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Description
Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering

Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering highly specific targets is the application of phage display utilizing single chain variable fragment antibodies (scFv). The aim of this research was to employ phage display to identify pathologies related to traumatic brain injury (TBI), particularly astrogliosis. A unique biopanning method against viable astrocyte cultures activated with TGF-β achieved this aim. Four scFv clones of interest showed varying relative affinities toward astrocytes. One of those four showed the ability to identify reactive astroctyes over basal astrocytes through max signal readings, while another showed a statistical significance in max signal reading toward basal astrocytes. Future studies will include further affinity characterization assays. This work contributes to the development of targeting therapeutics and diagnostics for TBI.
ContributorsMarsh, William (Author) / Stabenfeldt, Sarah (Thesis advisor) / Caplan, Michael (Committee member) / Sierks, Michael (Committee member) / Arizona State University (Publisher)
Created2013
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Description
Contrast agents in medical imaging can help visualize structural details, distributions of particular cell types, or local environment characteristics. Multi-modal imaging techniques have become increasingly popular for their improved sensitivity, resolution, and ability to correlate structural and functional information. This study addresses the development of dual-modality (magnetic resonance/fluorescence) and dual-functional

Contrast agents in medical imaging can help visualize structural details, distributions of particular cell types, or local environment characteristics. Multi-modal imaging techniques have become increasingly popular for their improved sensitivity, resolution, and ability to correlate structural and functional information. This study addresses the development of dual-modality (magnetic resonance/fluorescence) and dual-functional (thermometry/detection) nanoprobes for enhanced tissue imaging.
ContributorsHemzacek, Katherine Leigh (Author) / Kodibagkar, Vikram (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
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Description
Oxygen delivery is crucial for the development of healthy, functional tissue. Low tissue oxygenation, or hypoxia, is a characteristic that is common in many tumors. Hypoxia contributes to tumor malignancy and can reduce the success of chemotherapy and radiation treatment. There is a current need to noninvasively measure tumor oxygenation

Oxygen delivery is crucial for the development of healthy, functional tissue. Low tissue oxygenation, or hypoxia, is a characteristic that is common in many tumors. Hypoxia contributes to tumor malignancy and can reduce the success of chemotherapy and radiation treatment. There is a current need to noninvasively measure tumor oxygenation or pO2 in patients to determine a personalized treatment method. This project focuses on creating and characterizing nanoemulsions using a pO2 reporter molecule hexamethyldisiloxane (HMDSO) and its longer chain variants as well as assessing their cytotoxicity. We also explored creating multi-modal (MRI/Fluorescence) nanoemulsions.
ContributorsGrucky, Marian Louise (Author) / Kodibagkar, Vikram (Thesis director) / Rege, Kaushal (Committee member) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05
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Description
Traumatic brain injury (TBI) is a major concern in public health due to its prevalence and effect. Every year, about 1.7 million TBIs are reported [7]. According to the According to the Centers for Disease Control and Prevention (CDC), 5.5% of all emergency department visits, hospitalizations, and deaths from 2002

Traumatic brain injury (TBI) is a major concern in public health due to its prevalence and effect. Every year, about 1.7 million TBIs are reported [7]. According to the According to the Centers for Disease Control and Prevention (CDC), 5.5% of all emergency department visits, hospitalizations, and deaths from 2002 to 2006 are due to TBI [8]. The brain's natural defense, the Blood Brain Barrier (BBB), prevents the entry of most substances into the brain through the blood stream, including medicines administered to treat TBI [11]. TBI may cause the breakdown of the BBB, and may result in increased permeability, providing an opportunity for NPs to enter the brain [3,4]. Dr. Stabenfeldt's lab has previously established that intravenously injected nanoparticles (NP) will accumulate near the injury site after focal brain injury [4]. The current project focuses on confirmation of the accumulation or extravasation of NPs after brain injury using 2-photon microscopy. Specifically, the project used controlled cortical impact injury induced mice models that were intravenously injected with 40nm NPs post-injury. The MATLAB code seeks to analyze the brain images through registration, segmentation, and intensity measurement and evaluate if fluorescent NPs will accumulate in the extravascular tissue of injured mice models. The code was developed with 2D bicubic interpolation, subpixel image registration, drawn dimension segmentation and fixed dimension segmentation, and dynamic image analysis. A statistical difference was found between the extravascular tissue of injured and uninjured mouse models. This statistical difference proves that the NPs do extravasate through the permeable cranial blood vessels in injured cranial tissue.
ContributorsIrwin, Jacob Aleksandr (Author) / Stabenfeldt, Sarah (Thesis director) / Bharadwaj, Vimala (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Allogeneic islet transplantation has the potential to reverse Type 1 Diabetes in patients. However, limitations such as chronic immunosuppression, islet donor numbers, and islet survival post-transplantation prevent the widespread application of allogeneic islet transplantation as the treatment of choice. Macroencapsulation devices have been widely used in allogeneic islet transplantation due

Allogeneic islet transplantation has the potential to reverse Type 1 Diabetes in patients. However, limitations such as chronic immunosuppression, islet donor numbers, and islet survival post-transplantation prevent the widespread application of allogeneic islet transplantation as the treatment of choice. Macroencapsulation devices have been widely used in allogeneic islet transplantation due to their capability to shield transplanted cells from the immune system as well as provide a supportive environment for cell viability, but macroencapsulation devices face oxygen transport challenges as their geometry increases from preclinical to clinical scales. The goal of this work is to generate complex 3D hydrogel macroencapsulation devices with sufficient oxygen transport to support encapsulated cell survival and generate these devices in a way that is accessible in the clinic as well as scaled manufacturing. A 3D-printed injection mold has been developed to generate hydrogel-based cell encapsulation devices with spiral geometries. The spiral geometry of the macroencapsulation device facilitates greater oxygen transport throughout the whole device resulting in improved islet function in vivo in a syngeneic rat model. A computational model of the oxygen concentration within macroencapsulation devices, validated by in vitro analysis, predicts that cells and islets maintain a greater viability and function in the spiral macroencapsulation device. To further validate the computational model, pO2 Reporter Composite Hydrogels (PORCH) are engineered to enable spatiotemporal measurement of oxygen tension within macroencapsulation devices using the Proton Imaging of Siloxanes to map Tissue Oxygenation Levels (PISTOL) magnetic resonance imaging approach. Overall, a macroencapsulation device geometry designed via computational modeling of device oxygen gradients and validated with magnetic resonance (MR) oximetry imaging enhances islet function and survival for islet transplantation.
ContributorsEmerson, Amy (Author) / Weaver, Jessica (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Sadleir, Rosalind (Committee member) / Stabenfeldt, Sarah (Committee member) / Wang, Kuei-Chun (Committee member) / Arizona State University (Publisher)
Created2023
Description

Polymeric nanoparticles (NP) consisting of Poly Lactic-co-lactic acid - methyl polyethylene glycol (PLLA-mPEG) or Poly Lactic-co-Glycolic Acid (PLGA) are an emerging field of study for therapeutic and diagnostic applications. NPs have a variety of tunable physical characteristics like size, morphology, and surface topography. They can be loaded with therapeutic and/or

Polymeric nanoparticles (NP) consisting of Poly Lactic-co-lactic acid - methyl polyethylene glycol (PLLA-mPEG) or Poly Lactic-co-Glycolic Acid (PLGA) are an emerging field of study for therapeutic and diagnostic applications. NPs have a variety of tunable physical characteristics like size, morphology, and surface topography. They can be loaded with therapeutic and/or diagnostic agents, either on the surface or within the core. NP size is an important characteristic as it directly impacts clearance and where the particles can travel and bind in the body. To that end, the typical target size for NPs is 30-200 nm for the majority of applications. Fabricating NPs using the typical techniques such as drop emulsion, microfluidics, or traditional nanoprecipitation can be expensive and may not yield the appropriate particle size. Therefore, a need has emerged for low-cost fabrication methods that allow customization of NP physical characteristics with high reproducibility. In this study we manufactured a low-cost (<$210), open-source syringe pump that can be used in nanoprecipitation. A design of experiments was utilized to find the relationship between the independent variables: polymer concentration (mg/mL), agitation rate of aqueous solution (rpm), and injection rate of the polymer solution (mL/min) and the dependent variables: size (nm), zeta potential, and polydispersity index (PDI). The quarter factorial design consisted of 4 experiments, each of which was manufactured in batches of three. Each sample of each batch was measured three times via dynamic light scattering. The particles were made with PLLA-mPEG dissolved in a 50% dichloromethane and 50% acetone solution. The polymer solution was dispensed into the aqueous solution containing 0.3% polyvinyl alcohol (PVA). Data suggests that none of the factors had a statistically significant effect on NP size. However, all interactions and relationships showed that there was a negative correlation between the above defined input parameters and the NP size. The NP sizes ranged from 276.144 ± 14.710 nm at the largest to 185.611 ± 15.634 nm at the smallest. In conclusion, the low-cost syringe pump nanoprecipitation method can achieve small sizes like the ones reported with drop emulsion or microfluidics. While there are trends suggesting predictable tuning of physical characteristics, significant control over the customization has not yet been achieved.

ContributorsDalal, Dhrasti (Author) / Stabenfeldt, Sarah (Thesis director) / Wang, Kuei-Chun (Committee member) / Flores-Prieto, David (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
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Description
Cellular metabolism is an essential process required for tissue formation, energy production and systemic homeostasis and becomes dysregulated in many disease states. In the context of human cerebral cortex development, there’s a limited understanding of how metabolic pathways, such as glycolysis, impacts proliferation and differentiation of cortical cells. The technical

Cellular metabolism is an essential process required for tissue formation, energy production and systemic homeostasis and becomes dysregulated in many disease states. In the context of human cerebral cortex development, there’s a limited understanding of how metabolic pathways, such as glycolysis, impacts proliferation and differentiation of cortical cells. The technical challenges of studying primary in vivo cortical tissue at a cellular and molecular level led to the development of human pluripotent stem cell (PSC) derived cortical organoids. Cortical organoids are a highly tractable model system that can be used for high-throughput investigation of early stages of development and corresponding glycolytic programs. Through transplantation of cortical organoids into the developing mouse cortex, human cortical cells can also be studied in an in vivo environment that more closely resembles endogenous development where the impact of metabolism in typical developmental programs and disease states can be studied. While current data is preliminary, initial observations suggest that cortical populations increase glucose uptake over time and regulation of glucose uptake rates occur in cell type-specific manner. Additionally, mouse transplantation data suggests that glycolytic activity is downregulated post-transplantation, suggesting that the in vitro environment contributes metabolic state. The more dynamic range of metabolic states in vivo may impact the rate of differentiation and maturation in cellular populations in the transplant model. I hypothesize that the more endogenous-like regulation of glycolysis may impact the proliferative window and expansion of key progenitor cell types in the human brain, particularly the intermediate progenitor cells.
ContributorsMorales, Alexandria (Author) / Andrews, Madeline (Thesis advisor) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2023
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Description
APOE encodes for a lipid transport protein and has three allelic variants-APOE ε2, ε3 and ε4 each of which differentially modulate the risk for Alzheimer’s disease (AD). The presence of the ε4 allele of APOE greatly increases AD risk compared to the presence of the more prevalent and risk neutral

APOE encodes for a lipid transport protein and has three allelic variants-APOE ε2, ε3 and ε4 each of which differentially modulate the risk for Alzheimer’s disease (AD). The presence of the ε4 allele of APOE greatly increases AD risk compared to the presence of the more prevalent and risk neutral ε3 allele. An imbalance in the generation and clearance of amyloid beta (Aβ) peptides has been hypothesized to play a key role in driving the disease. APOE4 impacts several AD-relevant cellular processes. However, it is unclear whether these effects represent a gain of toxic function or a loss of function, specifically as it relates to modulating amyloid beta (Aβ) levels. Here, a set of APOE knockout (KO) and APOE4 isogenic human induced pluripotent stem cells (hiPSCs) were generated from a parental APOE3 hiPSC line with a highly penetrant familial AD (fAD) mutation to investigate this with respect to Aβ secretion in neural cultures and Aβ uptake in monocultures of microglia-like cells (iMGLs). Conversion of APOE3 to E4 as well as functionally knocking APOE out from the APOE3 parental line, result in elevated Aβ levels in neural cultures, likely through multiple mechanisms including the altered processing of the precursor protein to Aβ called amyloid precursor protein (APP). In pure neuronal cultures, a shift in the processing of APP was observed with the Aβ-generating amyloidogenic pathway being favored in both APOE3 as well as APOE4 neurons compared to APOE KO neurons, with APOE4 neurons exhibiting a greater shift. In iMGLs derived from the isogenic hiPSC lines, expression of APOE, regardless of the isoform, lowered the uptake of Aβ. Overall, APOE4 modulates Aβ levels through distinct loss of protective and gain of function effects. Dissecting these effects would contribute towards a better understanding of the design of potential APOE-targeted therapeutics in the future.
ContributorsRajaram Srinivasan, Gayathri (Author) / Brafman, David (Thesis advisor) / Plaisier, Christopher (Committee member) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2024
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Description
The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access

The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access of target DNA to Cas9. The Haynes lab has constructed fusions of enzymatic chromatin-modifying domains designed to remodel chromatin and increase Cas9 editing efficiency. With a library of PFs available, this research focuses on analyzing the behavior of Cas9 in chromatin that has been artificially opened by PFs. The types and frequency of INDELs (insertions & deletions) were determined after non-homologous end joining (NHEJ) in PF and Cas9-treated cells using quantitative Sanger sequencing and Synthego’s ICE software. Furthermore, NOME-seq analysis was carried out to map nucleosome position in PF and Cas9 treated cells. Although this experiment was unsuccessful, the heat map generated with data obtained from Synthego ICE predicts a possible presence of nucleosome in the vicinity suggesting that perhaps a fully open chromatin state was not achieved. Linear Regression analysis with certain assumptions confirms that with the increase in distance downstream of cut-site, the editing frequency decreases exponentially. Nevertheless, further experimental work should be carried out to investigate this hypothesis.
ContributorsHamna, Syeda Fatima (Author) / Haynes, Karmella A (Thesis advisor) / Stabenfeldt, Sarah (Thesis advisor) / Tian, Xiaojun (Committee member) / Arizona State University (Publisher)
Created2019
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Description
Achieving effective drug concentrations within the central nervous system (CNS) remains one of the greatest challenges for the treatment of brain tumors. The presence of the blood-brain barrier and blood-spinal cord barrier severely restricts the blood-to-CNS entry of nearly all systemically administered therapeutics, often leading to the development of peripheral

Achieving effective drug concentrations within the central nervous system (CNS) remains one of the greatest challenges for the treatment of brain tumors. The presence of the blood-brain barrier and blood-spinal cord barrier severely restricts the blood-to-CNS entry of nearly all systemically administered therapeutics, often leading to the development of peripheral toxicities before a treatment benefit is observed. To circumvent systemic barriers, intrathecal (IT) injection of therapeutics directly into the cerebrospinal fluid (CSF) surrounding the brain and spinal cord has been used as an alternative administration route; however, its widespread translation to the clinic has been hindered by poor drug pharmacokinetics (PK), including rapid clearance, inadequate distribution, as well as toxicity. One strategy to overcome the limitations of free drug PK and improve drug efficacy is to encapsulate drug within nanoparticles (NP), which solubilize hydrophobic molecules for sustained release in physiological environments. In this thesis, we will develop NP delivery strategies for brain tumor therapy in two model systems: glioblastoma (GBM), the most common and deadly malignant primary brain tumor, and medulloblastoma, the most common pediatric brain tumor. In the first research chapter, we developed 120 nm poly(lactic acid-co-glycolic acid) NPs encapsulating the chemotherapy, camptothecin, for intravenous delivery to GBM. NP encapsulation of camptothecin was shown to reduce the drug’s toxicity and enable effective delivery to orthotopic GBM. To build off the success of intravenous NP, the second research chapter explored the utility of 100 nm PEGylated NPs for use with IT administration. Using in vivo imaging and ex vivo tissue slices, we found the NPs were rapidly transported by the convective forces of the CSF along the entire neuraxis and were retained for over 3 weeks. Based on their wide spread delivery and prolonged circulation, we examine the ability of the NPs to localize with tumor lesions in a leptomeningeal metastasis (LM) model of medulloblastoma. NPs administered to LM bearing mice were shown to penetrate into LM mets seeded within the meninges around the brain. These data show the potential to translate our success with intravenous NPs for GBM to improve IT chemotherapy delivery to LM.
ContributorsHouseholder, Kyle Thomas (Author) / Sirianni, Rachael W. (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Vernon, Brent (Committee member) / Caplan, Michael (Committee member) / Wechsler-Reya, Robert (Committee member) / Arizona State University (Publisher)
Created2018