NM-MRI for treatment evaluation of Parkinson’s Disease patients

T1-weighted fast spin echo magnetic resonance imaging (MRI) sequences are able to depict neuromelanin (NM)-containing structures, such as the Substantia nigra (SN), as hyper-intense signal areas. NM-MRI can accurately discriminate Parkinson’s Disease (PD) patients from controls and could potentially be used to evaluate the effects of PD treatment - either surgery or medication. PD patients that are treated with Deep Brain Stimulation (DBS) can only undergo 1.5T MRI sequences with specific conditions that prevent the tissues surrounding the neurostimulators from overheating. However, NM-MRI sequences are usually not applied at 1.5T due to worse image quality. Nevertheless, it would be interesting to study how DBS and medication influence the NM signal as a path for a better understanding of the disease and to potentially evaluate the progression of PD after the surgical intervention. Firstly in this work, a NM-MRI sequence was adapted for scanning patients with implanted DBS systems at 1.5T. To evaluate the performance of the sequence, images were taken on the same day with 1.5T and 3T MRI systems. The contrast ratio of both sequences was evaluated and SN areas were measured resorting to a semi-automatic segmentation algorithm. The assessment of these measurements revealed a good agreement between the developed sequence and the original 3T sequence. A second study was carried out, in which SN areas of PD de novo patients were evaluated before and after two months of initiating pharmacological treatment. The median SN area tended to be increased after treatment, suggesting a potential increase of NM related to dopamine therapy. In conclusion, this work presented the first 1.5T NM-MRI sequence that enables SN area measurement of patients with implanted neurostimulators, for further investigation of this method as a diagnostic tool for assessment of disease progression and to better understand clinical effects on NM-MRI and PD itself

Functionalised silica nanoparticles for biomedical imaging

Magnetic resonance imaging is one of the most widely used diagnostic techniques in the clinic as it affords many of the attributes sought from a non-invasive imaging modality. The main limitation of MRI is its inherent insensitivity, and as a result only large-scale abnormalities can be detected from a scan. With an increasing demand for earlier cancer diagnosis there has been a move towards imaging the molecular biomarkers that are present from the beginning of the disease process. This thesis describes the development of highly fluorinated, silica nanoparticles to actively target cancer cells for imaging by 19F MRI. Silica nanoparticles were prepared, and their size optimised for the molecular imaging application. A method was developed to modify the nanoparticles with the highest possible number of surface amine groups. These amine groups were conjugated to fluorinated PEG chains, each containing six equivalent 19F nuclei, and the resulting particles had a high 19F content. To provide the particles with the properties required for a molecular imaging probe, a tenth of the surface bound 19F PEG chains were conjugated to targeting peptides and the remainder were coupled to stabilising ligands. Using quantitative characterisation techniques each modification step was optimised and the exact composition of the nanoparticles was determined. To complement 19F MRI, fluorophores were incorporated into the particles for optical detection as this modality offered an accessible, sensitive and inexpensive alternative. Several samples were prepared which incorporated fluorophores at different positions throughout the nanoparticle structure. Adding the fluorophores to the nanoparticle surface was found to produce the most sensitive optical probe. The final particles were used for in vitro targeting studies to assess their potential as molecular imaging probes. Preliminary in vitro assays demonstrated that these particles selectively targeted cancer cells in the M21 cell line when compared to a control.Open Acces

Harnessing the plasmonic properties of gold nanoparticles: functionalization strategies coupled with novel spectroscopic tools

Metallic plasmonic substrates such as gold nanoparticles (AuNPs) have fascinated researchers due to their usefulness in verious interdisciplinary studies at the interface between applied physics, biochemistry, engineering, and medicine. A good understanding of the physics of these noble nanostructures, particularly the plasmonic and optical properties, can be employed to improve a wide range of sensors and electronic devices. The relevance of molecular recognition and the binding of biological and chemical entities to diagnostics, biosensors, and drug delivery has attracted significant research interest. By addressing material functionalization design and advanced characterization methods, this doctoral work aims to highlight efforts to exploit the surface modification strategies to enhance the responsiveness of nanoparticle substrates for improved detection of health-relevant biomolecules. The self-assembly of small ligands, such as alkanethiols, and oligonucleotides on the surface of AuNPs provided a possible starting route for the preparation of bio-nanomaterials with precise physicochemical properties. The versatile AuNPs were optimized and thoroughly characterized by employing electron microscopy techniques such as transmission electron microscope (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), spectroscopic techniques, including ultraviolet/visible (UV/Vis), dynamic light scattering (DLS), and thermal lens spectrometry (TLS), and biochemical assays (gel electrophoresis, Dot plot, Western plot, and the Enzyme Linked Immunosorbent Assay (ELISA)). Subsequently, the molecular recognition capabilities of functionalized AuNPs were investigated using multiple techniques, including novel detection routes such as the electrophoresis approach coupled with online TLS. This work establishes a versatile platform for AuNP engineering with controlled size and surface functionality. The strategies presented in this thesis aim to improve medical diagnostics to make them affordable for point-of-care scenarios to enhance the quality of human health.wide range of sensors and electronic devices. The relevance of molecular recognition and the binding of biological and chemical entities to diagnostics, biosensors, and drug delivery has attracted significant research interest. By addressing material functionalization design and advanced characterization methods, this doctoral work aims to highlight efforts to exploit the surface modification strategies to enhance the responsiveness of nanoparticle substrates for improved detection of health-relevant biomolecules. The self-assembly of small ligands, such as alkanethiols and oligonucleotides on the surface of AuNPs provided a possible starting route for the preparation of bio-nanomaterials with precise physicochemical properties. The versatile AuNPs were optimized and thoroughly characterized by employing electron microscopy techniques such as transmission electron microscope (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), spectroscopic techniques, including ultraviolet/visible (UV/Vis), dynamic light scattering (DLS), and thermal lens spectrometry (TLS), and biochemical assays (gel electrophoresis, Dot plot, Western plot, and the Enzyme Linked Immunosorbent Assay (ELISA)). Subsequently, the molecular recognition capabilities of functionalized AuNPs were investigated using multiple techniques, including novel detection routes such as the electrophoresis approach coupled with online TLS. This work establishes a versatile platform for AuNP engineering with controlled size and surface functionality. The strategies presented in this thesis aim to improve medical diagnostics to make them affordable for point-of-care scenarios to enhance the quality of human health

Investigating Glymphatic Function In Alzheimer’s Disease Pathology

Alzheimer’s disease is fast becoming the greatest healthcare challenge of our time, with no known cure to-date. Brought about by the toxic formation of plaques of amyloid-β and tangles of tau in the brain, much is still unknown about the precise mechanisms that initiate these protein accumulations, thought to occur decades before clinical manifestation of symptoms. One theory is that an imbalance between the production of these proteins and their removal from the brain promotes retention that eventually aggregates into entities that devastate molecular and cellular machinery. Thus, targeting waste clearance mechanisms responsible for removing cerebral metabolites, including amyloid-β and tau, present novel, enthralling research targets. The glymphatic system is one such pathway that has been recently characterised. Considered a surrogate for lymphatics which are largely lacking in the brain, this fluid network relies on the water channel aquaporin-4, expressed highly on glia, thus being named “glymphatics”. In this work, first, a surgical protocol was established in the mouse brain to facilitate the delivery of tracer molecules into the cerebrospinal fluid. Direct, single time-point, histological assessment of fluorescent tracer distribution was performed to check consistency with previous characterisation of glymphatics in the mouse brain. Glymphatics were then visualised dynamically across the whole brain using magnetic resonance imaging. Glymphatic patterns were investigated in real-time by imaging fluid dynamics in the brain alongside a potent blocker of aquaporin-4. Next, imaging was used to characterise glymphatic changes and aquaporin-4 profiles in mouse models of Alzheimer’s pathology. This revealed a time-dependant relationship between glymphatics and tau accumulation. Finally, the findings were extrapolated onto humans by studying aquaporin-4 modifications in subjects with and without cognitive deficits. Here, the crucial relationship between aquaporin-4 and pathological aggregates of tau and amyloid-β was determined. Furthermore, dystrobrevin, a membrane protein linked to aquaporin-4, was also profiled in the setting of aging and amyloid-β pathology. The work presented herein elucidates the role of glymphatic perturbances in the context of Alzheimer’s disease and clarifies the implications of aquaporin-4 mediated clearance in neurodegeneration

To see or not to see: In vivo nanocarrier detection methods in the brain and their challenges

Nanoparticles have a great potential to significantly improve the delivery of therapeutics to the brain and may also be equipped with properties to investigate brain function. The brain, being a highly complex organ shielded by selective barriers, requires its own specialized detection system. However, a significant hurdle to achieve these goals is still the identification of individual nanoparticles within the brain with sufficient cellular, subcellular, and temporal resolution. This review aims to provide a comprehensive summary of the current knowledge on detection systems for tracking nanoparticles across the blood-brain barrier and within the brain. We discuss commonly employed in vivo and ex vivo nanoparticle identification and quantification methods, as well as various imaging modalities able to detect nanoparticles in the brain. Advantages and weaknesses of these modalities as well as the biological factors that must be considered when interpreting results obtained through nanotechnologies are summarized. Finally, we critically evaluate the prevailing limitations of existing technologies and explore potential solutions

The Development of Hyaluronan-Based Contract Agents for the Intraoperative Detection of Pancreatic Tumors

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. Tumor-specific intraoperative fluorescence imaging could improve staging and surgical resection, thereby improving prognosis. In the first study, hyaluronic acid derived NPs with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG accumulated significantly in an orthotopic pancreatic ductal adenocarcinoma model with safety profile both in vitro and in vivo. To maximize tumor signal, while minimizing signal in healthy pancreas and RES capture of macromolecules, in the next study, we describe the rational development of a series of hyaluronic acid (HA) conjugates that vary in molecular weight and are conjugated to near-infrared fluorescent (NIRF) dyes that have differences in hydrophilicity, serum protein binding affinity, and clearance mechanism. We systematically investigated the roles of each of these properties on tumor accumulation, relative biodistribution, and the impact of intraoperative imaging of orthotopic, syngeneic pancreatic cancer. Overall, each HA-NIRF conjugate displayed intra-pancreatic tumor enhancement compared to uninvolved pancreas at 24 and 96 h. Regardless of HA molecular weight, Cy7.5 conjugation directed biodistribution to the liver, spleen, and bowels. Conjugation of IRDye-800 to 5 and 20 kDa HA resulted in low liver and spleen signal, while preserving tumor contrast enhancement up to 14-fold compared to healthy pancreas. When IRDye800 was conjugated to 100 kDa HA, the conjugate preferentially distributed to RES organs. When assessing the imaging efficacy of HA-based conjugates in hepatic metastases, those that accumulated to the liver utmost (HA100k-Cy7.5, HA100k-IRDye800, NanoICG) turned to aid the identification of hepatic malignancy with hypo-contrast. These studies demonstrate that by tuning HA molecular weight and the physicochemical properties of the conjugated moiety, in this case a NIRF probe, peritoneal biodistribution can be substantially altered to achieve optimized delivery to tumors with robust contrast enhancement for intraoperative imaging to abdominal tumors. Aside from assisting the accurate delineation of primary tumor, HA-NIRF conjugates demonstrated potential for identification of occult metastases in the intraoperative setting, as a versatile tool for accurate staging

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

A review of imaging techniques for systems biology

This paper presents a review of imaging techniques and of their utility in system biology. During the last decade systems biology has matured into a distinct field and imaging has been increasingly used to enable the interplay of experimental and theoretical biology. In this review, we describe and compare the roles of microscopy, ultrasound, CT (Computed Tomography), MRI (Magnetic Resonance Imaging), PET (Positron Emission Tomography), and molecular probes such as quantum dots and nanoshells in systems biology. As a unified application area among these different imaging techniques, examples in cancer targeting are highlighted