Background Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can... Show moreBackground Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. Results We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. Conclusions Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs. Show less
In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the... Show moreIn this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR receptor, necessary for infection. We developed a linker protein consisting of the virus-binding moiety of CAR genetically fused to the chicken protein avidin. Biotinylated ligands for cell specific receptors are bound to the linker protein via the avidin-biotin interaction. This now targeting protein is used to redirect adenovirus vectors to previously refractory cell types. Using this technology endothelial cell lines as well as primary endothelial cells can by infected at low MOI__s using an biotinylated cyclic RGD peptide. Primary bone marrow derived macrophages and macrophage cell lines are easily infected using a biotinylated dA6dG10 oligo nucleotide ligand. In vivo experiments showed a marked reduction of adenovirus mediated transgene expression by the liver, the organ responsible for virus uptake when unmodified adenovirus vectors are administered, after addition of several different ligands to the virus via the linker protein. Show less
This thesis centers on the mechanisms of estrogen action and the effects on the development of atherosclerosis. We have focused on the liver as central organ in lipid and glucose metabolism and the... Show moreThis thesis centers on the mechanisms of estrogen action and the effects on the development of atherosclerosis. We have focused on the liver as central organ in lipid and glucose metabolism and the vessel wall as the actual site where the injury occurs. To gain insight in tissue-specific actions of estrogens, we have spent considerable effort to develop tools for liver and blood vessel specific modulation of the estrogen receptor (ER) signaling cascade. The generation, characterization and application of these tools in vitro and in vivo will be described in the different chapters of this thesis. Show less
