Matching Items (7)
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- All Subjects: Central Nervous System
- Creators: Stabenfeldt, Sarah
- Creators: Sirianni, Rachael
Description
One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs that could be used to treat diseases in the CNS. Previous studies have shown that activation of the adenosine receptor signaling pathway through the use of agonists has been demonstrated to increase BBB permeability. For example, regadenoson is an adenosine A2A receptor agonist that has been shown to disrupt the BBB and allow for increased drug uptake in the CNS. The goal of this study was to verify this property of regadenoson. We hypothesized that co-administration of regadenoson with a non-brain penetrant macromolecule would facilitate its entry into the central nervous system. To test this hypothesis, healthy mice were administered regadenoson or saline concomitantly with a fluorescent dextran solution. The brain tissue was either homogenized to measure quantity of fluorescent molecule, or cryosectioned for imaging with confocal fluorescence microscopy. These experiments did not identify any significant difference in the amount of fluorescence detected in the brain after regadenoson treatment. These results contradict those of previous studies and highlight potential differences in injection methodology, time windows, and properties of brain impermeant molecules.
ContributorsWohlleb, Gregory Michael (Author) / Sirianni, Rachael (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
ContributorsPetty, Francis (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2016
Description
Stromal cell-derived factor-1α (SDF-1α) and its key receptor, CXCR4 are ubiquitously expressed in systems across the body (e.g. liver, skin, lung, etc.). This signaling axis regulates a myriad of physiological processes that range from maintaining of organ homeostasis in adults to, chemotaxis of stem/progenitor and immune cell types after injury. Given its potential role as a therapeutic target for diverse applications, surprisingly little is known about how SDF-1α mediated signaling propagates through native tissues. This limitation ultimately constrains rational design of interventional biomaterials that aim to target the SDF-1α/CXCR4 signaling axis. One application of particular interest is traumatic brain injury (TBI) for which, there are currently no means of targeting the underlying biochemical pathology to improve prognosis.
Growing evidence suggests a relationship between SDF-1α/CXCR4 signaling and endogenous neural progenitor/stem cells (NPSC)-mediated regeneration after neural injury. Long-term modulation of the SDF-1α/CXCR4 signaling axis is thus hypothesized as a possible avenue for harnessing and amplifying endogenous regenerative mechanisms after TBI. In order to understand how the SDF-1α/CXCR4 signaling can be modulated in vivo, we first developed and characterized a sustained protein delivery platform in vitro. We were the first, to our knowledge, to demonstrate that protein release profiles from poly(D,L,-lactic-co-glycolic) acid (PLGA) particles can be tuned independent of particle fabrication parameters via centrifugal fractioning. This process of physically separating the particles altered the average diameter of a particle population, which is in turn was correlated to critical release characteristics. Secondly, we demonstrated sustained release of SDF-1α from PLGA/fibrin composites (particles embedded in fibrin) with tunable burst release as a function of fibrin concentration. Finally, we contrasted the spatiotemporal localization of endogenous SDF-1α and CXCR4 expression in response to either bolus or sustained release of exogenous SDF-1α. Sustained release of exogenous SDF-1α induced spatially diffuse endogenous SDF-1/CXCR4 expression relative to bolus SDF-1 administration; however, the observed effects were transient in both cases, persisting only to a maximum of 3 days post injection. These studies will inform future systematic evaluations of strategies that exploit SDF-1α/CXCR4 signaling for diverse applications.
Growing evidence suggests a relationship between SDF-1α/CXCR4 signaling and endogenous neural progenitor/stem cells (NPSC)-mediated regeneration after neural injury. Long-term modulation of the SDF-1α/CXCR4 signaling axis is thus hypothesized as a possible avenue for harnessing and amplifying endogenous regenerative mechanisms after TBI. In order to understand how the SDF-1α/CXCR4 signaling can be modulated in vivo, we first developed and characterized a sustained protein delivery platform in vitro. We were the first, to our knowledge, to demonstrate that protein release profiles from poly(D,L,-lactic-co-glycolic) acid (PLGA) particles can be tuned independent of particle fabrication parameters via centrifugal fractioning. This process of physically separating the particles altered the average diameter of a particle population, which is in turn was correlated to critical release characteristics. Secondly, we demonstrated sustained release of SDF-1α from PLGA/fibrin composites (particles embedded in fibrin) with tunable burst release as a function of fibrin concentration. Finally, we contrasted the spatiotemporal localization of endogenous SDF-1α and CXCR4 expression in response to either bolus or sustained release of exogenous SDF-1α. Sustained release of exogenous SDF-1α induced spatially diffuse endogenous SDF-1/CXCR4 expression relative to bolus SDF-1 administration; however, the observed effects were transient in both cases, persisting only to a maximum of 3 days post injection. These studies will inform future systematic evaluations of strategies that exploit SDF-1α/CXCR4 signaling for diverse applications.
ContributorsDutta, Dipankar (Author) / Stabenfeldt, Sarah E (Thesis advisor) / Kleim, Jeffrey (Committee member) / Nikkhah, Mehdi (Committee member) / Sirianni, Rachael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2016
Description
An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells.
ContributorsRaman, Sreedevi (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2017
Description
Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method due to their biocompatibility and ability to be modified by cell penetrating peptides, such as transactivating transciptor (TAT) peptide, which has been shown to increase efficiency of delivery. There are multiple proposed mechanisms of TAT-mediated delivery that also have size restrictions on the molecules that can undergo each BBB crossing mechanism. The effect of nanoparticle size on TAT-mediated delivery in vivo is an important aspect to research in order to better understand the delivery mechanisms and to create more efficient NPs. NPs called FluoSpheres are used because they come in defined diameters unlike polymeric NPs that have a broad distribution of diameters. Both modified and unmodified 100nm and 200nm NPs were able to bypass the BBB and were seen in the brain, spinal cord, liver, and spleen using confocal microscopy and a biodistribution study. Statistically significant differences in delivery rate of the different sized NPs or between TAT-modified and unmodified NPs were not found. Therefore in future work a larger range of diameter size will be evaluated. Also the unmodified NPs will be conjugated with scrambled peptide to ensure that both unmodified and TAT-modified NPs are prepared in identical fashion to better understand the role of size on TAT targeting. Although all the NPs were able to bypass the BBB, future work will hopefully provide a better representation of how NP size effects the rate of TAT-mediated delivery to the CNS.
ContributorsCeton, Ricki Ronea (Author) / Stabenfeldt, Sarah (Thesis director) / Sirianni, Rachael (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
There is growing interest in intranasal delivery of therapeutics because of direct nose-to-brain pathways which are able to bypass biological barriers, such as the blood-brain barrier (BBB), that have historically limited our ability to effectively deliver drugs to the central nervous system (CNS). Since these pathways were first discovered, there has been significant preclinical success in delivering a wide range of therapeutics to the CNS with additional growing efforts to further improve delivery through nanoparticle drug delivery systems. Here we sought to improve intranasal delivery of DiR, a lipophilic small molecule cyanine dye, to the CNS by surface modifying a poly (lactic-co-glycolic acid) (PLGA) nanoparticle with a short peptide derived from the rabies virus glycoprotein (RVG). The specific aims of this thesis were to evaluate administration route-dependent delivery of RVG nanoparticles to the CNS, and to identify anatomical transport pathways by which nanoparticles facilitate transport of small lipophilic molecules. Route-dependent delivery kinetics and distribution were studied by administering DiR loaded nanoparticles to healthy Balb/C mice. Specific tissues were homogenized and the fluorescent intensity of DiR was measured and compared to control tissue spiked with known amounts of dye. While bioavailability of DiR after intranasal administration was near 0% with minimal exposure to peripheral organs, quick and efficient delivery to the CNS was still observed. CNS delivery after intranasal administration was rapid with peak concentrations at 30 minutes post-administration followed by broad clearance by 2 hours. Regional differences of delivery of DiR to the CNS demonstrated engagement of direct nose-to-brain transport pathways with high delivery being observed to the olfactory bulb, brain stem, and trigeminal nerve. RVG modification however presented only modest targeting benefits. In conclusion, the biodistribution of DiR after intranasal administration of DiR loaded nanoparticles showed high potential for the direct nose-to-brain delivery while limiting peripheral exposure of lipophilic small molecule drugs.
ContributorsChung, Eugene Paul (Author) / Kodibagkar, Vikram (Thesis director) / Sirianni, Rachael (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug screening, or patient specific disease modeling. Human induced pluripotent stem cells (hiPSC)-derived neural progenitor cells (hNPCs) have the ability to self-renew indefinitely and differentiate into the various neuronal and glial cell types of the CNS. In order to realize the potential of hNPCs, it is necessary to develop a xeno-free scalable platform for effective expansion and differentiation. Previous work in the Brafman lab led to the engineering of a chemically defined substrate—vitronectin derived peptide (VDP), which allows for the long-term expansion and differentiation of hNPCs. In this work, we use this substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Finally, VDP was shown to support the differentiation of hNPCs into functional astrocytes. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their derivatives in quantities necessary for basic and translational applications.
ContributorsMorgan, Daylin (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2018