Diffusion tensor imaging (DTI) provides an indirect measure of tissue structure on microscopic scales. To date, DTI is the only imaging method that provides such information in vivo, and it has proven to be a valuable tool in both research and clinical settings. In this study, A multi-step procedure was developed to register diffusion tensor imaging (DTI) and histological data in the light microscopy image space, with the ultimate goal of allowing quantitative comparisons of the two datasets. The registration procedure was utilized to investigate the relationship between white matter structures and diffusion parameters measured by DTI. We used micrographs from light microscopy of fixed, myelin stained brain sections as a gold standard for direct comparison with data from DTI. Relationships between microscopic tissue properties observed with light microscopy - fiber orientation, density, and coherence - and fiber properties observed by DTI tensor orientation and fractional anisotropy (FA) - were investigated. Agreement between the major eigenvector of the tensor and myelinated fibers was excellent in voxels with high fiber coherence. However, the diffusion tensor was not a reliable indicator of fiber geometry where fibers crossed or diverged.

<p>Macrophages have been proposed as a potential therapeutic target because of their central role in the progression of a number of debilitating diseases, such as cancer and cardiovascular diseases. However, macrophages are resident in almost all healthy tissues. Therefore, immunotherapies targeted to macrophages in pathologic tissues require the development of site-specific techniques to target these pathologic macrophages while leaving healthy ones unaffected.</p> <p>In this work, I pursued the design and validation of macrophage-targeted biomaterials, including the following steps: (1) Optimization of nanoparticle characteristics that would reduce non-specific recognition of the particles by macrophages that may exist outside of the desired site of intervention, (2) Optimization of nanoparticle characteristics that would increase site-specific recognition of pathologic macrophages, (3) Design of a localized delivery platform that may serve as an implantable patch for delivery of nanoparticles to a site of pathologic inflammation, and (4) Identification of potential molecular and gene pathway targets for biomaterials-mediated, therapeutic intervention in vivo. Finally, I also demonstrate some work in the development of assays to quantify T-cell-mediated immune responses in human patients following immunotherapy. This is important because most immunotherapies are designed with the ultimate goal of effecting T-cell responses, whether by generating T-cells reactive against the pathologic site (in the context of cancer), or generating regulatory T-cells and other immunosuppressive T-cells (in the context of autoimmune diseases).</p> <p>Looking forward, significant challenges exist for future work by my successors and other future lab members, including (1) the identification of immunotherapeutic agentssmall molecules or peptides or nucleic acids, that can reprogram tumor-associated macrophages into anti-tumor drug depots, (2) optimization of nanoparticle properties for efficient in vivo targeting of TAMs within primary and metastatic tumor sites, (3) the creation of new in vitro assays to evaluate and characterize the serum stability of nanoparticles and their interactions with blood cells in circulation, and (4) the optimization of cell-based assays to facilitate their adoption into other immunotherapeutic clinical trials and studies.</p>

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is a method that can be used to quantitatively and qualitatively assess physiological characteristics of tissue. Quantitative DCE-MRI requires an estimate of the time rate of change of the concentration of the contrast agent in the blood plasma; the so-called vascular input function, or VIF. Measuring the VIF is notoriously difficult as it requires high temporal resolution images limiting the achievable number of slices, field-of-view, spatial resolution, and signal-to-noise. Alternatively, a population-averaged VIF could be used to mitigate the acquisition demands in studies aimed to investigate, for example, tumor vascular characteristics. Eight rats inoculated with C6 glioma cells underwent DCE-MRI at 9.4T. A set of dynamic spoiled gradient-echo images was acquired before, during, and after a bolus injection of Gd-DTPA (Magnevist, Wayne, NJ). Pharmacokinetic parameters (Ktrans, ve, and vp) were extracted from signal-time curves of tumor tissue using both individual and population-averaged VIFs. Standard model voxel values of Ktrans and ve estimated using a population-averaged VIF have a high correlation (concordance correlation coefficient > 0.7852) and a strong linear relationship (Pearson correlation coefficient > 0.9802) with Ktrans and ve values estimated using an individual VIF. Additionally, the extended model voxel values of Ktrans, ve, and vp also showed a high correlation (concordance correlation coefficient > 0.6931) and a strong linear relationship (Pearson correlation coefficient > 0.9159) supporting the use of a population based VIF in DCE-MRI.

More than 700,000 deaths are attributed to malarial infection annually in the rural areas of Sub-Saharan Africa. Effective and reliable early detection is inadequate with currently available technology. Development of a low-cost, sensitive and simple-to-use diagnostic assay appropriate for the point-of-care setting is a critical component of malaria containment efforts. This work has demonstrated a diagnostic assay based on the same mechanism that causes a ring to form in an evaporating coffee drop. This novel method for noninstrumented signal visualization is potentially well-suited for low-resource applications that require a simple-to-use, low cost method for pathogen detection. After exploring and optimizing basic design parameters, we demonstrated proof-of-principle for malaria testing by detecting poly-l-histidine, a biomolecular mimic of pfHRP2. The proposed assay was found to have a limit of detection in the mid-nanomolar range, approximately one order of magnitude higher than the clinically relevant range.

<p>The central goal of this dissertation is to develop a drug carrier capable of retaining the benefits of high molecular weight drug carriers but improving on the intratumoral spatial distribution of drug by engineering the carrier to disassemble in response to the tumor-specific endogenous signal of low pH. High molecular weight drug carriers improve clinical outcomes for cytotoxic chemotherapeutics by preventing their accumulation in healthy tissues, extending their circulation time, and allowing for gradual accumulation in solid tumors due to the leaky tumor vasculature. These carriers increase the maximum tolerated dose that can be administered, and as a result of these characteristics can exhibit improved antitumor effectiveness with reduced toxicity compared with free drug. One drawback of these formulations is that they exacerbate a problem inherent to tumor drug delivery, the poor penetration of drug into the tumor tissue due to the poor vascularization in many tumor regions coupled with the dense extracellular matrix, dense cell packing, and high interstitial fluid pressure of the tumor tissue. Therefore, a high molecular weight drug carrier that disassembles in response to a tumor-specific stimulus could retain the benefits detailed above but release the highly diffusive small molecule drug within the tumor site, improving its ability to penetrate into the tumor and treat the whole tumor volume.</p><p>This work addresses this problem by synthesizing a pH-sensitive drug carrier capable of disassembling in response to the low extracellular pH that exists in many solid tumors. The drug carrier design is a polypeptide block copolymer with a histidine-rich hydrophobic block that self-assembles at pH 7.4 but disassembles at slightly low pH as the histidine residues become charged and the hydrophobic block becomes increasingly hydrophilic. Chapter 1 briefly establishes key features of cancer and solid tumors that impact drug delivery, and then reviews the central findings of the field of tumor drug delivery. After an extensive discussion of pH-sensitive drug carriers that have been synthesized to date, an extended description of the proposed design is given.</p><p>Chapter 2 describes the synthesis and characterization of histidine-rich Elastin-Like Polypeptides (ELPs), the pH-responsive component of the material design. These polymers are synthesized using molecular biological methods and their transition temperatures are characterized using absorbance spectroscopy, demonstrating remarkable dependence on small pH changes in the physiological range of interest (pH 6-8). This chapter further demonstrates that histidine-rich ELPs are sensitive to transition metal ions known to coordinate with histidine, exhibiting a precipitous drop in transition temperature in the presence of µM concentrations of these ions. Chapter 3 characterizes histidine-rich ELP block copolymers (ELPBCs), demonstrating by dynamic light scattering that these polymers form micellar nanoparticles at body temperature and pH 7.4 that disassemble at pH 6.4. These micelles are stabilized by ZnCl2 as demonstrated by a reduced critical micelle temperature observed by light scattering and critical micelle concentration shown by pyrene fluorescence. Static light scattering, Atomic Force Microscopy, and freeze-fracture Transmission Electron Microscopy confirm the presence of these particles and demonstrate that the ELPBCs form uniform spherical micelles.</p><p>Chapter 4 attempts to develop these materials as drug delivery vehicles in several different schemes. First, drug encapsulation experiments are conducted to serve the initial design of pH-sensitive drug release, but these micelles have not yet demonstrated an ability to encapsulate chemotherapeutic drugs. An alternative scheme of disassembly-induced display of a ligand for cell entry is investigated by the inclusion of the LHRH peptide in the micelle core. Disassembly at low pH indeed increases cell uptake of the ELPBCs, but this effect does not depend on ligand presentation. Finally, based on the hypothesis that metal coordination resulted in the formation of crosslinks between ELP chains, histidine-rich ELPs are investigated as a depot formulation for intratumoral drug delivery.</p><p>Chapter 5 returns to the original ELPBC micelle design and addresses the core questions of this work: does the histidine-rich ELPBC retain the benefits of high molecular weight drug carriers, and does low pH-induced disassembly enhance the intratumoral spatial distribution? A low pH mouse tumor model is established to test the effects of pH-sensitive materials, and then fluorescently and radioactively labeled pH-sensitive and pH-insensitive ELPBCs are injected to determine their pharmacokinetics, biodistribution, and intratumoral spatial distribution. The histidine-rich ELPBCs exhibit extended blood circulation and tumor accumulation typical of high molecular weight carriers, although some healthy tissues take up significant amounts of both ELPBCs. The pH-sensitive histidine-rich ELPBCs demonstrate a more even distribution throughout the tumor volume than their pH-insensitive counterparts, indicating that stimulus-responsive nanoparticles can improve tumor penetration through a tumor-specific material response.</p><p>Finally, Chapter 6 summarizes the main findings of the work as noted here, and discusses potential future directions. The main accomplishment of this work is the demonstration that histidine-rich ELPs exhibit triple stimulus-responsiveness and achieve an improved intratumoral spatial distribution.</p> / Dissertation

Thesis (M.App.Sc.) - Carleton University, 2009. / Includes bibliographical references (p.137-146). Also available in electronic format on the Internet.

<p> Diagnostic ultrasound is a safe, inexpensive and highly portable real-time imaging modality for viewing the human body. For over two decades, lipid-coated fluorocarbon microbubble contrast agents have been developed to help improve the diagnostic and therapeutic capabilities of ultrasound, but they have certain limitations. Recently, it was found that the microbubbles can be condensed into superheated liquid nanodrops capable of being vaporized by external optical or acoustic triggers. The compact form and vaporization effects of these phase-shift nanodrops may offer advantages over microbubbles for a number of current and future therapeutic and diagnostic applications. The goal of this dissertation work was to study the molecular thermodynamics and interfacial phenomena of these superheated phase-shift nanodrops.</p><p> In the first part of this work, a custom microscopy pressure chamber with control over temperature and pressure was used to observe microbubbles during condensation. Compression behaviors of fluorocarbon microbubbles constructed with lipid shells of varying acyl chain lengths were quantified over a broad temperature range. Microbubbles containing lipids of longer acyl chains were found to resist ideal compression and condensation. Dissolution was found to dominate as temperature approached the lipid main phase transition temperature, resulting in incomplete condensation. However, successful condensation of gas-filled microbubbles to liquid-filled nanodrops could be achieved at lower temperatures, and fluorescence microscopy showed that the lipid monolayer shell buckles and folds into surface-attached bilayer strands. The nanodrops were found to be remarkably stable when brought back to standard temperature and pressure. The temperature-pressure data were used to construct condensation phase diagrams to determine the thresholds for successful nanodrop formation. </p><p> In the second part of this study, the superheated nanodrops were vaporized back into microbubbles by changes in temperature and pressure. A custom optical chamber with control over temperature and pressure was used to track the kinetics of condensation, vaporization and dissolution of microbubble suspensions with varying fluorocarbon core and lipid shell compositions. A simple model was used to extract kinetic rates from the optical data, and Arrhenius plots were used to determine activation energies. The activation energy for thermal vaporization was found to vary with lipid acyl chain length, and a simple model of lipid intermolecular forces was used to explain this effect. Additionally, thermal vaporization was found to occur near 90% of the critical temperature of the fluorocarbon core, indicating that metastability of the superheated droplets was due to the low probability of homogenous nucleation rather than a Laplace overpressure. The superheated droplets could be reversibly vaporized and condensed to at least ten cycles, showing remarkable stability. </p><p> In the final part of this study, the tunability of vaporization was examined through the mixing of fluorocarbon gases in droplet core. A clinical ultrasound imaging system was used to track vaporization as a function of temperature and mechanical index. Discrepancies were found in the vaporization thresholds owing to mass transfer; the high solubility of the lower fluorocarbon caused it to rapidly deplete. However, a successful acoustic temperature probe was demonstrated. The experimental data from all three parts of this study were examined and explained by conventional molecular thermodynamics theory, providing new insights into the behavior and properties of these novel theranostic agents. </p>

<p>From gene expression to protein abundance, biological measurements have traditionally been taken as population averages. Studies over the past decade, however, have established the biological relevance of population heterogeneity, even for cells with identical genetic background. The advancements of our understanding, albeit transformative, have been almost exclusively focused on the generation and the regulation of population heterogeneity and its implications in cell-fate decisions. What’s less appreciated is the potential of using population heterogeneity measurements as an information source of cellular physiology and network dynamics. While gene expression from a signaling network in a single cell is stochastic and unpredictable, the distribution of the gene expression in a sufficiently large cell population is uniquely determined by the signaling network and is deterministic. This distribution represents a quantitative fingerprint for the cell population under a specific environmental condition. I established and characterized a computational platform using stochastic modeling of the Myc/Rb/E2F network that supports the analysis of distributional data—how it changes as a function of perturbation and how it can be used to infer cellular or external variables of interest. I then demonstrated that a viral-mediated gene expression probe can be effectively and efficiently employed to generate heterogeneity fingerprints of cell populations and differentiate different cell lines as well as characterize drug activities.</p> / Dissertation

With the advent of bionanotechnology, bioengineers utilize nanoscale tools to better characterize and modify biological processes in an effort to enhance medical applications. Most of these applications are single-purpose and utilize only a single property of the biofunctionalized nanomaterial. This work is focused on improvements in nanomaterial design, through variation in fabrication technique, that allow multiple characteristics and properties to be combined into a single, theranostic, nanoscale entity. Acinetobacter baumannii has emerged as a bacterial species of interest due to its significant virulence and enhanced persistence in combat and healthcare environments. Acinetobacter species cause a multitude of ailments which contribute to the incidence of bloodstream infections. The mortality rate associated with A. baumannii bacteremia is 52%. Treatment options are increasingly limited due to the rapid acquisition of multi-drug resistance to the few antibiotics readily available. Due to this, the development of a nanotechnology-mediated adjuvant treatment strategy for A. baumannii is paramount. As outlined in the chapters of this dissertation, novel, multilayered, magneto-metallodielectric multistrata nanoparticles which possess theranostic characteristics were developed (Chapter II). Thereafter, a synthesis strategy was implemented in order to enhance the magnetic properties of the composite material while decreasing the synthesis duration such that freshly synthesized materials could be rapidly utilized in a biological application (Chapter III). The central hypothesis of this dissertation was confirmed using superparamagnetic FeOx/Au core/shell nanoparticles that were used to magnetically capture, visualize and separate Acinetobacter baumannii using an antibiotic, polymyxin E, as the microbial targeting ligand (Chapter IV). Further development of core/shell nanotechnology will bolster the technological impact of biomedical applications thus improving the way we deliver medical care to patients across the globe and beyond.

<p>Targeted drug delivery to solid tumors aims to increase the accumulation of drug at the site of disease while limiting accumulation in healthy tissues. Thus, targeted delivery serves to enhance therapeutic efficacy while minimizing off-target side effects. Targeting drug to the site of disease is especially important for many current anti-cancer therapeutics whose cytotoxic effects are not exclusive to cancer cells. Drug carriers can improve tumor targeting of drug cargo by either passive or active mechanisms. Passive targeting of drug carriers occurs by the enhanced permeability and retention effect, whereby long circulating drug carriers can accumulate in the tumor by extravasation from the tumor's leaky vasculature and be retained in the tumor due to the lack of an organized tumor lymphatic system. Alternatively, active targeting can improve drug delivery to the tumor by means of functionalizing a drug carrier such that it interacts specifically with the tumor tissue. Traditionally, actively targeted drug carriers rely on intrinsic features of the tumor such as upregulated cell receptors, overexpressed extracellular enzymes, or depressed tissue pH. These intrinsic targets, however, are heterogeneous across cancer classes and between patients with a single tumor type. Therefore traditional active targeting cannot be applied to a breadth of cancers or patients without prior knowledge of the cancer phenotype.</p><p>Active targeting can alternatively be achieved by an extrinsic trigger, independent of the characteristics of the tumor. This approach could thereby achieve targeted drug delivery in a breadth of tumor types and cancer patients. This dissertation describes one such approach that exploits cell-penetrating peptides (CPPs) to achieve receptor-independent and non-specific uptake in a variety of cancer cells. The function of this non-specific CPP is controlled by an extrinsic trigger by means of the modulation of its local interfacial density with temperature-triggered micelle assembly. Elastin-like polypeptide diblock copolymers (ELP_BCs) were used as the drug carrier platform, as their lower critical solution temperature phase transition behavior permits their controlled self-assembly from unimer to micelle in response to a thermal stimulus. CPP-ELP_BCs were recombinantly synthesized in <italic>E. coli</italic> with CPP-functionalization at their hydrophilic terminus, such that temperature-triggered micelle assembly would result in the decoration of CPP on the micelle corona. The CPP-ELP_BC design was carefully optimized to permit micelle self-assembly in response to the clinically relevant trigger of mild hyperthermia.</p><p>Temperature-triggered micelle assembly of CPP-ELP_BCs achieved controlled cellular uptake <italic>in vitro</italic> by means of their CPP density modulation, such that cellular uptake was minimized at physiologic temperature and was greatly enhanced at conditions of mild hyperthermia. This effect was achieved in multiple cell lines, albeit with variable magnitude. Controlled uptake of the CPP-ELP_BC carrier could control the intracellular delivery of appended drug cargo. This controlled intracellular delivery was translated to controlled therapeutic effect when the CPP-ELP_BC was genetically appended to a proapoptotic peptide drug cargo. These drug-loaded CPP-ELP_BCs achieved controlled cytotoxicity in cancer cells, whereby significant cell death was induced at conditions of mild hyperthermia, but cells at physiologic temperature were spared. </p><p>For use as targeted drug carriers <italic>in vivo</italic>, CPP-ELP_BCs would be systemically administered and circulate throughout the body in their soluble state. It would only be at the site of the solid tumor that local mild hyperthermia would be applied and induce the self-assembly of CPP-ELP_BC micelles that could induce internalization into cancer cells. Translation of CPP-ELP_BC function from <italic>in vitro</italic> to <italic>in vivo</italic> environments proved to be quite challenging. Issues such as perturbation of temperature-triggered assembly in serum and interference of CPP-ELP_BC internalization by serum proteins likely played a role in preventing the extrinsically targeted accumulation of CPP-ELP_BCs in hyperthermia treated tumors, as investigated by intravital tumor microscopy and biodistribution studies. Further optimization of the CPP-ELP_BC platform is thus required to achieve extrinsically targeted drug carrier delivery <italic>in vivo</italic>.</p> / Dissertation