Modular Multimodal Iron Oxide-Based Nanocarriers for Image-Guided dsRNA Immunostimulation and Platinum Anticancer Drug Design

237 p.El objetivo de este proyecto se centra en la síntesis de nanopartículas inorgánicas de óxido de hierro con una composición, superficie y tamaño especialmente diseñados para activar el sistema inmune y tener capacidad anticancerígena y/o para su utilización como agente de imagen multimodal.Para la activación del sistema inmune se ha biofuncionalizado el sistema con un patrón molecular asociado a patógeno (PAMPs) de interés clínico, llamado poly (I:C) (Polyinosinic:polycytidylic acid). Este PAMP es capaz de activar un receptor tipo Toll (TLR3) presente en las células del sistema inmune.La capacidad anticancerígena, se consigue mediante a la incorporación en el sistema de un complejo de Pt (IV) inerte que actúa como profármaco de cisplatino y que junto con el poly (I:C) como agente inmunoestimulador se usan para combinar los efectos de dos terapias complementarias para matar células tumorales in vitro e in vivo.La incorporación de fluorescencia en el sistema mediante un fosfolípido modificado con rodamina B disponible en el mercado, así como el uso del agente radioactivo fac-[99mTc(OH2)3(CO)3]+ y las propiedades magnéticas intrínsecas del sistema, permitieron la visualización del mismo in vitro e in vivo mediante diferentes técnicas de imagen molecular

Innovating surgery for oral squamous cell carcinoma with targeted fluorescent and magnetic tracers

Oral squamous cell carcinoma (OSCC) is characterised by its locally aggressive nature and its high propensity to metastasise to regional lymph nodes (LN)s. It is well-established that margin status and the presence of LN metastases are two of the most important factors affecting prognosis. While patient and tumour factors cannot be changed after presentation, margin control during primary tumour resection and staging of the clinically occult neck are two components of treatment that are within the control of the head and neck surgeon. Despite this, positive margin rates have not improved over the last three decades and staging of the clinically negative neck continues to rely on elective neck dissection (END), which results in overtreatment of up to 75% of patients. Molecular imaging, with tracers designed to target specific tissue and tumour ligands, is a growing field that has significant potential to improve outcomes for patients with OSCC. This thesis evaluates how two different tracer technologies could be used to aid either margin assessment during resection of the primary tumour, and/or staging of the clinically negative neck with sentinel lymph node biopsy (SLNB). The tracers studied within this thesis are Panitumumab-IRDye800CW, an EGFR-targeting fluorophore used for fluorescence molecular imaging, and FerroTrace, a superparamagnetic iron oxide nanoparticle (SPION) that has been engineered with mannose end targets for macrophage-specific binding to aid SLNB.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

Magnetic Hyperthermia for the Treatment of Glioblastoma

Introduction: Glioblastoma, the most common primary adult brain malignancy, is an aggressive tumour with median survival of around one year. Despite extensive research there has been minimal improvement in prognosis and innovative new treatments are urgently required. The research within this thesis focussed on designing a novel therapeutic approach using nanotechnology to achieve in-situ immune stimulation mediated by localised hyperthermia and characterising the effects of hyperthermia within the tumour microenvironment (TME). Methods: In-situ heating was generated using superparamagnetic iron-oxide nanoparticles (SPIONs) stimulated by an alternating magnetic field (AMF); a combined process known as magnetic hyperthermia. Candidate SPIONs were first tested for biocompatibility and favourable heating properties. In-vivo experiments utilised the immunocompetent GL261 glioblastoma model and included: (i) Testing reticuloendothelial system blocking, and direct intratumoural injection to obtain sufficient intratumoural SPION concentrations; (ii) Utilising 89Zr-labelled SPIONs to evaluate in-vivo fate using PET-CT Imaging; (iii) Evaluation of SPION in-vivo heating ability using thermal imaging; (iv) Tumour growth and timed immunohistochemical (IHC) response analysis; (v) Flow cytometry analysis of the tumour infiltrating lymphocyte (TIL) populations following treatment and (vi) testing a combination therapeutic approach combining magnetic hyperthermia with immune checkpoint inhibition. Results: Perimag-COOH was identified as the lead candidate SPION, and intratumoural injection chosen as the optimal method to obtain sufficient intratumoural SPION concentrations. Perimag-COOH remained within the tumour following injection and retained ability to generate AMF-induced heat for at least 72 h post injection. Digital image analysis of IHC demonstrated a specific, localised, heat-shock protein response following hyperthermia. Tumour growth inhibition was observed up to one week following treatment and tumour flow cytometry analysis revealed changes in TIL populations suggestive of an immune response, providing a rational for a combination approach with immune checkpoint inhibition. Conclusions: SPION mediated hyperthermia is achievable in-vivo and can generate TME changes suggestive of an anti-tumour immune response

Monitoring of Immune Cell Response to B Cell Depletion Therapy and Nerve Root Injury Using Spio Enhanced MRI

Magnetic resonance (MR) is a robust platform for non-invasive, high-resolution anatomical imaging. However, MR imaging lacks the requisite sensitivity and contrast for imaging at the cellular level. This represents a clinical impediment to greater diagnostic accuracy. Recent advances have allowed for the in vivo visualization of populations and even of individual cells using superparamagnetic iron oxide (SPIO) MR contrast agents. These nanoparticles, commonly manifested as a core of a single iron oxide crystal or cluster of crystals coated in a biocompatible shell, function to shorten proton relaxation times. In MR imaging these constructs locally dephase protons, resulting in a decrease in signal (hypointensity) localized to the region of accumulation of SPIO. In the context of immune cell imaging, SPIO can provide insight into the cellular migration patterns, trafficking, temporal dynamics and progression of diseases and their related pathological states. Furthermore, by visualizing the presence and activity of immune cells, SPIO-enabled cellular imaging can help evaluate the efficacy of therapy in immune disorders. This thesis examines the production, modification and application of SPIO in a range of in vitro and in vivo immune-response-relevant cellular systems. The role of different nanoparticle characteristics including diameter, surface charge and concentration are investigated in the labeling of T cells in culture. Following optimization of SPIO loading conditions for lymphocytes, the effect these particles have on the activation of primary B cells are elucidated. B cells are tracked using a variety of modalities, with and without the application of B cell depleting therapy. This is to evaluate the efficacy of SPIO as in vivo marker for B cell distribution. Unmodified SPIO were applied to monitor macrophage infiltration in a transient nerve root compression model, with implications for neck pain diagnosis and treatment. Nanoparticle accumulation and MR hypointensity was correlated to the presence of activated macrophage at the site of injury. Taken together, the application of SPIO to study nanoparticle uptake in vitro and visualization of immune cells in vivo provide a basis for advanced study and diagnosis of diverse pathologies

Quantitative analysis of superparamagnetic contrast agent in sentinel lymph nodes using ex vivo vibrating sample magnetometry

As the first step in developing a new clinical technique for the magnetic detection of colorectal sentinel lymph nodes (SLNs), a method is developed to measure the magnetic content in intact, formalin fixated lymph nodes using a vibrating sample magnetometer (VSM). A suspension of superparamagnetic nanoparticles is injected ex vivo around the tumor in the resected colon segments. A selection of three lymph nodes is excised from the region around the tumor and is separately measured in the VSM. The iron content in the lymph nodes is quantified from the magnetic moment curve using the Langevin model for superparamagnetism and a bimodal particle size distribution. Adverse, parasitic movements of the sample were successfully reduced by tight fixation of the soft tissue and using a small vibration amplitude. Iron content in the lymph nodes is detected with 0.5 μg accuracy and ranged from 1 to 51 μg. Histological staining confirmed iron presence. The current method of measuring intact biological tissue in a VSM is suitable to show the feasibility and merit of magnetic detection of SLNs in colorectal cancer. For clinical validation of magnetic SLN selection in colorectal cancer, a new magnetometer with high specificity for superparamagnetic nanoparticles is required. © 1964-2012 IEEE

ΔE-Effect Magnetic Field Sensors

Many conceivable biomedical and diagnostic applications require the detection of small-amplitude and low-frequency magnetic fields. Against this background, a magnetometer concept is investigated in this work based on the magnetoelastic ΔE effect. The ΔE effect causes the resonance frequency of a magnetoelastic resonator to detune in the presence of a magnetic field, which can be read-out electrically with an additional piezoelectric phase. Various microelectromechanical resonators are experimentally analyzed in terms of the ΔE effect and signal-and-noise response. This response is highly complex because of the anisotropic and nonlinear coupled magnetic, mechanical, and electrical properties. Models are developed and extended where necessary to gain insights into the potentials and limits accompanying sensor design and operating parameters. Beyond the material and geometry parameters, we analyze the effect of different resonance modes, spatial property variations, and operating frequencies on sensitivity. Although a large ΔE effect is confirmed in the shear modulus, the sensitivity of classical cantilever resonators does not benefit from this effect. An approach utilizing surface acoustic shear-waves provides a solution and can detect small signals over a large bandwidth. Comprehensive analyses of the quality factor and piezoelectric material parameters indicate methods to increase sensitivity and signal-to-noise ratio significantly. First exchange-biased ΔE-effect sensors pave the way for compact setups and arrays with a large number of sensor elements. With an extended signal-and-noise model, specific requirements are identified that could improve the signal-to-noise ratio. The insights gained lead to a new concept that can circumvent previous limitations. With the results and models, important contributions are made to the understanding and development of ΔE-effect sensors with prospects for improvements in the future