Lipid-based nanoparticles for magnetic resonance molecular imaging : design, characterization, and application

In this thesis research is described which was aimed to develop lipidic nanoparticles for the investigation and visualization of atherosclerosis and angiogenesis with both magnetic resonance molecular imaging and optical techniques. The underlying rationale for this is that conventional MR imaging techniques are only capable of visualizing physiological and morphological changes, while magnetic resonance molecular imaging aims to depict cellular and molecular processes that are associated with or lie at the basis of pathological processes. This may lead to earlier detection, and improved diagnosis and prognosis of disease processes. Furthermore this technique may be very useful for the evaluation of a given therapy. The introduction of MRI as a molecular imaging modality is hampered by its low sensitivity compared to nuclear methods like PET and SPECT. With recent developments in chemistry and the synthesis of powerful, innovative, specific, and multimodal contrast agents, e.g. by introducing fluorescent properties as well, MRI is becoming increasingly important for molecular imaging. Therefore, the first aim of the research described in this thesis was to develop biocompatible nanoparticles that can be made target specific and can be detected by both MRI and optical techniques to allow the investigation of disease processes with two highly complementary imaging methods. Chapter 1 gives a general introduction in magnetic resonance molecular imaging and its potential use for the investigation of several pathological processes. Furthermore, contrast enhanced MRI based on differences in T1 and T2 relaxation times is explained. Lastly, different classes of contrast agents and their contrast generating properties are described. Amphphilic molecules are widely applied to serve as building blocks for nanoparticles in biomedical applications. In the field of drug targeting for example, liposomes comprised of amphiphilic molecules hold great promise and have been used extensively the last several decades. Furthermore, micelles, microemulsions, and other amphiphilic aggregates are also under investigation to serve as drug carriers. A relatively new application of lipidic nanoparticles is their use as contrast generating materials for MRI. In Chapter 2 the properties of amphiphilic molecules and their assembly in a wide range of aggregated structures are described. This is followed by an overview of different strategies that are employed to conjugate targeting ligand to such lipid based nanoparticles. The emphasis of this chapter is a literature overview of what has been realized in this research field thus far. Chapter 3 describes the physical characterization of novel liposomal contrast agents. The morphology of different formulations was investigated with electron microscopy, which revealed the necessity of incorporating cholesterol in the liposomal bilayer. Furthermore the relaxation properties of these contrast agent were measured as a function of temperature and magnetic field strength. In Chapter 4 a liposomal contrast agent with both fluorescent and magnetic properties is described. The liposomes were made target specific by conjugating multiple E-selectin specific antibodies to the surface of the nanoparticle. Its feasibility to serve as molecular imaging contrast agent for the detection of the inflammation marker E-selectin is demonstrated in vitro. The specific uptake of the liposomes by human endothelial cells stimulated to express E-selectin was visualized by MRI and fluorescence microscopy. Chapter 5 describes a superparamagnetic nanoparticle encapsulated in a micellular shell. Fluorescent properties were introduced to this contrast agent by the incorporation of fluorescent lipids in the lipid layer. The contrast agent has a very high r2/r1 ratio and therefore is especially suitable to be used for T2 (*) enhanced MRI. The nanoparticle can be made target specific by covalently linking targeting ligands distally to the PEG chains of lipids incorporated in the micellular shell via maleimide-sulfhydryl coupling. Specificity for apoptotic cells was realized by conjugating multiple Annexin A5 proteins. The feasibility to use this contrast agent for molecular imaging purposes was demonstrated in vitro on apoptotic Jurkat cells. In Chapter 6 the synthesis and characterization of a novel bimodal nanoparticle based on semiconductor nanocrystals encapsulated within the corona of paramagnetic micelles is described. The CdSe nanoparticle, also referred to as quantum dot, serves as the contrast generating material for fluorescence imaging, while the paramagnetic micellular coating is employed for contrast enhanced MRI. The in vitro association of this nanoparticle with isolated cells by either conjugating multiple avß3-integrin specific RGDpeptides or multiple phosphatidyl serine specific Annexin A5 proteins was demonstrated with both fluorescence microscopy and MRI. The second aim of the research described in this thesis was to apply the novel nanoparticles for the investigation of atherosclerosis and tumor angiogenesis in mouse models with magnetic resonance molecular imaging. Chapter 7 describes the application of non targeted paramagnetic liposomes for the improved and sustained visualization of neointimal lesions induced after placing a constrictive collar around the right carotid artery of apoE-KO mice. Commercially available Gd-DTPA (Magnevist) showed little potential for the detection of such lesion. In Chapter 8 pegylated micelles conjugated with macrophage scavenger receptor (MSR) specific antibodies were employed for improved atherosclerotic plaque detection and characterization. Existing nanoparticulate agents that are used to detect macrophages, such as USPIO or lipophilic micelles, show little specificity. The micelles used for this study have a hydrophilic PEG coating, and therefore show minimal non-specific interaction with plaque, which results in negligible background signal. In case of the MSR micelles a pronounced enhancement of atherosclerotic plaque was observed. Furthermore, the micelles exhibit fluorescent properties by the incorporation of either quantum dots or fluorescent lipids. This allowed the detection of macrophages with optical techniques as well. Chapter 9 and Chapter 10 describe the application of avß3 targeted bimodal liposomes for the visualization of angiogenically activated tumor blood vessels with both MRI in vivo and fluorescence microscopy ex vivo. The specificity of the contrast agent was demonstrated with an MRI competition experiment, while the exclusive association with endothelial cells was demonstrated with fluorescence microscopy. The follow-up study demonstrates the usefulness of contrast enhanced MRI after applying this contrast agent for the evaluation of angiostatic therapies, i.e. using endostatin and anginex, at two time points after onset of therapy. Most importantly, the in vivo MRI data show very good correlation with ex vivo microvessel density determinations. In the last experimental Chapter 11 of this thesis a sophisticated method for the parallel visualization of angiogenic tumor blood vessels with both intravital microscopy (IVM) and MRI is described. The nanoparticulate contrast agent conjugated with avß3-specific RGDpeptides described in Chapter 6 was administrated to tumor bearing mice. IVM allowed the investigation of the disease process at the cellular level, while MRI was used to investigate angiogenesis at the anatomical level. The contrast agent possesses excellent contrast generating properties for these complementary imaging techniques. Widespread angiogenic activity within the rim of the tumor, and up to 1 cm from the tumor boundary could be observed by using both techniques

Monitoring Veenterpen : Effectmonitoring van waterberging in herinrichting Matsloot-Roderwolde en Peizer- & Eeldermaden, 2009-2014

In 2012 is een waterbergingsgebied binnen de herinrichting Peize- en Roderwoldermaden in gebruik genomen. De effecten hiervan op de conservering van veenterpen zijn gemeten. Tussen 2009 en 2014 zijn daartoe 15 veenterpen gemonitord op o.a. grondwaterstand en redoxpotentiaal, bodemvocht, bodemtemperatuur op verschillende diepten en zijn vegetatie-opnamen gedaan. Vermoedelijk door hogere grondwaterstanden, geringere -fluctuatie en ander landgebruik zijn de redoxpotentialen gedaald, wat duidt op betere conservering van archeologica in de veenterpen. De veenterpen zijn qua vegetatie wel sterk verruigd, met grotere en diepwortelende soorten. Een vorm van actief graslandbeheer wordt sterk aanbevolen. Verder is aangegeven dat voor een beter inzicht in de veranderde conservering en het effect van de vegetatie-ontwikkeling op de veenterpen het nodig is om in afgeslankte vorm nog enkele jaren door te meten en de vegetatie-ontwikkeling te blijven volgen

Contrast enhancement by differently sized paramagnetic MRI contrast agents in mice with two phenotypes of atherosclerotic plaque

Interest in the use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of three differently sized gadolinium-based contrast agents to permeate different mouse plaque phenotypes was evaluated with MRI. A tapered cast was implanted around the right carotid artery of apoE-/- mice to induce two different plaque phenotypes: a thin cap fibroatheroma (TCFA) and a non-TCFA lesion. Both plaques were allowed to develop over 6 and 9 weeks, leading to an intermediate and advanced lesion, respectively. Signal enhancement in the carotid artery wall, following intravenous injection of Gd-HP-DO3A as well as paramagnetic micelles and liposomes was evaluated. In vivo T1-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy and correlated to lesion phenotype. The two smallest contrast agents, i.e. Gd-HP-DO3A and micelles, were found to enhance contrast in T1-weighted MR images of all investigated plaque phenotypes. Maximum contrast enhancement ranged between 53 and 70% at 6¿min after injection of Gd-HP-DO3A with highest enhancement and longest retention in the non-TCFA lesion. Twenty-four hours after injection of micelles maximum contrast enhancement ranged between 24 and 35% in all plaque phenotypes. Administration of the larger liposomes did not cause significant contrast enhancement in the atherosclerotic plaques. Confocal fluorescence microscopy confirmed the MRI-based differences in plaque permeation between micelles and liposomes. Plaque permeation of contrast agents was strongly dependent on size. Our results implicate that, when equipped with targeting ligands, liposomes are most suitable for the imaging of plaque-associated endothelial markers due to low background enhancement, whereas micelles, which accumulate extravascularly on a long timescale, are suited for imaging of less abundant markers inside plaques. Low molecular weight compounds may be employed for target-specific imaging of highly abundant extravascular plaque-associated target

Q-Curves with Complex Multiplication

The Hecke character of an abelian variety A/F is an isogeny invariant and the Galois action is such that A is isogenous to its Galois conjugate A^σ if and only if the corresponding Hecke character is fixed by σ. The quadratic twist of A by an extension L/F corresponds to multiplication of the associated Hecke characters. This leads us to investigate the Galois groups of families of quadratic extensions L/F with restricted ramification which are normal over a given subfield k of F. Our most detailed results are given for the case where k is the field of rational numbers and F is a field of definition for an elliptic curve with complex multiplication by K. In this case the groups which occur as Gal(L/K) are closely related to the 4-torsion of the class group of K. We analyze the structure of the local unit groups of quadratic fields to find conditions for the existence of curves with good reduction everywhere. After discussing the question of finding models for curves of a given Hecke character, we use twists by 3-torsion points to give an algorithm for constructing models of curves with known Hecke character and good reduction outside 3. The endomorphism algebra of the Weil restriction of an abelian variety A may be determined from the Grössencharacter of A. We describe the computation of these algebras and give examples in which A has dimension 1 or 2 and its Weil restriction has simple abelian subvarieties of dimension ranging between 2 and 24

Liposomal prednisolone promotes macrophage lipotoxicity in experimental atherosclerosis

Atherosclerosis is a lipid-driven inflammatory disease, for which nanomedicinal interventions are under evaluation. Previously, we showed that liposomal nanoparticles loaded with prednisolone (LN-PLP) accumulated in plaque macrophages, however, induced proatherogenic effects in patients. Here, we confirmed in low-density lipoprotein receptor knockout (LDLr−/−) mice that LN-PLP accumulates in plaque macrophages. Next, we found that LN-PLP infusions at 10 mg/kg for 2 weeks enhanced monocyte recruitment to plaques. In follow up, after 6 weeks of LN-PLP exposure we observed (i) increased macrophage content, (ii) more advanced plaque stages, and (iii) larger necrotic core sizes. Finally, in vitro studies showed that macrophages become lipotoxic after LN-PLP exposure, exemplified by enhanced lipid loading, ER stress and apoptosis. These findings indicate that liposomal prednisolone may paradoxically accelerate atherosclerosis by promoting macrophage lipotoxicity. Hence, future (nanomedicinal) drug development studies are challenged by the multifactorial nature of atherosclerotic inflammation

Multifunctional Magnetic-fluorescent Nanocomposites for Biomedical Applications

Nanotechnology is a fast-growing area, involving the fabrication and use of nano-sized materials and devices. Various nanocomposite materials play a number of important roles in modern science and technology. Magnetic and fluorescent inorganic nanoparticles are of particular importance due to their broad range of potential applications. It is expected that the combination of magnetic and fluorescent properties in one nanocomposite would enable the engineering of unique multifunctional nanoscale devices, which could be manipulated using external magnetic fields. The aim of this review is to present an overview of bimodal “two-in-one” magnetic-fluorescent nanocomposite materials which combine both magnetic and fluorescent properties in one entity, in particular those with potential applications in biotechnology and nanomedicine. There is a great necessity for the development of these multifunctional nanocomposites, but there are some difficulties and challenges to overcome in their fabrication such as quenching of the fluorescent entity by the magnetic core. Fluorescent-magnetic nanocomposites include a variety of materials including silica-based, dye-functionalised magnetic nanoparticles and quantum dots-magnetic nanoparticle composites. The classification and main synthesis strategies, along with approaches for the fabrication of fluorescent-magnetic nanocomposites, are considered. The current and potential biomedical uses, including biological imaging, cell tracking, magnetic bioseparation, nanomedicine and bio- and chemo-sensoring, of magnetic-fluorescent nanocomposites are also discussed

Lipid-based nanoparticles for magnetic resonance molecular imaging : design, characterization, and application

In this thesis research is described which was aimed to develop lipidic nanoparticles for the investigation and visualization of atherosclerosis and angiogenesis with both magnetic resonance molecular imaging and optical techniques. The underlying rationale for this is that conventional MR imaging techniques are only capable of visualizing physiological and morphological changes, while magnetic resonance molecular imaging aims to depict cellular and molecular processes that are associated with or lie at the basis of pathological processes. This may lead to earlier detection, and improved diagnosis and prognosis of disease processes. Furthermore this technique may be very useful for the evaluation of a given therapy. The introduction of MRI as a molecular imaging modality is hampered by its low sensitivity compared to nuclear methods like PET and SPECT. With recent developments in chemistry and the synthesis of powerful, innovative, specific, and multimodal contrast agents, e.g. by introducing fluorescent properties as well, MRI is becoming increasingly important for molecular imaging. Therefore, the first aim of the research described in this thesis was to develop biocompatible nanoparticles that can be made target specific and can be detected by both MRI and optical techniques to allow the investigation of disease processes with two highly complementary imaging methods. Chapter 1 gives a general introduction in magnetic resonance molecular imaging and its potential use for the investigation of several pathological processes. Furthermore, contrast enhanced MRI based on differences in T1 and T2 relaxation times is explained. Lastly, different classes of contrast agents and their contrast generating properties are described. Amphphilic molecules are widely applied to serve as building blocks for nanoparticles in biomedical applications. In the field of drug targeting for example, liposomes comprised of amphiphilic molecules hold great promise and have been used extensively the last several decades. Furthermore, micelles, microemulsions, and other amphiphilic aggregates are also under investigation to serve as drug carriers. A relatively new application of lipidic nanoparticles is their use as contrast generating materials for MRI. In Chapter 2 the properties of amphiphilic molecules and their assembly in a wide range of aggregated structures are described. This is followed by an overview of different strategies that are employed to conjugate targeting ligand to such lipid based nanoparticles. The emphasis of this chapter is a literature overview of what has been realized in this research field thus far. Chapter 3 describes the physical characterization of novel liposomal contrast agents. The morphology of different formulations was investigated with electron microscopy, which revealed the necessity of incorporating cholesterol in the liposomal bilayer. Furthermore the relaxation properties of these contrast agent were measured as a function of temperature and magnetic field strength. In Chapter 4 a liposomal contrast agent with both fluorescent and magnetic properties is described. The liposomes were made target specific by conjugating multiple E-selectin specific antibodies to the surface of the nanoparticle. Its feasibility to serve as molecular imaging contrast agent for the detection of the inflammation marker E-selectin is demonstrated in vitro. The specific uptake of the liposomes by human endothelial cells stimulated to express E-selectin was visualized by MRI and fluorescence microscopy. Chapter 5 describes a superparamagnetic nanoparticle encapsulated in a micellular shell. Fluorescent properties were introduced to this contrast agent by the incorporation of fluorescent lipids in the lipid layer. The contrast agent has a very high r2/r1 ratio and therefore is especially suitable to be used for T2 (*) enhanced MRI. The nanoparticle can be made target specific by covalently linking targeting ligands distally to the PEG chains of lipids incorporated in the micellular shell via maleimide-sulfhydryl coupling. Specificity for apoptotic cells was realized by conjugating multiple Annexin A5 proteins. The feasibility to use this contrast agent for molecular imaging purposes was demonstrated in vitro on apoptotic Jurkat cells. In Chapter 6 the synthesis and characterization of a novel bimodal nanoparticle based on semiconductor nanocrystals encapsulated within the corona of paramagnetic micelles is described. The CdSe nanoparticle, also referred to as quantum dot, serves as the contrast generating material for fluorescence imaging, while the paramagnetic micellular coating is employed for contrast enhanced MRI. The in vitro association of this nanoparticle with isolated cells by either conjugating multiple avß3-integrin specific RGDpeptides or multiple phosphatidyl serine specific Annexin A5 proteins was demonstrated with both fluorescence microscopy and MRI. The second aim of the research described in this thesis was to apply the novel nanoparticles for the investigation of atherosclerosis and tumor angiogenesis in mouse models with magnetic resonance molecular imaging. Chapter 7 describes the application of non targeted paramagnetic liposomes for the improved and sustained visualization of neointimal lesions induced after placing a constrictive collar around the right carotid artery of apoE-KO mice. Commercially available Gd-DTPA (Magnevist) showed little potential for the detection of such lesion. In Chapter 8 pegylated micelles conjugated with macrophage scavenger receptor (MSR) specific antibodies were employed for improved atherosclerotic plaque detection and characterization. Existing nanoparticulate agents that are used to detect macrophages, such as USPIO or lipophilic micelles, show little specificity. The micelles used for this study have a hydrophilic PEG coating, and therefore show minimal non-specific interaction with plaque, which results in negligible background signal. In case of the MSR micelles a pronounced enhancement of atherosclerotic plaque was observed. Furthermore, the micelles exhibit fluorescent properties by the incorporation of either quantum dots or fluorescent lipids. This allowed the detection of macrophages with optical techniques as well. Chapter 9 and Chapter 10 describe the application of avß3 targeted bimodal liposomes for the visualization of angiogenically activated tumor blood vessels with both MRI in vivo and fluorescence microscopy ex vivo. The specificity of the contrast agent was demonstrated with an MRI competition experiment, while the exclusive association with endothelial cells was demonstrated with fluorescence microscopy. The follow-up study demonstrates the usefulness of contrast enhanced MRI after applying this contrast agent for the evaluation of angiostatic therapies, i.e. using endostatin and anginex, at two time points after onset of therapy. Most importantly, the in vivo MRI data show very good correlation with ex vivo microvessel density determinations. In the last experimental Chapter 11 of this thesis a sophisticated method for the parallel visualization of angiogenic tumor blood vessels with both intravital microscopy (IVM) and MRI is described. The nanoparticulate contrast agent conjugated with avß3-specific RGDpeptides described in Chapter 6 was administrated to tumor bearing mice. IVM allowed the investigation of the disease process at the cellular level, while MRI was used to investigate angiogenesis at the anatomical level. The contrast agent possesses excellent contrast generating properties for these complementary imaging techniques. Widespread angiogenic activity within the rim of the tumor, and up to 1 cm from the tumor boundary could be observed by using both techniques