The blood brain barrier (BBB) remains a major challenge to the advancement and application of systemic anti-cancer therapeutics into the central nervous system. The structural and physiological delivery constraints of the BBB significantly limit the effectiveness of conventional chemotherapy, thereby making systemic administration a non-viable option for the vast majority of chemotherapy agents. Furthermore, the lack of specificity of conventional systemic chemotherapy when applied towards malignant brain tumors remains a major shortcoming. Hence novel therapeutic strategies that focus both on targeted and enhanced delivery across the BBB are warranted. In recent years nanoparticles (NPs) have emerged as attractive vehicles for efficient delivery of targeted anti-cancer therapeutics. In particular, gold nanoparticles (AuNPs) have gained prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within permissive tumor microvasculature provide a selective advantage for targeting. Malignant brain tumors also exhibit transport-permissive microvasculature secondary to blood brain barrier disruption. Hence AuNPs may have potential relevance for brain tumor targeting. However, the permeation of AuNPs across the BBB has not been well characterized, and hence is a potential limitation for successful application of AuNP-based therapeutics within the central nervous system (CNS). In this dissertation, we designed and characterized AuNPs and assessed the role of polyethylene glycol (PEG) on the physical and biological properties of AuNPs. We established a size-dependent permeation profile with respect to core size as well as PEG length when AuNPs were assessed through a transport-permissive in-vitro BBB. This study was the first of its kind to systematically examine the influence of design on permeation of AuNPs through transport-permissive BBB. Given the significant delivery limitations through the non-transport permissive and intact BBB, we also assessed the role of magnetic resonance imaging (MRI) guided focused ultrasound (MRgFUS) disruption of the BBB in enhancing permeation of AuNPs across the intact BBB and tumor BBB in vivo. MRgFUS is a novel technique that can transiently increase BBB permeability thereby allowing delivery of therapeutics into the CNS. We demonstrated enhanced delivery of AuNPs with therapeutic potential into the CNS via MRgFUS. Our study was the first to establish a definitive role for MRgFUS in delivering AuNPs into the CNS. In summary, this thesis describes results from a series of research projects that have contributed to our understanding of the influence of design features on AuNP permeation through the BBB and also the potential role of MRgFUS in AuNP permeation across the BBB.

<p><span style="font-size: 11.5pt">The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB. </span></p> <div><span style="font-size: 11.5pt">The aim of this study was to investigate <i>in vitro </i>effects of pure and street MA &ldquo / tik&rdquo / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. </span></div> <div><span style="font-size: 11.5pt">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. </span></div> <div><span style="font-size: 11.5pt">Results of this study showed that: </span></div> <div><span style="font-size: 11.5pt">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05)&nbsp / </span><span style="font-size: 11.5pt">&nbsp / </span></div> <div><span style="color: windowtext / font-size: 11.5pt">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). </span></div> <div style="margin: 0cm 0cm 25pt"><span style="color: windowtext / font-size: 11.5pt">3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) </span><span style="color: windowtext / font-size: 11.5pt">4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. </span><span style="color: windowtext / font-size: 11.5pt">5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase.&nbsp / </span></div> <div style="margin: 0cm 0cm 25pt"><span style="line-height: 115% / font-size: 11.5pt">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.</span></div>

<p><span style="font-size: 11.5pt">The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB. </span></p> <div><span style="font-size: 11.5pt">The aim of this study was to investigate <i>in vitro </i>effects of pure and street MA &ldquo / tik&rdquo / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. </span></div> <div><span style="font-size: 11.5pt">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. </span></div> <div><span style="font-size: 11.5pt">Results of this study showed that: </span></div> <div><span style="font-size: 11.5pt">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05)&nbsp / </span><span style="font-size: 11.5pt">&nbsp / </span></div> <div><span style="color: windowtext / font-size: 11.5pt">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). </span></div> <div style="margin: 0cm 0cm 25pt"><span style="color: windowtext / font-size: 11.5pt">3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) </span><span style="color: windowtext / font-size: 11.5pt">4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. </span><span style="color: windowtext / font-size: 11.5pt">5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase.&nbsp / </span></div> <div style="margin: 0cm 0cm 25pt"><span style="line-height: 115% / font-size: 11.5pt">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.</span></div>

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

Little is known about potential influences of kinase pathway modulation on expression and activity of P-glycoprotein (P-gp). A protein kinase inhibitor (PKI) library was screened, to determine its effects on activity and expression of P-gp, in various cell lines. Cell lines were incubated with PKI for 24 h. Subsequent P-gp substrate accumulation studies were performed. Changes in P-gp activity and/or expression ≥ 25% compared to control were considered hits. Kinase pathways identified as P-gp activity hits were examined for their ability to modulate permeability. PKI families GSK-3, Craf1 and VEGFR2 and Tie-2, significantly modulated P-gp activity in the MDCK cell line. PKI families GSK-3, Iκκ and Jnk2/3 significantly modulated P-gp activity in the Caco-2 cell line. Few P-gp activity hits significantly modulated P-gp expression. PKIs modulate P-gp activity more than P-gp expression in a cell line dependent manner, excluding GSK-3 PKI family, which appears to be cell line independent.

The blood brain barrier (BBB) remains a major challenge to the advancement and application of systemic anti-cancer therapeutics into the central nervous system. The structural and physiological delivery constraints of the BBB significantly limit the effectiveness of conventional chemotherapy, thereby making systemic administration a non-viable option for the vast majority of chemotherapy agents. Furthermore, the lack of specificity of conventional systemic chemotherapy when applied towards malignant brain tumors remains a major shortcoming. Hence novel therapeutic strategies that focus both on targeted and enhanced delivery across the BBB are warranted. In recent years nanoparticles (NPs) have emerged as attractive vehicles for efficient delivery of targeted anti-cancer therapeutics. In particular, gold nanoparticles (AuNPs) have gained prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within permissive tumor microvasculature provide a selective advantage for targeting. Malignant brain tumors also exhibit transport-permissive microvasculature secondary to blood brain barrier disruption. Hence AuNPs may have potential relevance for brain tumor targeting. However, the permeation of AuNPs across the BBB has not been well characterized, and hence is a potential limitation for successful application of AuNP-based therapeutics within the central nervous system (CNS). In this dissertation, we designed and characterized AuNPs and assessed the role of polyethylene glycol (PEG) on the physical and biological properties of AuNPs. We established a size-dependent permeation profile with respect to core size as well as PEG length when AuNPs were assessed through a transport-permissive in-vitro BBB. This study was the first of its kind to systematically examine the influence of design on permeation of AuNPs through transport-permissive BBB. Given the significant delivery limitations through the non-transport permissive and intact BBB, we also assessed the role of magnetic resonance imaging (MRI) guided focused ultrasound (MRgFUS) disruption of the BBB in enhancing permeation of AuNPs across the intact BBB and tumor BBB in vivo. MRgFUS is a novel technique that can transiently increase BBB permeability thereby allowing delivery of therapeutics into the CNS. We demonstrated enhanced delivery of AuNPs with therapeutic potential into the CNS via MRgFUS. Our study was the first to establish a definitive role for MRgFUS in delivering AuNPs into the CNS. In summary, this thesis describes results from a series of research projects that have contributed to our understanding of the influence of design features on AuNP permeation through the BBB and also the potential role of MRgFUS in AuNP permeation across the BBB.

West Nile Virus (WNV) has emerged as a major cause of viral encephalitis. Since its outbreak in the United States 27,000 people have presented with clinical WNV disease resulting in 1074 fatalities. WNV causes a range of disease from mild febrile illnesses to severe and fatal encephalitis. To date, there are currently no therapeutic agents or vaccines available to treat WNV infection in humans. In order to address this, a better understanding of the mechanisms responsible for viral neuroinvasion and neuropathology are required. Using a range of in vitro and in vivo studies, we have investigated the routes by which WNV enters the CNS. Virus replication was observed in the brain microvascular endothelial cells in mice that succumbed to WNV encephalitis. Moreover, we demonstrated that infection of a polarized HBMEC with WNV induced apoptosis. Microarray analysis of WNV-infected HBMEC’s revealed that WNV elicited the expression of cytokines that have been shown to contribute to permeablization of the BBB. These findings suggest that WNV can enter the CNS through the BBB via multiple mechanisms. Real-time RT-PCR performed on WNVinfected HBMECs identified two host genes involved in the host cellular anti-oxidant response that were differentially regulated during viral infection. Furthermore, the addition of the antioxidant, N-acetylcysteine, restored cell viability and decreased viral replication, indicating that oxidative stress contributes to WNV-induced pathogenesis. The current state of knowledge regarding the pathogenesis of WNV encephalitis is based on studies that have defined the role of systemic immune responses to WNV. Limited investigations have been undertaken to determine the contribution of brain cells in the defence, or damage to the brain once WNV has gained access to the CNS. Real-time RT-PCR results in conjunction with in vivo CBA assay data, suggested several candidate host genes that could contribute to the pathogenesis of WNV. Thus, it is necessary to further define the mechanisms of WNV induced pathogenesis as this will aid in the development of targeted strategies to prevent neurological infection and mitigate neurological diseases in affected individuals.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 98-114).

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 4 uppsatser.