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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
The object of this study is to charac terize the effect of focused ultrasound stimulation (FUS) on the rat ce rvix which has been observed to speed its ripening during pregnancy. Ce rvical ripening is required for successful fetal delivery. Timed-pregnant Sprague-Dawley rats (n=36) were used. On day 14 of

The object of this study is to charac terize the effect of focused ultrasound stimulation (FUS) on the rat ce rvix which has been observed to speed its ripening during pregnancy. Ce rvical ripening is required for successful fetal delivery. Timed-pregnant Sprague-Dawley rats (n=36) were used. On day 14 of gestation, the FUS system was placed on the body surface of the rat over the cervix and ultrasound energy was applied to cervix for variable times up to 1 hour in the control group, the FUS system was placed on rats but no energy was applied. Daily measurement of cervix light-induced florescence (LIF, photon counts of collagen x-bridge fluorescence) were made on days 16 of gestation and daily until spont-aneous delivery (day22) to estimate changes in cervical ripening. We found that pulses of 680 KHz ultrasound at 25 Hertz, 1 millisecond pulse duration at 1W/cm^2 applied for as little as 30 minutes would immediately afterwards show the cervix to hav e ripened to the degree seen just before delivery on day 22. Delivery times, fetal weights and viability were unaffected in the FUS-treated animals.
ContributorsLuo, Daishen (Author) / Towe, Bruce C (Thesis advisor) / Wang, Xiao (Committee member) / Caplan, Michael (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge

Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens.
ContributorsPatel, Chetan (Author) / Muthuswamy, Jitendran (Thesis advisor) / Helms Tillery, Stephen (Committee member) / Jain, Tilak (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Flow measurement has always been one of the most critical processes in many industrial and clinical applications. The dynamic behavior of flow helps to define the state of a process. An industrial example would be that in an aircraft, where the rate of airflow passing the aircraft is used to

Flow measurement has always been one of the most critical processes in many industrial and clinical applications. The dynamic behavior of flow helps to define the state of a process. An industrial example would be that in an aircraft, where the rate of airflow passing the aircraft is used to determine the speed of the plane. A clinical example would be that the flow of a patient's breath which could help determine the state of the patient's lungs. This project is focused on the flow-meter that are used for airflow measurement in human lungs. In order to do these measurements, resistive-type flow-meters are commonly used in respiratory measurement systems. This method consists of passing the respiratory flow through a fluid resistive component, while measuring the resulting pressure drop, which is linearly related to volumetric flow rate. These types of flow-meters typically have a low frequency response but are adequate for most applications, including spirometry and respiration monitoring. In the case of lung parameter estimation methods, such as the Quick Obstruction Method, it becomes important to have a higher frequency response in the flow-meter so that the high frequency components in the flow are measurable. The following three types of flow-meters were: a. Capillary type b. Screen Pneumotach type c. Square Edge orifice type To measure the frequency response, a sinusoidal flow is generated with a small speaker and passed through the flow-meter that is connected to a large, rigid container. True flow is proportional to the derivative of the pressure inside the container. True flow is then compared with the measured flow, which is proportional to the pressure drop across the flow-meter. In order to do the characterization, two LabVIEW data acquisition programs have been developed, one for transducer calibration, and another one that records flow and pressure data for frequency response testing of the flow-meter. In addition, a model that explains the behavior exhibited by the flow-meter has been proposed and simulated. This model contains a fluid resistor and inductor in series. The final step in this project was to approximate the frequency response data to the developed model expressed as a transfer function.
ContributorsHu, Jianchen (Author) / Macia, Narciso (Thesis advisor) / Pollat, Scott (Committee member) / Rogers, Bradley (Committee member) / Arizona State University (Publisher)
Created2013
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Description
Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs

Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging.
ContributorsRamos, James (Author) / Rege, Kaushal (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Garcia, Antonio (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Aqueous solutions of temperature-responsive copolymers based on N-isopropylacrylamide (NIPAAm) hold promise for medical applications because they can be delivered as liquids and quickly form gels in the body without organic solvents or chemical reaction. However, their gelation is often followed by phase-separation and shrinking. Gel shrinking and water loss is

Aqueous solutions of temperature-responsive copolymers based on N-isopropylacrylamide (NIPAAm) hold promise for medical applications because they can be delivered as liquids and quickly form gels in the body without organic solvents or chemical reaction. However, their gelation is often followed by phase-separation and shrinking. Gel shrinking and water loss is a major limitation to using NIPAAm-based gels for nearly any biomedical application. In this work, a graft copolymer design was used to synthesize polymers which combine the convenient injectability of poly(NIPAAm) with gel water content controlled by hydrophilic side-chain grafts based on Jeffamine® M-1000 acrylamide (JAAm). The first segment of this work describes the synthesis and characterization of poly(NIPAAm-co-JAAm) copolymers which demonstrates controlled swelling that is nearly independent of LCST. The graft copolymer design was then used to produce a degradable antimicrobial-eluting gel for prevention of prosthetic joint infection. The resorbable graft copolymer gels were shown to have three unique characteristics which demonstrate their suitability for this application. First, antimicrobial release is sustained and complete within 1 week. Second, the gels behave like viscoelastic fluids, enabling complete surface coverage of an implant without disrupting fixation or movement. Finally, the gels degrade rapidly within 1-6 weeks, which may enable their use in interfaces where bone healing takes place. Graft copolymer hydrogels were also developed which undergo Michael addition in situ with poly(ethylene glycol) diacrylate to form elastic gels for endovascular embolization of saccular aneurysms. Inclusion of JAAm grafts led to weaker physical crosslinking and faster, more complete chemical crosslinking. JAAm grafts prolonged the delivery window of the system from 30 seconds to 220 seconds, provided improved gel swelling, and resulted in stronger, more elastic gels within 30 minutes after delivery.
ContributorsOverstreet, Derek (Author) / Caplan, Michael (Thesis advisor) / Massia, Stephen (Committee member) / Mclaren, Alexander (Committee member) / Vernon, Brent (Committee member) / McLemore, Ryan (Committee member) / Arizona State University (Publisher)
Created2012
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Description
The effects of specific histone deacetylase inhibitors (HDACi) on transgene expression in combination with a novel polymer as a delivery vehicle are investigated in this research. Polymer vectors, although safer than viruses, are notorious for low levels of gene expression. In this investigation, the use of an emerging chemotherapeutic anti-cancer

The effects of specific histone deacetylase inhibitors (HDACi) on transgene expression in combination with a novel polymer as a delivery vehicle are investigated in this research. Polymer vectors, although safer than viruses, are notorious for low levels of gene expression. In this investigation, the use of an emerging chemotherapeutic anti-cancer drug molecule, HDACi, was used to enhance the polymer-mediated gene expression. HDACi are capable of inhibiting deacetylation activities of histones and other non-histone proteins in the cytoplasm and nucleus, as well as increase transcriptional activities necessary for gene expression. In a prior study, a parallel synthesis and screening of polymers yielded a lead cationic polymer with high DNA-binding properties, and even more attractive, high transgene expressions. Previous studies showed the use of this polymer in conjunction with cytoplasmic HDACi significantly enhanced gene expression in PC3-PSMA prostate cancer cells. This led to the basis for the investigation presented in this thesis, but to use nuclear HDACi to potentially achieve similar results. The HDACi, HDACi_A, was a previously discovered lead drug that had potential to significantly enhance luciferase expression in PC3-PSMA cells. The results of this study found that the 20:1 polymer:plasmid DNA weight ratio was effective with 1 uM and 2 uM HDACI_A concentrations, showing up to a 9-fold enhancement. This enhancement suggested that HDACi_A was effectively aiding transfection. While not an astounding enhancement, it is still interesting enough to investigate further. Cell viabilities need to be determined to supplement the results.
ContributorsLehrman, Jennifer (Author) / Rege, Kaushal (Thesis advisor) / Caplan, Michael (Committee member) / Pizziconi, Vincent (Committee member) / Arizona State University (Publisher)
Created2012
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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
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Description
Annually, approximately 1.7 million people suffer a traumatic brain injury (TBI) in the United States. After initial insult, a TBI persists as a series of molecular and cellular events that lead to cognitive and motor deficits which have no treatment. In addition, the injured brain activates the regenerative niches of

Annually, approximately 1.7 million people suffer a traumatic brain injury (TBI) in the United States. After initial insult, a TBI persists as a series of molecular and cellular events that lead to cognitive and motor deficits which have no treatment. In addition, the injured brain activates the regenerative niches of the adult brain presumably to reduce damage. The subventricular zone (SVZ) niche contains neural progenitor cells (NPCs) that generate astrocytes, oligodendrocyte, and neuroblasts. Following TBI, the injury microenvironment secretes signaling molecules like stromal cell derived factor-1a (SDF-1a). SDF-1a gradients from the injury contribute to the redirection of neuroblasts from the SVZ towards the lesion which may differentiate into neurons and integrate into existing circuitry. This repair mechanism is transient and does not lead to complete recovery of damaged tissue. Further, the mechanism by which SDF-1a gradients reach SVZ cells is not fully understood. To prolong NPC recruitment to the injured brain, exogenous SDF-1a delivery strategies have been employed. Increases in cell recruitment following stroke, spinal cord injury, and TBI have been demonstrated following SDF-1a delivery. Exogenous delivery of SDF-1a is limited by its 28-minute half-life and clearance from the injury microenvironment. Biomaterials-based delivery improves stability of molecules like SDF-1a and offer control of its release. This dissertation investigates SDF-1a delivery strategies for neural regeneration in three ways: 1) elucidating the mechanisms of spatiotemporal SDF-1a signaling across the brain, 2) developing a tunable biomaterials system for SDF-1a delivery to the brain, 3) investigating SDF-1a delivery on SVZ-derived cell migration following TBI. Using in vitro, in vivo, and in silico analyses, autocrine/paracrine signaling was necessary to produce SDF-1a gradients in the brain. Native cell types engaged in autocrine/paracrine signaling. A microfluidics device generated injectable hyaluronic-based microgels that released SDF-1a peptide via enzymatic cleavage. Microgels (±SDF-1a peptide) were injected 7 days post-TBI in a mouse model and evaluated for NPC migration 7 days later using immunohistochemistry. Initial staining suggested complex presence of astrocytes, NPCs, and neuroblasts throughout the frontoparietal cortex. Advancement of chemokine delivery was demonstrated by uncovering endogenous chemokine propagation in the brain, generating new approaches to maximize chemokine-based neural regeneration.
ContributorsHickey, Kassondra (Author) / Stabenfeldt, Sarah E (Thesis advisor) / Holloway, Julianne (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Newbern, Jason (Committee member) / Arizona State University (Publisher)
Created2021
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Description
Aortic pathologies such as coarctation, dissection, and aneurysm represent a

particularly emergent class of cardiovascular diseases and account for significant cardiovascular morbidity and mortality worldwide. Computational simulations of aortic flows are growing increasingly important as tools for gaining understanding of these pathologies and for planning their surgical repair. In vitro experiments

Aortic pathologies such as coarctation, dissection, and aneurysm represent a

particularly emergent class of cardiovascular diseases and account for significant cardiovascular morbidity and mortality worldwide. Computational simulations of aortic flows are growing increasingly important as tools for gaining understanding of these pathologies and for planning their surgical repair. In vitro experiments are required to validate these simulations against real world data, and a pulsatile flow pump system can provide physiologic flow conditions characteristic of the aorta.

This dissertation presents improved experimental techniques for in vitro aortic blood flow and the increasingly larger parts of the human cardiovascular system. Specifically, this work develops new flow management and measurement techniques for cardiovascular flow experiments with the aim to improve clinical evaluation and treatment planning of aortic diseases.

The hypothesis of this research is that transient flow driven by a step change in volume flux in a piston-based pulsatile flow pump system behaves differently from transient flow driven by a step change in pressure gradient, the development time being substantially reduced in the former. Due to this difference in behavior, the response to a piston-driven pump can be predicted in order to establish inlet velocity and flow waveforms at a downstream phantom model.

The main objectives of this dissertation were: 1) to design, construct, and validate a piston-based flow pump system for aortic flow experiments, 2) to characterize temporal and spatial development of start-up flows driven by a piston pump that produces a step change from zero flow to a constant volume flux in realistic (finite) tube geometries for physiologic Reynolds numbers, and 3) to develop a method to predict downstream velocity and flow waveforms at the inlet of an aortic phantom model and determine the input waveform needed to achieve the intended waveform at the test section. Application of these newly improved flow management tools and measurement techniques were then demonstrated through in vitro experiments in patient-specific coarctation of aorta flow phantom models manufactured in-house and compared to computational simulations to inform and execute future experiments and simulations.
ContributorsChaudhury, Rafeed Ahmed (Author) / Frakes, David (Thesis advisor) / Adrian, Ronald J (Thesis advisor) / Vernon, Brent (Committee member) / Pizziconi, Vincent (Committee member) / Caplan, Michael (Committee member) / Arizona State University (Publisher)
Created2015