Treatment of neurological disease has made little progress due to the inability of many therapeutics to access the brain environment. However, delivery vehicles like nanoparticles can allow therapeutics to overcome brain-specific biological barriers including the blood-brain barrier (BBB), the dense extracellular space (ECS), and cellular targeting. The ability of nanoparticles to overcome these barriers is influenced by surface properties which can be modified through the formulation process. One understudied parameter is the choice of surfactant, molecules which stabilize nanoparticle formation and likely form an interface between the nanoparticle and brain environment. First, we investigated the potential toxicity of several commonly used surfactants on brain cells and slices. We added surfactant solutions to mouse microglial cells (BV2) or cultured brain slices and assessed cell viability two days later with colorimetric assays. Our results showed that while surfactants cholic acid (CHA) and polysorbate 80 (P80) caused toxicity at high doses, they were nontoxic at the low doses involved with nanoparticle formulation. Other surfactants, including Pluronic® F127 (F127) and poly(vinyl alcohol) (PVA), were nontoxic throughout the tested dose range. Interestingly, although the F127 compound is nontoxic on its own, nanoparticles formulated with F127 reduced cell viability. This result was not observed with any other nanoparticle-surfactant combination. Confocal microscopy indicated higher intracellular accumulation of the nanoparticles formulated with F127 compared to all other formulations, suggesting that toxicity is mediated by nanoparticle internalization and surfactant choice. Finally, we used a live cell imaging technique to capture videos of the nanoparticle internalization process. Building off these results, ongoing experiments will evaluate several nanoparticle-surfactant formulations on their ability to accumulate within brain tissue after in vivo administration. Findings from this work will guide nanoparticle design for future clinical translation.

Alzheimer’s Disease (AD) is a progressive, debilitating, neurodegenerative disorder where patients lose their ability to think and carry out tasks. This disease is characterized by aggregation of the β-amyloid (Aβ) peptide. Cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) are derivatives of marijuana which have been shown to possess neuroprotective properties. Experimental work in this field, is limited in its scope when probing mechanisms driving the phenomenon of Aβ peptide aggregation. Molecular dynamics (MD) simulations have been used to understand the intra-peptide interactions and potential impact of cannabinoids. In order to understand the effects of cosolvent structure on the mechanism of amyloid aggregation, we used classical molecular dynamics simulations of Aβ derived switch-peptides in the presence of model cannabinoids (i.e. CBD and THC). Aβ peptides transform from functional peptides into beta-sheets and therefore impact function within the brain. We tracked beta-sheet formation as a function of time to understand if cannabinoids sterically inhibit interactions between and within peptides. Preliminary results indicate that CBD and THC demonstrate a trapping effect on aggregated peptides. The impact of synthetic cannabinoids are much less understood, prompting additional interest in investigating the interactions among these molecules.

Brain extracellular matrix (ECM) structure mediates many aspects of neuronal function. When ECM structure becomes dysregulated in neurological disease, one resulting impact is impaired neuronal function. Therefore, probing changes in ECM structure could provide insights into disease mechanisms and expose potential therapeutic pathways. Previous work in our group determined that degrading neural ECM structures, including perineuronal nets (PNNs), leads to a significant increase in the diffusive ability of nanoparticles navigating the brain extracellular space. However, this diffusion-based analysis provides little insight into changes in PNN-specific morphology or structure; it only predicts whether or not they are present and the degree to which they may be altered from normal. With this project, we aim to quantify changes in PNN structure with high spatial resolution. PNNs are stained using a fluorescently labeled lectin (Wisteria floribunda agglutinin) and images are acquired via confocal microscopy. Using Python, a coding language, we developed an automated image processing workflow to characterize morphological and structural features associated with PNNs, including total number of branches, average branch length, average mesh size of the net, and the areal density of fluorescence. This approach was applied to brains that span a range of chronological ages, from 14 days old to adult. PNNs are known to increase in counts early on in life, so this age-based study served as a proof of concept for our methodology. This same approach can be applied to study the effect of various neurological diseases on PNN structure. Collectively, this work aims to enhance our understanding of neurological disease mechanisms and open new avenues of therapeutic intervention.

Extracellular Vesicles (EVs) are group of cell-derived structures including exosomes, microvesicles, and apoptotic bodies, which have been found to play a key role in intercellular communication, through the biological cargo that these EVs can carry. Their ability to deliver proteins and nucleic acids from donor cells to their target cells has led to growing interest in the potential of EVs being used as biomarkers for disease. But, a comprehensive understanding of EVs behavior is lacking, especially in neuroscience, which may hinder the development for further application of the EVs. Thus, we are interested in investigating the effect of brain-derived EVs (bEVs) on brain cells, especially microglia, the brain’s primary resident immune cells. To do this, we first extracted the bEVs from the rat brain through ultracentrifugation and purified them through size exclusion chromatography (SEC). We then applied the bEVs to cultured mouse BV-2 microglial cells and incubated for 24 hours before performing quantitative reverse transcription PCR (RT-qPCR) on the treated BV-2 cells to explore any bEV induced inflammation response. Preliminary results have shown that bEVs play a role in inducing both pro and anti-inflammatory responses in microglial cells, both to varying degrees in the cytokine markers expressed after incubation for 24 hrs. Furthermore, to better understand the interaction between bEVs and microglial cells, we labeled bEVs with fluorescent nano-sized semiconductor quantum dots (QDs). Through fluorescent confocal microscopy and time-lapse imaging, we were able to explore the time-dependent interaction of bEVs and BV-2 cells at high resolution. Our study can provide insights into bEV behavior, which can be used to better understand their potential use as biomarkers for specific brain disease models. 

The most effective semiconductors used as absorber layers for solar cells have concerns regarding earth abundance, toxicity, cost-volatility of the materials, or high capital expenditure (CapEx) for new manufacturing facilities. Solution processing is a low cost, low temperature development method leading to lower CapEx. The exploration of "new" photovoltaic materials seeks to develop an earth abundant, non-toxic semiconductor via solution processing with efficiencies comparable to market-leading materials like silicon or CdTe. Bismuth rudorffites (chemical formula A_aBi_bX_a+3b) are a category of new materials proven to be solution processable, to have high absorption, and to be capable of cell efficiencies over 5%. One of the limitations of bismuth rudorffites thus far is current flow; the electrons that are capable of providing electrical power are not being extracted from the absorber material effectively before they return to their stable low-energy state. A way in which this limitation can be explored is via photoconductivity, the difference between material conductivity under illumination versus in the dark. My project seeks to characterize the photoconductivity of silver bismuth iodide (Ag_aBi_bI_a+3b) as a function of the ratio a/b, identifying the composition(s) that best facilitate electron transport and the crystal phases to which they correspond. Results indicate that ion migration within the crystal lattice occurs when a/b is large, and that high a/b ratios introduce AgI impurities in the film that dramatically increase the photoconductivity, among other important phenomena. This presentation gives procedures, results, and analyses from this photoconductivity exploration, working toward a more advanced understanding of bismuth rudorffite material properties.

In trying to understand biology’s dynamic heterogeneity, scientists have sought to recapitulate the spatial complexity and temporal presentation in which proteins are naturally presented to cells. Currently, the most promising strategies in this regard exploit sequential ligation/cleavage reactions, each controlled in time and space using light so as to reversibly immobilize proteins within synthetic biomaterials. Though our lab has utilized these approaches to spatially control complex biological fates with micron-scale resolutions, previous methods suffer from complex syntheses, as well as requirements for specialized equipment and skillsets rarely available in bio-based laboratories. Improving upon these fundamental limitations, our group has developed a scalable system wherein proteins can be bound/released from hydrogels using light, without the need for such expertise/equipment, by being fully genetically encodable. In this approach, biology performs all the modifications necessary to photopattern protein binding to gels, as well as install the reactive species requisite for the protein’s photo-mediated release. We have accomplished this using a photoactivatable protein-peptide ligation reaction developed by our lab, wherein UV irradiation “activates” the protein to ligate specifically with the peptide tag. Additionally, we exploit co-translational chemoenzymatic modification strategies to install a functional handle for tethering the protein into polymeric hydrogels during protein expression. To the peptide tag, we append a photocleavable protein that cleaves when irradiated by visible light, fused to a protein of interest (POI) to be tethered to the hydrogel. Expressing these proteins in E. coli yields the first-ever fully genetically encodable system which can reversibly pattern proteins into hydrogels, by first shining UV light to tether POIs into biomaterials, then subsequently shining visible light to photocleave the protein and trigger POI release. Highlighting the system’s versatility, we demonstrate that the approach is compatible with fluorescent proteins and bioactive growth factors to direct 4D cell fate.

The human body does not fully metabolize a pharmaceutical dose. Consequently, these stable compounds are excreted through human waste, and many pass through wastewater treatment plants untreated, polluting aquatic ecosystems and drinking water supplies. Point-source electrochemical oxidation of fresh human urine can be a low cost, versatile option for eliminating these biologically active compounds before their discharge into the environment. To design an effective device to do this, it is important to understand the mechanisms of pharmaceutical degradation in a complex system containing solution-phase and interfacial chemistry as well as kinetic and mass transport limited degradation rates. We used the limiting current technique to characterize the mass transport conditions in a cylindrical electrochemical cell stirred by a magnetic stir bar. By conducting a steady state potential scan on electrolyte containing a kinetically facile redox couple, [Fe(CN)₆]^3-/[Fe(CN)₆]^4-, we measured the mass transport limiting current. The limiting current reflects the reaction rate of an electroactive compound with the electrode surface, and this rate is limited by the diffusion of the compound through a boundary layer with a thickness defined by the system’s convective conditions. We derived a Sherwood number correlation as a function of Reynolds number and Schmidt number by varying stir rate, stir bar dimensions and shape and measured the corresponding limiting current. Knowledge of the mass transport coefficient demonstrated that pharmaceutical degradation can be mass transport limited on boron-doped diamond (BDD) anodes in full synthetic urine matrixes. For the same stir conditions, observed rate constants on iridium(IV) oxide (IrO₂) anodes fell below the mass transport limiting rate indicating a kinetic limit for pharmaceutical degradation. The limiting current technique is an easy method to characterize and predict mass transport conditions for any geometry and helps differentiate between interfacial and solution-phase degradation pathways for environmental pollutants.

Water-swollen polymeric networks (i.e., hydrogels) provide a structural platform for the manipulation of chemical and mechanical signals that mimics the complex heterogeneous environment experienced by cells in vivo. Photoresponsive chemistries have been of particular interest to this end, as they allow for precise spatiotemporal control of physiochemical properties and, thus, cell behavior. Here, we present a novel protein-based network that will allow for the photo-mediated stiffening of genetically-encoded hydrogels. In this system, we exploit a biochemical technology recently pioneered by our lab in which two pairs of proteins undergo irreversible, covalent heterodimerization after photoactivation. Through the incorporation of an inert, unstructured polypeptide backbone, we have exploited the aforementioned reaction to induce gelation in response to light through the formation of four-arm protein crosslinks. Unlike previous synthetic polymer-based hydrogel systems, this system is entirely genetically encoded, which provides significant advantages in terms of cost, time, and production simplicity. As we intend to demonstrate through photorheometry, this reaction proceeds in a dose-dependent manner, providing step-wise control of both where and when gel stiffening occurs. Such 4D control of a gel’s mechanical properties can be used to influence cell migration, growth, and differentiation, and, thus, could have applications in tissue engineering. Furthermore, we anticipate our system could be utilized to model the stiffening of the extracellular matrix, which is commonly associated with pathologies such as cancer, fibrosis, and cardiovascular disease.

More than 442 million people worldwide have been diagnosed with diabetes, many of which regulate their glucose levels using the pump/catheter system. However, just 2-3 days after the catheter is inserted into the body, the tissue clogs due to the foreign body reaction (FBR), an immune reaction elicited by the body in response to any foreign material injected in the body. At this point, the patient must remove the catheter and insert a new device into fresh skin elsewhere, resulting in excess scar tissue. Our project focuses on preventing the FBR by reducing its triggering event--protein attachment--so that insulin catheters can last longer (2-3 weeks) and can reduce fibrotic accumulation in patients. To combat the frequency of delivery site changes, we have designed a nonfouling zwitterionic polymeric brush coating for the surface of the catheter to reduce protein attachment. For the coating, zwitterionic sulfobetaine methacrylate (SBMA) was surface-polymerized onto the catheter using atom transfer radical polymerization (ATRP). SBMA has been shown to resist protein adsorption down to less than 1ng/cm². The ATRP initiator was plasma-deposited to robustly adhere to the unique geometry of the catheter. In this work, we used a full factorial design of experiment (DOE) to determine significant experimental factors to the polymerization protocol to maximize the amount of SBMA on the surface. The coating was characterized using x-ray photoelectron spectroscopy (XPS) to confirm the presence of SBMA and the radiolabeled protein adsorption assay to measure the amount of protein adsorbed to the coating. We plan to use the results of the DOE screening to further optimize the nonfouling coating and ultimately plan to test this coating on insulin-delivering catheters in a diabetic mouse model to observe sustained lowered blood sugar levels and histologically review the extent of the FBR through collagen accrual.

Taking advantages of advances in controlled drug delivery, modern medicine now has the ability to access and treat disease within many parts of the human body not previously possible. Where current medicine lacks, however, is the ability to treat complex disorders that require specific control over the timing, order, and sequential dosing of active therapeutics. To access these systems, we have attempted to create a library of light-sensitive compounds that will release protein therapeutics orthogonally in the presence of different wavelengths of tissue-penetrating light. These compounds are based on ruthenium polypyridyl linker complexes and can be structurally tuned to respond to visible and NIR light irradiation, leading to exchange of a ligand with water and rapid cleavage. We have modified the complexes with a reactive azide handle for site-specific incorporation into hydrogel biomaterials that can be transplanted to or formulated within specific bodily locations. Varying the ligands in the complex gives rise to different photocleavable crosslinkers that cleave in response to up to 715 nm light, well into the therapeutic window for in vivo applications. In this poster, I will describe the synthesis and characterization of one model Ru-based linker (RuPhen), including its photolysis, stability, and applications of the complex in the development of dynamic biomaterials.

Conjugated polymers (CPs) are used in a wide variety of organic electronics such as photovoltaics, organic thin-film transistors (OFET), and flexible displays because of the increased flexibility of polymer-based electronics. However, CPs often have significant downsides such as being prone to environmental degradation, lacking mechanical robustness, and being overall very expensive to create and use. To combat these limitations, CPs can be blended with lower-cost commodity engineering plastics (CEP), such as polystyrene, to create a blended composite that forms nanoscale structures of CP in a CEP matrix. To characterize the blends, we use small-angle neutron scattering (SANS) and wide-angle x-ray scattering (WAXS), which are techniques that provide information in the form of a scattering pattern. After data reduction and background removal, SANS data can then be modeled to extract information about the structures that develop from 1 nm – 1000 nm. We have decided to focus on using the sphere, fractal, and parallelepiped models since those geometries often form from self-assembled CPs. The scattering pattern from WAXS can be used to determine the preferential growth direction of CP self-assembly, either along the pi-pi stacking or lamellar directions. Through the combined use of these techniques, we are able to characterize structural dependence on the choice of solvent including both moderate and good solvents for the CPs. We also tested different side-chain lengths on the CP which will affect solubility and the ability to self-assemble. From these experiments, we found that a moderate solvent (such as toluene) will encourage nanofiber formation growth at lower concentrations of CP. Control over nanofiber formation could potentially lead to more favorable electrical performance for these materials. Conductivity and rheology tests will be conducted which will allow us to determine how much of an effect solvent choice has on the mechanical and conductive properties of these blends.