Applications of nanotechnology in neuroscience : Functionalized superparamagnetic nanoparticles as contrast enhancers in experimental magnetic resonance imaging

Abstract: Magnetic resonance imaging (MRI) is a noninvasive imaging technique that helps clinicians not only to diagnose but also to plan the treatment of invasive surgery and actually treat medical conditions. Superparamagnetic iron oxide nanoparticles (SPIONs) are a family of MRI contrast agents that improves MRI sensitivity/specificity. The aim of this thesis was to caracterize newly developed target specific MRI contrast enhancers using SPIONs. The specific objectives were: Functionalization of SPIONs; Characterization of the functionalized SPIONs with the properties of MR image, biocompatibility and in vivo interactions. Chemical surface modifications of SPIONs were carried out in three ways: (a) by coating polymeric starch, ethanolamine or aminopropyltrimethoxysilane with co-precipitation of magnetite in the matrix of these substances; or (b) by coating gold with coprecipitation of magnetite and subsequent reduction of Au on the surface; or (c) conjugate bovine serum albumin (BSA) to the surface of the particles using the zero-length cross-linker 1-ethyl-3-(3 dimethylaminopropyl) carbodiimde hydrochloride (EDC). A method based on capillaryelectrophoresis with laser-induced fluorescence detection (CE/LIF) was developed to determine the conjugation efficiency of proteins or other primary amino group containing molecules linked to SPIONs. Monocrystalline iron oxide nanoparticles (MIONs) are a type of SPIONs with an optimal size of 10-30 nm, when starch coated MIONs (Starch-MIONs) were infused into the striatum of the rats, T2'-imaging showed that Starch-MIONs were capable of diffusing in the brain parenchyma. Additional studies showed when Dextran or gold coated MIONs (Dextran-MIONs) or (Au-MIONs) were infused into the striatum of the rats. T2-weighted imaging revealed that increasing the infusion volume resulted in a dramatic expansion of the labeled area of Dextran-MIONs. Similar effects were observed when the infusion dose or retention time was increased. In contrast, Au-MIONs were static at the local injection area and no diffusion was observed. In addition, after two weeks, the signal of Dextran-MIONs was much attenuated whereas Au-MIONs still produced strong signals. Au-MIONs were incubated with mouse neural stem cells at a dose of 10 mug Fe/ml without a transfection agent, and Dextran-MIONs were used at a dose of 10/100 mug Fe/ml as a control. A 100% labeling efficiency was observed with Au-MIONs 24 hours after incubation, but no uptake was detected for Dextran-MIONs even at a dose of 100 mug Fe. Green fluorescent protein positive neural stem cells (GFP-NSCs) were labeled with Au-MIONs. Without any transfection agent, an 80% labeling efficiency was observed 24 hours after incubation. The Au-MIONs labeled GFP-NSCs were tested in agar medium and a dose-dependent attenuation of MRI signals was observed for the labeled cells in samples containing as few as only 20 cells. The labeled cells were infused into the spinal cord of rats and tracked by MRI for a period of one month. Histological analysis revealed that MRI correlated well with gold-positive staining of transplanted cells. In summary, SPIONs can be functionalized with certain substances. The coating efficiency can be monitored by CE/LIF via primary amino groups within the coating substances. Both Starch-MIONs and Dextran-MIONs were capable of diffusing through the interstitial space of brain parenchyma and were progressively cleared out, which can be beneficially used in drug or molecule target delivery and MRI applications for tracing and therapy. Au-MIONs possess superior stability and surface properties, the latter enabling rapid cellular uptake of the particles under physiological conditions. These properties make Au-MIONs especially suitable for tracking transplated cells by MRI.