH4 ratio. The
correlation between in situ X-ray absorption spectroscopy (XAS) and catalytic behavior suggests
that the switching between two activity regimes is caused by two nonequivalent oxidation state
of Pd and Ag as well as slight structure changes in oxidized state. In the reduced state, Pd and Ag
form a random mixed alloy, while in the oxidized state, a sandwich oxide between Pd and Ag is
possibly formed.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Catalyst Development for an Advanced Oxygen Recovery System
Wesley W. Luc
Advisor: Dr. Feng Jiao
Committee Members: Dr. Bingjun Xu, Dr. Yushan Yan
In order to enable future NASA deep space human exploration missions, research towards
the development of Advanced Oxygen Recovery Systems (AORS) is crucial. The current air
revitalization system in operation on the International Space Station is a partially-closed system
that only recovers about 50% of oxygen (O2) from carbon dioxide (CO2). A novel AORS that is
fully-closed will increase oxygen recovery rate from the current state-of-the-art, ultimately
reducing mission life-cycle costs and mass of consumables on board spacecrafts [1]. One
promising technology is aqueous-based CO2 electrochemical reduction. However, recent research
focuses primarily on the development of efficient CO2 reduction electrocatalysts, while little
attention has been given to the development of an effective oxygen evolution anode that is
compatible for practical CO2 electrolyzers. Here, we investigated potential anode candidates that
are highly active for oxygen evolution and structurally stable under near-neutral operation
conditions for a typical CO2 electrolyzer. The majority of non-precious metal-based catalysts are
unstable in neutral bicarbonate electrolytes during CO2 electrolysis. In contrast, iridium-coated
porous titanium electrodes exhibited decent oxygen evolution reaction activities as well as
excellent stability under near-neutral conditions. Furthermore, by spray coating iridium black
nanoparticles onto a cation exchange membrane (Nafion 211) and coupling it with a state-of-theart nanoporous Ag catalyst as the CO2 reduction cathode, a remarkable total current of ~375 mA
at an applied cell voltage of 2.6 V in the span of 24 hours was achieved using a 25 cm 2 CO2
electrolyzer flow cell.
[1] National Aeronautics and Space Administration, "Game changing development program,
advanced oxygen recovery for spacecraft life support systems appendix," NASA Headquarters,
Washington, DC, Tech. Rep. NNH14ZOA001N-14GCD-C2, 2014.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Symbiotic Growth of the Photoautotrophic Microalga Chlorella vulgaris
with Heterotrophic Microbes
Brian McConnell
Advisor: Maciek Antoniewicz
Committee Members: Wilfred Chen and Kelvin Lee
Climate change due to rising atmospheric greenhouse gas (GHG) concentrations is one of
the most threatening problems facing humanity. Methods for capturing GHGs and producing
biofuels have gained much attention. Chlorella vulgaris, a unicellular, photosynthetic, eukaryotic
microalga, can be used to capture carbon dioxide from point-source emitters and produce biodiesel
feedstock at high levels [1]. The presence of bacteria in large-scale open bioreactors has been
shown to significantly impact the growth of microalgae both positively and negatively [2]. The
understanding of these interactions at the molecular and biochemical levels is limited [3], but
nutrient exchange is thought to be the basis of the positive interactions [4].
My experimental work began with the determination C. vulgaris’s growth rate and biomass
composition during photoautotrophic growth (14 hours of light per day with atmospheric carbon
dioxide as the only carbon substrate). There was a lack of mass balance closure for the biomass
composition (~75%) which raised questions about my centrifugation method and the potential for
cell breakage during centrifugation. Protein and carbohydrate quantification of whole culture
samples (cells + medium), cell pellets, supernatants, and filter permeates showed little cell
breakage but a significant amount of biomolecules in the supernatant (explained the lack of mass
balance closure for cell pellets). Growing unlabeled C. vulgaris on a 13C labeled spent medium
showed that it does not significantly uptake its released biomolecules. Repeating this experiment
with unlabeled E. coli showed that E. coli does uptake the microalgal biomolecules. Taking a step
forward, co-cultures with (a) C. vulgaris and E. coli and (b) C. vulgaris and S. cerevisiae were
performed. The co-cultures showed decreased levels of biomolecules in the medium compared to
an algal monoculture. Currently, soil microbes are being grown on spent algal medium to select
for a co-culture partner which significantly boosts C. vulgaris’s growth and lipid production. Once
a highly beneficial partner has been identified, 13C tracer experiments will be performed to explain
why C. vulgaris naturally fixes inorganic carbon for another microorganisms’ consumption. This
understanding should help lead to increased lipid productivity during large scale microalgal
cultivation and a greater fundamental understanding of microalgal metabolism.
[1] Liu, J., Chen, F., 2014. Biology and Industrial Applications of Chlorella: Advances and
Prospects. Adv. Biochem. Eng. Biotechnol. 153, 1-35.
[2] Wang, H., Hill, R., Zheng, T., Hu, X., Wang, B., 2016. Effects of bacterial communities on
biofuel-producing microalgae: stimulation, inhibition and harvesting. Crit. Rev. Biotechnol.
32, 341-352.
[3] Natrah, F., Bossier, P., Sorgeloos, P., Yusoff, F., Defoirdt, T., 2014. Significance of
microalgal-bacterial interactions for aquaculture. Rev. Aquaculture. 6, 48-61.
[4] Cooper, M., Smith, A., 2015. Exploring the mutualistic interactions between microalgae and
bacteria in the omics age. Curr. Opin. Plant. Biol. 26, 147-153.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Effect of pH on the activity of hydrogen oxidation reaction/hydrogen evolution reaction
over PtRu bimetallic catalysts
Jared Nash
Advisor: Bingjun Xu, Yushan Yan
Committee Members: Feng Jiao, Raul Lobo
Hydrogen fueled proton exchange membrane fuel cell (PEMFC) powered automobiles have
become commercially available, but are still too expensive for widespread adoption due to their
use of Pt catalysts. Hydroxide exchange membrane fuel cells (HEMFCs) offer the possibility of
cheaper non-precious metal catalysts. However, the hydrogen oxidation reaction (HOR) is
significantly slower in base than in acid for Pt, Pd, Ir, and Rh[1-4]. PtRu shows both higher HOR
and hydrogen evolution reaction (HER) activity than Pt[5, 6], so understanding the mechanism
and origin of the enhanced activity of the HOR/HER on PtRu is key to developing non-precious
metal catalysts for HEMFCs. Using rotating disk electrode, bimetallic PtRu showed a minimum
in activity-pH relationship at intermediate pHs, which is not seen on monometallic catalysts. The
hydrogen binding energy and the hydroxide binding energy measured from the hydrogen
underpotential desorption peak and the CO stripping peak, respectively, did not change
significantly with pH. This indicates that neither the hydrogen binding energy, nor the hydroxide
binding energy are sufficient to describe the trend in activity with pH. The additional variable is
the bimetallic nature of the catalyst, which can be evaluated by changing the catalyst
composition. By cycling to high potential, Ru is leached from the bimetallic catalyst. These
leached catalysts show that the activity remains approximately constant while the Ru content is
reduced. Only after about 20% of Ru has been removed does the activity change. Using Cu
underpotential deposition and EDX from SEM respectively, both the surface and bulk
composition of the catalyst were determined. The charge transfer coefficient, α, decreases with
the Ru content going from 0.8 to 0.5. Fitting the kinetic data with the dual pathway model[7]
shows that below pH 11, the Heyrovsky and Volmer reaction barriers are comparable and above
pH 11, the Volmer step becomes rate determining. This shift in rate-determining step is a
plausible explanation for the change in trend in activity as a function of pH. It is proposed that
Ru can align more oxygen species (either water or hydroxide) on the surface giving a more
favorable orientation for the oxygen-hydrogen interaction that is necessary for the Volmer step to
proceed. This suggests that alloying other HOR catalysts with more oxophilic catalysts might
improve the activity.
References:
[1]
W. Sheng, Z. Zhuang, M. Gao, J. Zheng, J.G. Chen, Y. Yan, Nature communications 6 (2015).
[2]
J. Zheng, W. Sheng, Z. Zhuang, B. Xu, Y. Yan, Science Advances 2 (2016).
[3]
J. Durst, C. Simon, F. Hasché, H.A. Gasteiger, J Electrochem Soc 162 (2015) F190-F203.
[4]
W. Sheng, H.A. Gasteiger, Y. Shao-Horn, J Electrochem Soc 157 (2010) B1529.
[5]
Y. Wang, G. Wang, G. Li, B. Huang, J. Pan, Q. Liu, J. Han, L. Xiao, J. Lu, L. Zhuang, Energy
Environ. Sci. 8 (2014) 177-181.
[6]
J.X. Wang, Y. Zhang, C.B. Capuano, K.E. Ayers, Scientific Reports 5 (2015) 12220.
[7]
K. Elbert, J. Hu, Z. Ma, Y. Zhang, G. Chen, W. An, P. Liu, H.S. Isaacs, R.R. Adzic, J.X. Wang,
ACS Catal (2015) 6764-6772.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Effects of Surfactant Degradation on Solution Physiochemical Properties Relevant for
Protein Formulations used in Cancer Treatment
Jannat Nayem
Advisors: Dr. Norman Wagner, Dr. Yun Liu
Committee Members: Dr. Abraham Lenhoff, Dr. Christopher Roberts
To stabilize monoclonal antibodies (mAbs) against surface-induced denaturation and
aggregation, pharmaceutical industries add surfactants like polysorbate (PS) 20 in the drug
product formulations1. However, recent findings in bio-pharmaceutical research confirm that PS
20 can degrade via ester hydrolysis and auto-oxidation at pharmaceutically relevant conditions1,2.
While hydrolysis preferentially cleaves off the head groups of monoesters, oxidation leads to the
cleavage of the head or tail groups off of the higher order esters1,2. Prior industrial research at
Genentech suggests that these two degradation mechanisms lead to different formulation
stabilities. Consequently, we hypothesize that the two mechanism lead to differences in
nanoscale solution microstructure and develop an experimental program to test it. A
combination of techniques including small angle neutron scattering (SANS), small angle x-ray
scattering (SAXS), dynamic light scattering (DLS), and cryogenic transmission electron
microscopy (cryo-TEM) provides a comprehensive and quantitative characterization of the
solution microstructure at appropriate spatial and temporal scales. The results for the multi-ester
and pure laurate ester PS 20 show that the chemical diversity of PS 20 affects the hydrolytic
stability significantly. Additionally, SANS data confirm that both hydrolysis and oxidation lead
to different nanoscale solution structure, where the hydrolysis data indicate formation of micelles
of different morphologies in solutions prepared at different times and the oxidation data
consistently demonstrate difference in micellar size. Both SANS and DLS data of hydrolyzed
and oxidized mixed ratio samples show that the higher order esters can affect the micellar
conformation more than the monoesters implying that degradation via hydrolysis may impact the
micellar conformation more than oxidation. This work confirms that these two distinct
degradation mechanisms result in different solution microstructures. The next step is to
determine how these microstructures affect the stability of mAb drug formulations. The
industrial benefits of this project include improvements in the stability of mAb formulations at
therapeutic conditions relevant for oncology and related biopharmaceutical applications.
References:
1. Hewitt, Daniel, Taylor Zhang, and Yung-Hsiang Kao. "Quantitation of polysorbate 20 in
protein solutions using mixed-mode chromatography and evaporative light scattering
detection." Journal of Chromatography A 1215.1 (2008): 156-160
2. Kishore, Ravuri SK, et al. "The degradation of polysorbates 20 and 80 and its potential
impact on the stability of biotherapeutics." Pharmaceutical research 28.5 (2011): 1194-1210.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Engineering a native E. coli formaldehyde-inducible promoter for the development of
synthetic methylotrophy
Julia Rohlhill
Advisor: Eleftherios T. Papoutsakis
Committee Members: Maciek Antoniewicz and Wilfred Chen
Industrial microbial fermentation is frequently used to produce a wide array of desirable
products, including amino acids, vitamins, recombinant proteins, pharmaceuticals, and
alternative renewable fuels such as ethanol or butanol. Production of these compounds is most
commonly accomplished utilizing glucose or other sugar substrates. Methanol is emerging as an
attractive non-food feedstock option due to its high degree of reduction, low contamination risk,
an increasing and sustainable supply, and a corresponding declining price. Initial attempts to
generate a strain of the model organism Escherichia coli capable of efficiently utilizing methanol
as a substrate have been met with various bottlenecks.
The first step of methanol utilization involves its conversion to formaldehyde, a toxic
intermediate. We use a native E. coli formaldehyde-inducible promoter to drive expression of
key methanol assimilation genes, imitating native methylotrophic regulation. This approach
avoids the need to add inducers and high constitutive expression levels, and instead allows for
optimal expression levels driven directly in response to cell needs. Promoter-driven gene
expression was evaluated using a GFP reporter plasmid and fluorescence-activated cell sorting
(FACS). Promoter performance for the purpose of synthetic methylotrophy was assessed by
monitoring methanol consumption in E. coli strains with methylotrophic genes under the control
of the promoter of interest.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Design of RNA-responsive Polymeric Nanocapsules for Personalized Therapeutic Delivery
Rashida E.N. Ruddock
Advisors: Millicent O. Sullivan and Thomas H. Epps, III
Committee Members: Wilfred Chen and Christopher J. Kloxin
Therapeutic delivery systems designed on the basis of patient-specific biomarkers, such
as nucleic acids, are widely thought of as a promising approach to address the complex genetics
that underlie human disease, thereby improving the low efficacy rates associated with the current
usage of generalized biomarkers. To this end, our long term goal is to develop DNA-block
copolymer micelles that utilize DNA strand displacement reactions to enable RNA-responsive
micelle disassembly and then the subsequent release of encapsulated therapeutic cargoes.
Herein, we describe the development of a DNA hybrid capable of strand displacement triggered
by Akt1 mRNA, which in its overexpressed state, is correlated to the invasiveness of
inflammatory breast cancer (IBC). The DNA hybrid was designed to utilize Förster Resonance
Energy Transfer (FRET) as an indicator of strand displacement, through the inclusion of a
fluorophore-quencher pair on the proximal end of each DNA strand within the DNA hybrid. We
demonstrate the highly sequence-specific and sensitive nature of the designed DNA hybrid. In
particular, we show that the DNA hybrid is able to identify sequence mismatches in a synthetic
analog of the Akt1 mRNA sequence at the single-nucleotide level. The FRET signal induced by
the single-nucleotide mismatched sequence was 50% less intense than that of the fully
complementary sequence due to a hindrance to strand displacement by the mismatch.
Additionally, the DNA hybrid is capable of detecting the fully complementary sequence at
concentrations as low as 250 nM. Rapid DNA strand displacement times, on the order of
seconds, were measured in the presence of the fully complementary sequence in the cell-free
environment. Ongoing work is focused on studying the specificity and sensitivity of the DNA
hybrid in the intracellular environment. The development and characterization of these DNAblock copolymer micelles will provide a modular framework for incorporating nucleic acid
responsive release mechanisms into therapeutic delivery systems.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Development of a dynamic, 3D blood-brain barrier model to study drug transport
John Ruano-Salguero
Advisor: Kelvin H. Lee
Committee Members: Wilfred Chen, April Kloxin
The blood-brain barrier (BBB) is the generally impermeable network of cerebral
capillaries that tightly regulates molecular trafficking between the blood and the brain. Models of
the BBB have been developed to study the transport of molecules, such as medicines treating
neurodegenerative diseases, but currently no model recapitulates the microvascular
characteristics of the BBB known to alter transport. To improve on current BBB models, we aim
to develop a stem cell-based human BBB model that incorporates the dynamic and
microarchitectural nature of native BBBs. This talk presents the preliminary progress to develop
and assay a physiologically relevant dynamic, 3D BBB model.
Using live-cell fluorescent microscopy on a novel 2D BBB model, techniques to
visualize and quantify molecular transport were established. Calculated permeability values
show good agreement with values obtained via conventional in vitro permeability assays as well
as reported in vivo values. To enable 3D cell culture, perfusable microchannels within
biomimetic hydrogels were fabricated. 3D BBBs demonstrated similar protein localization
reported for in vitro and in vivo BBBs.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Enhanced affinity precipitation of antibodies using functionalized protein nanoscaffolds
Andrew Swartz
Advisor: Wilfred Chen
Committee Members: Abraham Lenhoff and Chris Roberts
Recent advancements in upstream therapeutic monoclonal antibody production have posed
significant challenges for platform downstream purification. Protein A chromatography has been
identified as a potential bottleneck due to limitations in throughput, scale-up, and cost. This has
generated increased interest in non-chromatographic capture technologies. Affinity precipitation
is a promising alternative because it combines high specificity with multiple operational benefits.
An environmentally responsive, recombinant elastin-like polypeptide (ELP) genetically fused to
an antibody affinity domain (Z-domain) has been shown to make an ideal candidate for antibody
purification. However, elevated temperature and salt concentrations necessary to induce
aggregation can diminish product quality and operational efficiency. To minimize the requirement
of these harsh conditions, we propose to enlarge the scale of ELP aggregation via conjugation to a
novel protein nanoscaffold. The Z domain-ELP fusion (Z-ELP) was conjugated to a modified, 25
nm E2 core from the pyruvate dehydrogenase enzyme complex using post-translational Sortase A
mediated ligation (Z-ELP-E2). Z-ELP-E2 cages demonstrated superior aggregation properties
compared to Z-ELP, requiring significantly less applied heat to precipitate the complex out of
solution. The functionalized nanoscaffold was shown to effectively bind, precipitate, and elute
human IgG antibodies (HIgG) with high recovery (>95%). This process was evaluated using a
mock industrial HIgG culture supernatant and high yield and purity was obtained. Affinity
precipitation using the Z-ELP-E2 nanoscaffold exhibited a significant improvement over the
existing ELP-based system and can be implemented as an easily scalable, cost-effective alternative
to platform Protein A chromatography.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Highly Stable Anion-Exchange Membranes for High-Voltage Redox-Flow Batteries
Hao Wang
Advisor: Yushan Yan
Committee Members: Thomas H. Epps, Arthi Jayaraman
As a reversible fuel cell, redox-flow batteries (RFBs) are one of the most promising
electrochemical technologies with the great scalability and durability (e.g., 20 years) required for
intermittent renewable energy storage. In particular, cerium redox pair [Ce(IV)/Ce(III)]-based
RFBs are appealing because of their unprecedented high cell voltages (1.87 V−3.08 V). High cell
voltage is a key factor in achieving high energy and power densities, which lead to low storage
cost. An anion-exchange membrane (AEM) is needed as a key component in cerium RFBs to
achieve stable cell voltage and high coulombic efficiency. However, existing ammonium cationbased AEMs have very limited stability when working with Ce(IV) electrolytes (e.g., less than
200 h of durability). The lack of stable AEMs causes cerium RFBs to suffer from either high
self-discharge rate or low coulombic efficiency. The development of highly stable and
conductive AEMs has become one of the most urgent challenges for cerium RFBs to become a
viable electricity storage solution.
An oxidative resistant phosphonium cation [i.e., tris(2,4,6-trimethylphenyl)
phosphonium, or 9MeTTP+], with the oxidative stability a factor of 1500+ better than the
conventional trimethyl ammonium cation and a factor of 25+ better than our previous-generation
phosphonium cation [i.e., tris(2,4,6-trimethoxyphenyl) phosphonium, or 9MeOTTP+]. The
excellent oxidation resistance of 9MeTTP+ cation is attributed to the protection from
substantially improved steric hindrance. In order to develop highly stable AEMs functionalized
with the 9MeTTP+ cation, a two-step synthesis route was first proposed: (1) acylation reaction
between benzene ring of commercial polymer backbone and the carbonyl chloride group of
linkage molecule (iodoalkylene-carbonyl chlorides); and (2) quaternization reaction between
iodoalkylene group and tris(2,4,6-trimethylphenyl) phosphine. However, the direct
quanterniaztion through center phosphorus atom is proven to be infeasible due to the high steric
hindrance effect from the three trimethylphenyl rings. A second method of synthesizing the
9MeTTP+ functionalized polymer is to first synthesize brominated 9MeTTP+ [i.e., Br9MeTTP+],
and then connecting the Br9MeTTP+ to a polymer backbone. 9MeTTP+ functionalized
polysulfone [i.e., 9MeTTPPSf] was first synthesized through this bromination method. However,
the polysulfone backbone showed poor oxidative stability after the hydrophilicity change. Thus,
a more oxidative resistant polymer backbone need to be considered. hexafluoro
polybenzimidazole [i.e., 6FPBI] membrane is hydrophilic and present oxidation stability in
Ce(IV) test. 9MeTTP+ cation functionalized hexafluoro polybenzimidazole [i.e., 9MeTTPPBI] is
under investigation.
-----------------------------------------------------------------------------------------------------Summer Research Review 2016
Development of dynamic hydrogels to understand cell response to matrix remodeling
Katherine L. Wiley
Advisor: Dr. April M. Kloxin
Committee Members: Dr. Wilfred Chen and Dr. Millicent O. Sullivan
Two-dimensional (2D) in vitro disease models are often utilized to support in vivo studies due to
their low cost and consistency1. However, 2D in vitro models lack complexity that may be
critical to understanding certain aspects of disease development and behavior. In efforts to create
models more representative of the in vivo microenvironment, hydrogels have been used to
fabricate three-dimensional (3D) cell culture environments able to recreate physical aspects of
native tissue such as elasticity, which have been shown to more accurately mimic cell behavior
in vivo2. In particular synthetic hydrogels have demonstrated consistency and tunability of
physical and biochemical properties3. Synthetic hydrogels have to potential to create welldefined in vitro disease models that can assist in answering more complex questions about
disease progression. Specifically, we are interested in developing a synthetic hydrogel that can
mimic key aspects of matrix remodeling to understand its effects on the recurrence of breast
cancer. It is hypothesized that matrix remodeling caused by aging or injury could trigger the
recurrence of breast cancer at a metastatic site after a long period of dormancy4,5. During the
matrix remodeling process extracellular matrix proteins are degraded and deposited by cells to
create a microenvironment protein composition that changes over time.
In order to understand the impact of this remodeling process on cell behavior, our goal is to
design a synthetic extracellular matrix for the reversible presentation of biochemical cues to
mimic matrix remodeling and to study its effect on cell fate, specifically breast cancer cell
dormancy and activation. Towards this goal we have synthesized a molecule that becomes
reactive upon irradiation with ultra-violet (UV, 365 nm) light and have demonstrated its
functionalization to polyethylene glycol. We hypothesize that this will allow us to create a
hydrogel with control over gelation without exposure to harmful free radicals. Additionally, we
have demonstrated the use of sortase A, an enzyme utilized in biotechnology for the ligation of
proteins, for the removal of a fluorescent peptide in a model system. We hypothesize that by
incorporating appropriate peptide sequences for recognition this enzyme can be utilized within
our hydrogel system for the removal of peptide sequences to mimic the changing extracellular
matrix protein composition in time during cell culture.
(1)
(2)
(3)
(4)
(5)
Ma, Q. and Lu, A. Drug Metabolism and Deposition. 35, 1009-1016 (2007).
Tibbitt, M. W. & Anseth, K. S. Biotechnol. Bioeng. 103, 655–63 (2009).
Mosiewicz, K. A. et al. Nature Materials. 12, 1072-1078 (2013).
Barkan, D. et al. Cancer Res. 70, 5706–5716 (2010).
Demicheli, R. et al. Ann. Oncol. 19, 1821–1828 (2008).
Equal Opportunity Employer
The University of Delaware is an equal opportunity/affirmative action
employer. For the University’s complete non-discrimination statement, please
visit http://www.udel.edu/aboutus/legalnotices.html