The ability to recapitulate the dynamic presentation of signals in a stem cell’s microenvironment remains a major hurdle in tissue engineering. By controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) heterogeneous synthetic tissues could be produced that match the complexity of their native counterparts. Combining strategies in both light-programmable hydrogels and recombinant protein engineering, we control the cellular microenvironment. Previously, we have demonstrated the ability to photopattern gels with fluorescent proteins by introducing a biorthogonal synthetic handle site-specifically modified onto our proteins via the sortase enzyme, and a photocaged reactive group in the hydrogrels. The photocaged group uncages upon UV light exposure, allowing for a reaction to occur and a covalent bond to form between the synthetic handle and the hydrogel. While covalent interactions form strong bonds, they are generally irreversible using mild, cytocompatible chemistries. In addition, this technique uses UV light, which may be harmful to cells. By incorporating LOVTRAP into the photopatterning system, we introduce a greater amount of control by allowing for full reversibility. In the dark, LOV2 and Zdk1 dimerize to form a protein-protein bond with similar stability to an antibody-antigen interaction. Upon exposure to blue light, the dimer dissociates, allowing for fully reversible control over conjugation. The Zdk1 domain can be incorporated into a protein of interest using genetic engineering, while still maintaining its binding affinity for LOV2. LOV2 can then be conjugated throughout a hydrogel to make a light responsive protein binding material. By utilizing a light controlled dimerization event between proteins, the LOVTRAP system is fully reversible using only blue light. This technique could be used for tissue engineering applications that require mild treatment conditions and full dynamic control over the presentation of biochemical cues.

Recreating digital logic with cellular components has been a newly charted area of engineering research. This has applications in cellular diagnostics and expands our fundamental knowledge of cellular decision making processes. We tested a variety of digital logic systems of dCas9 repression networks in S. cerevisiae (yeast). dCas9 has be re-purposed to be a nucleus localized protein which function as a modular transcription factor, which when bound to specific guide RNA (gRNA) is directed to a distinct 20 base pair genomic sequence determined by the gRNA’s target sequence. Understanding the activity of the dCas9+gRNA complex, allowed us to use gRNA with a particular sequence to repress the expression of other gRNA. Using this insight, multiple repression cascades have been made with varying complexities to build unique logic pathways, also known as Boolean Expressions.  The expression of all gRNA and dCas9 proteins impact cell fitness because they divert carbon and energy for reproduction. The effects of metabolic load these exogenous components add to the cell is a factor that was investigated through cell growth. Cell growth was determined by fitting a logistic population model to a time-series of experimentally measured cell densities. We compared distinct strains of yeast with stable genomic modifications that explored the impact of increasing the network complexity and varying dCas9 expression levels.  We anticipate the results to concur with this statement and realize that a gRNA+dCas9 complex may further increase the metabolic burden of the cell from off target repression. Realizing how dCas9 repression networks lead to metabolic load will help guide their applied use in cellular applications ranging from developing programs for crop species to gene therapies.

Renewable energy harvesting technologies such as wind and solar cannot provide on-demand sources of power unless they are paired with energy storage technologies (e.g., batteries). Flow batteries are the best candidate for grid-scale load shifting, bridging and power management due to their safety, efficiency and scalability. Flow batteries operate by pumping liquid-based electrolytes into a two-part cell where charging/discharging occurs through ion exchange across a membrane. Unfortunately, the current membrane technology (Nafion©, a Chemours fluoro-polymer) is expensive to synthesize and cannot be optimized to improve performance. This presentation will highlight the development of a new, low-cost membrane based on ceramic materials. Previous work using the sol-gel method of curing sodium silicate in acid has shown a promising membrane structure (with pore sizes of 0.5-2nm) for selective proton transport. My work investigates variations in the properties and structures that arise from using different acid solutions for the curing agent in this process. I made samples using six different acids and then characterized the pore size, shape and network structure through fitting of small angle x-ray scattering (SAXS) profiles. I studied macroscopic membrane features such as defects using scanning electron microscopy (SEM). Key membrane performance attributes such as proton conductivity and vanadium ion permeability I tested with experimental set-ups involving a Potentiostat and a UV-vis measure of ion concentration over time, respectively. I found that acid chemistry did have an effect on pore size, shape and network structure, however, this structural information could not be directly correlated to key flow battery performance attributes. SEM investigations indicate that membranes contain inherent stress fractures leading to macroscopic defects. These defects likely dominate the observed performance attributes. Future investigations will focus on developing new methods to eliminate these defects and create uniform structures.

Less than 3% of therapeutics can cross the blood-brain barrier, and the lack of penetration of therapeutics into the brain is the most cited failure for neurological clinical trials. In order to assess the efficacy of developing therapeutics which have the potential to address these issues, we are working towards establishing a high-throughput organotypic ex vivo brain slice model. We are specifically interested in developing a model that allows us to study and characterize the brain in the presence of glutamate excitotoxicity. Glutamate excitotoxicity is cell death due to an excess of the excitatory neurotransmitter glutamate, and is a common disease hallmark in neurological injury. The slice model we developed consists of cultured newborn rat brain slices to simulate an in vivo brain environment. However, in slice form, we have the ability to systematically study key pathophysiological variables, such as exposure to various excitotoxins that induce glutamate excitotoxicity. We use quantitative assessments of cytotoxicity (cell death) through cellular staining and imaging, as well as assays for lactate dehydrogenase (LDH), an enzyme released from the cytoplasm during cell death. We have established percent cytotoxicity profiles for both healthy untreated and maximum death control brain slices, and developed a protocol for testing excitotoxins to simulate glutamate excitotoxicity. With this slice platform, we will be able to compare the efficacy of potential therapeutics based on the decreases in rates of cell death they produce in brain slices with induced glutamate excitotoxicity. This research represents a promising platform to assay behavior, mechanism, and efficacy of therapeutics in development for the treatment of neurological disorders associated with glutamate excitotoxicity.

Quantum dots (QDs), fluorescent semiconductor nanocrystals, can be used as a biomarker and diagnostic tool because of their unique optical and electrical properties, including high luminescence, resistance to photobleaching, and broad excitation and emission spectrums. But in order to use QDs as biomedical imaging devices, particularly in sensitive organs such as the developing brain, toxicity must be adequately characterized. Little is known about the toxic effect of QDs on the brain, especially on the neonatal brain. QDs consist of a metal core, typically cadmium selenide, and a shell to protect the core. Cadmium is known to be highly toxic to many of the body’s systems, including the central nervous system. However, expanding the shell and attaching functional biomolecules prevents cadmium release and changes the characteristics of the particles. In our study, several surface ligands were tested at variable concentrations and periods of exposure to measure the toxic potential of QDs on an ex vivo organotypic brain slice model. Three methods of characterization were used: a lactate dehydrogenase (LDH) assay, a propidium iodide stain, and a fluoro-jade C stain. The cellular stains were evaluated using confocal microscopy. Results showed that toxicity is a function of QD exposure time, surface chemistry, and concentration. Several QD conjugations displayed minimal or no toxic effect after 24 h of exposure. The results of this study will be useful in identifying a QD conjugate as nontoxic in a biological setting. QDs can then be tailored to site and cell-specific uptake in the brain, thereby improving the selectivity of current imaging techniques and providing a powerful diagnostic with regards to diseased cell fate.

Hydrogen fuel is a non-polluting, sustainable energy source that is a very attractive alternative to fossil fuels. The most efficient way to produce it is to split water with a semiconductor photocatalyst (a semiconductor photocatalyst is a catalyst that uses visible light, mostly sunlight, to work). A team in China has shown that Cadmium Sulfide nanocrystals effectively split water to produce hydrogen fuel, meaning that semiconductor photocatalysts in nanocrystal form are a very good means of producing hydrogen fuel. There are still several problems with the team’s results, however. For example, nanocrystals agglomerate, which means they stick together in unusable clumps, very easily. Additionally, many people are uncomfortable using cadmium sulfide because other cadmium compounds are carcinogenic. Instead of storing crystals in a way that allows them to stick together, we have suspended the crystals in the holes of a porous fiber. To put crystals in the fiber matrix, we soaked the fiber in a solution of a soluble semiconductor photocatalyst. Once the semiconductor saturated the fiber, the crystals began to precipitate.The crystals were now stored in a way that separated them from one another. The goal of this research is to find a photocatalyst that is both more effective and more positively viewed than cadmium sulfide. In this project, we have explained the advantages and disadvantages of several semiconductor photocatalylsts that we believe to be the most viable options to find the most effective compound that can use visible light to produce elemental hydrogen from water. The commercial production of hydrogen fuel using this method would be much more effective than current methods, and would be very inexpensive, because the fiber we use is nothing more than pulp from Alder tree -- which is available commercially at a very low price.

For a nanoparticle to become a pharmaceutical transport device, it must have shelf life stability and avoid aggregation. Spontaneous aggregate formation, or particle sedimentation, can render a therapeutic nanoparticle ineffective and potentially harmful as a pharmaceutical. Using a biologically safe material that provides steric stabilization (i.e Pluronic F127) may slow or eliminate aggregation, improving shelf life and potential for pharmaceutical use. The use of a surfactant coating instead of a chemically conjugated method will improve ease of nanoparticle formulation. To determine aggregation kinetics, various surfactant-coated nanoparticles (SCNP) have been compared in a number of medias, including standard measuring media (10mM NaCl), known aggregate media (30:70 methanol:water solution by volume), as well as in complex media such as artificial cerebral spinal fluid (aCSF) and high ion concentration media like phosphate buffered saline (PBS). These SCNP’s have been compared to stock carboxylated-polystyrene nanoparticles, as well as polyethylene glycol-coated polystyrene nanoparticles. Characterizing SCNP behavior can have significant implications for nanoparticle design for drug delivery.

Water-absorbent polymer networks known as hydrogels are attractive materials from which functional tissue substitutes could be developed using patient-derived stem cells. While hydrogels demonstrate biocompatibility conducive to harboring cells, they lack mechanical and biochemical aspects of the native extracellular matrix (ECM), the intricate microenvironment about which cells function. Conventional photopolymerization-based techniques provide an avenue for incorporating these stimuli within hydrogels, but at the expense of introducing propagating free radicals that are prone to non-specific reactions with biological systems. In light of these limitations, we have developed a strategy for hydrogel formation and modification absent of propagating free radicals, proceeding through oxime ligation moderated by a photocaged alkoxyamine. Upon mild UV light exposure, the photocage is cleaved, liberating the alkoxyamine and permitting localized condensation with an aldehyde to form an oxime-based linkage. After synthesizing multi-arm crosslinkers, functionalized with either benzaldehydes or photocaged alkoxyamines, I demonstrated successful formation of oxime-based hydrogels within minutes of light exposure in the presence of live cells. Through a series of rheological studies, I found polymerization rates and final mechanical properties of these gels could be systematically tuned by varying crosslinker concentrations, light intensity, aniline catalyst concentration, and pH. Harnessing the light-driven aspect of this chemistry, I controlled hydrogel geometry and final mechanical properties by dictating the location and extent of UV exposure, respectively. I then translated photomediated oxime ligation toward the biochemical modification of hydrogels, where full-length proteins containing photocaged alkoxyamines were immobilized in user-defined regions exposed to UV light. The programmability afforded by photomediated oxime ligation can recapitulate mechanical and biochemical stimuli found throughout the ECM, which are dynamic and heterogeneous in their presentation. Consequently, photopolymerized oxime-based hydrogels will enable an enhanced understanding of cell-matrix interactions by serving as improved 3D cell culture platforms, thereby leading to advancements in tissue engineering and regenerative medicine.

Neural injuries (either peripheral or central nervous systems) affect a large number of the human population world-wide and in general are considered irreparable due to the lack of regenerative capacity of the neural tissues. Various neural tissue engineering approaches have been employed to differentiate human neural stem cells (hNSCs) into neurons for the purpose of using them in regenerating the damaged neural tissues. Recently, electrical stimulation was found effective to enhance neuronal differentiation of hNSCs. Light-triggered generation of free electrons on the surface of semiconducting materials is an exciting option to achieve electrical stimulation on hNSCs. However, the band gap of most light-harvesting semiconductors falls in the UV range which is harmful to cells. The main hypothesis of this study is that hNSCs will preferentially differentiate into neurons when cultured on dopamine-modified titanium oxide (TiO2) substrates followed by irradiation by visible light (~ 420nm). We have discovered that TiO2 grown on polydopamine (PDA) coated substrates exhibits an absorption peak in the visible light range. The surface chemistry was confirmed by various surface characterization methods including UV-Vis, SEM, EDS, and XPS. Using this substrate, we investigated the effect of laminin on hNSC adhesion, surface free electron generation on TiO2 by visible light irradiation, and preferential differentiation of hNSCs into neurons. The results from this research open up a new possibility for implantable neural tissue engineering scaffolds that can be activated by light irradiation.

The blood-brain barrier is a very selective membrane that works to protect the brain from most pathogens; however, this means that it is a difficult therapeutic target for drug targeting. Cutting-edge nanotechnology research is being performed by Dr. Elizabeth Nance on targeted drug delivery mechanisms to combat brain tumors such as glioblastomas, neuromas etc. Nance’s group has found and supported good drug release results with poly-ethylene glycol coating (PEG) for which they hope to perform controlled and targeted release. Currently, there is a lack of molecular level understanding of the drug-polymer interactions that dictate controlled release. Better computational models are needed to understand release kinetics and facilitate rational design of biomaterials to control the release or retention of a particular drug or enzyme. In the present work, molecular dynamics (MD) simulations were used with GROMACS software to study glutamate diffusion in water to validate our method and set a baseline for more advanced systems. Diffusion constants calculated with the Einstein equation based on glutamate diffusion in MD were consistent with experimental values of ∼0.75 μm2/msec. We are currently extending this approach to probe glutamate diffusion in the presence of biocompatible polymers such as PLGA (poly(lactic-co-glycolic acid)) and PEG. Then, we compared and contrasted the diffusion constants to make sure they were on the same order of magnitude as glutamate. We also studied the system kinetics to discover the polymeric matrix that allows the system to get closest to a diffusion-limited rate. We will relay this to Dr. Nance to modify her experiments to lead to improved drug delivery systems that help fight off brain cancers.

Autism Spectrum Disorder (ASD) affects 1 in 68 children and has been steadily growing in incidence in recent years. As a developmental disability, ASD causes social, communication, and behavioral challenges. ASD is as mysterious as it is diverse—no two cases are the same. This not only complicates treatment methods but also makes physical characterization of the autistic brain a tedious challenge. As the number of ASD cases begins to increase, so does the urgency for answers at the developmental and biological level that may unravel this condition and lead to more effective and targeted treatment. Microglia and astrocytes are key non-neuronal cells that exhibit altered activity levels in the autistic brain. The changes in behavior and the role these cells play in ASD, and many other neurological diseases, make them potential therapeutic targets. Leveraging this fact, the focus of my research is to study the morphology and distribution of glial cells in wild type and knock-out (autistic-like) mGluR5 rats using confocal microscopy. Regions of interest for changes in cell behavior and morphology include the hippocampus, cortex, and thalamus. Rats with ASD have microglial cells with enlarged soma, retracted and thickened processes, and processes encircling neurons. Fluorescent antibody stains are used to visualize these cells and structures. By defining a physical distinction between healthy and autistic brains, a new wave of therapeutics that target and mitigate the over-activation and inflammation of microglia and astrocytes can be explored.

Quantum dot (QD) semiconductor nanocrystals offer significant advantages over conventional fluorescent markers due to broad excitation spectrum, narrow emission spectrum, and very high photo stability, which enables long term visualization. Because of this, there is great interest in using QDs as biomarkers, where cellular uptake of QDs play an important role. In this study, we aim to characterize QD uptake in neuronal and glial populations in the developing brain as a function of QD surface functionality. Previous research has shown that the behavior of QDs in biological systems can be dictated by surface functionality; however, this has not been systematically studied, particularly in the developing brain. Here, we used cadmium-selenide (CdSe) QDs with either cadmium-sulfide (CdS) or zinc-sulfide (ZnS) shell. These QDs are coated with a layer of surface ligands to protect the core-shell and minimize their hydrophobicity. We set out to examine five different surface coatings, including: mercaptoundecanoic acid (MUA), mercaptopropionic acid (MPA), polyethylene glycol-amine (PEG-NH2), polyethylene glycol-methoxy (mPEG) and carboxylic acid (COOH). We incubated organotypic neonatal rat brain slices in 0.1 and 0.01 µM of QDs over a 24 h period. Using immunohistochemistry and co-localization analysis, we observed QDs with PEG-NH2 functionality localize in neurons, whereas QDs with MUA or MPA surface functionality remain in the brain extracellular space. High resolution confocal imaging is used to visually assess the stained brain slices, and the degree of colocalization with cell stains can be quantified using ImageJ. Furthermore, we investigated the region-dependent of QD-PEG-NH2 localization in neurons. This provides insight into the mechanism of uptake for future studies, which include using QDs as biomarkers of inflammatory processes, mediated by glia, in the developing brain.

Development of antibody-based diagnostics, biomolecular sensors, and biomaterial with reactive surface requires the ability to control the adsorption of bioactive protein at a surface. All medical devices exposed in an in vivo biological environment are immediately coated with a layer of adsorbed protein that plays an important role in the body’s behaviors towards the devices and the effectiveness of the devices themselves. In this study, we use a multi-technique approach to characterize the effect of protein G B1 (6 kDa) orientation on multi-layer antibody binding. By conjugating a cysteine group at different positions along the protein’s surface, we can immobilize five variants of the protein (V21C, D35C, E42C, T11C, and WT) onto maleimide and bare gold surfaces. We have confirmed through X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry experiments that the proteins adsorb onto these surfaces. In addition, we introduced primary IgG and secondary F(ab’)2 antibodies to investigate if the protein’s orientation will affect the binding of the antibodies. The adsorbed mass are measured using quartz crystal microbalance with dissipation monitoring. We have demonstrated that the cysteine mutants orient better on maleimide surfaces and encourage more IgG binding. We will also study the effect of temperature on adsorption mass and extend the experiments to amine and carboxyl functionalized surfaces. Fully developed techniques to accurately characterize the orientation and quantify the amount of protein and secondary adsorption will significantly contribute to understanding the interactions of biomaterials with the biological environment.

Due to their luminescent properties, semiconductor nanocrystals are promising tools for optical imaging of biological samples. This experiment explores how the surface chemistry of CdSe/CdS core-shell semiconductor nanocrystals impacts cell targeting and cytotoxicity. Growing a CdS shell on top of a CdSe core and then altering the surface chemistry allows functional control of how these nanocrystals interact with the surrounding environment. Cytotoxicity was analyzed as a function of surface chemistry, in order to identify biocompatible surface chemistries. Surface chemistries were also altered to allow targeting of different cell types. CdSe core nanocrystals were produced using a hot injection method, and UV-vis spectroscopy was used to relate peak absorbance wavelength to nanocrystal core size. To increase the quantum yield, a uniform CdS shell was grown using a slow-growth process with cadmium and sulfur precursors. A ligand exchange was performed to transfer the nanocrystals from an organic solvent to an aqueous solvent. A fluorimeter was used to determine the nanocrystal quantum yields after synthesis of cores, shell growth, and surface functionalization with transfer to aqueous solvent. Quantum yields increased after the shell growth, but decreased in general after surface functionalization and transfer to aqueous solvent. Decreased cytotoxicity was observed for surface chemistries with longer ligands, such as functionalized polyethylene glycol (PEG) chains. Further research will aim to tune the properties of CdSe/CdS core-shell nanocrystals and target a variety of cell types.

Peptoids are a novel polymer with a molecular structure similar to proteins. They can be used for numerous applications such as drug delivery agents, scaffold material, and nucleation agents. Certain peptoids can also self-assemble into functional and tunable structures such as nanosheets and nanofibers. Recent experimental research uncovered that chains of N-ethylcarboxylate – N-ethylphenylchloryl (Nce-Npe) peptoids can aggregate to form clusters in a calcium chloride solution and form nanofibers in the presence of a mica surface. It was also found that calcium ions facilitate the aggregation of these peptoids. In order to characterize the solution-state behavior of these peptoids on the atomic scale, we used molecular dynamics simulations. Molecular dynamics simulations utilize Newton’s laws and fitted empirical equations to produce the trajectory of the peptoids over time. We setup several systems of varying size and ion concentration and ran these simulations using a three-site water model and an AMBER99sb-ildn forcefield with parameters optimized to describe peptoid systems more accurately. We ran these simulations on Hyak, a high-performance computing cluster at UW, using the GROMACS software package. Through analysis of the peptoid trajectory, we found that aromatic pi-stacking drives clustering and ion-carboxylate interactions dictate the structure and stability of the peptoid clusters. We also observed an increase in both the rate of aggregation and number of ion interactions as the concentration of calcium ions increased. These simulations are essential to understanding the self-assembly of peptoids and similar polymers in complex environments.