Bioresponsive molecular imaging probes

Molecular imaging is recognized as a powerful tool to visualize and characterize biological processes at the cellular and molecular level in vivo. In most imaging approaches, molecular probes are used to bind to disease-specific biomarkers highlighting disease target sites. In recent years, a new subset of molecular imaging probes, known as bioresponsive molecular probes, has been developed. Chapter 1 reviews the several types of these activatable imaging probes and its potential in vivo applicability. The goal of this thesis was the design, synthesis, and in vitro and in vivo characterization of novel bioresponsive imaging probes based on the elegant concept of activatable cell penetrating peptides (ACPPs). The experimental part of this thesis starts with the development of radiolabeled matrix metalloproteinase-2, -9 (MMP-2/9) activatable cell penetrating peptides. The matrix metalloproteinases -2 and -9 play an important role in angiogenesis and metastasis in cancer, and in adverse cardiac remodeling after myocardial infarction. In Chapter 2, it is shown that the proposed MMP-2/9 sensitive peptide-based imaging probes were successfully synthesized on the solid phase, and could be efficiently labeled with radio-isotopes. A dual-isotope labeled ACPP is presented that could discriminate between uptake of the activated probe and the integral probe and this ACPP was used to follow the activation process in vivo. Despite the probe showed specific sensitivity towards MMP-2 and MMP-9 in vitro, in vivo studies in tumor-bearing mice demonstrated that the ACPP was not activated in MMP-expressing tumor tissue, but most likely already in the circulation. Chapter 3 describes the in vivo characterization of the radiolabeled MMP-2/9 ACPPs in a mouse model of myocardial infarction. In this model, infarct-specific activation and retention of the MMP-2/9 ACPP was observed, as was assessed by biodistribution studies using the dual-isotope labeled ACPP. A significant correlation was found between MMP-2/9 expression and the degree of probe activation in infarcted and remote areas of the hearts. Furthermore, ACPP retention in infarcted regions was successfully visualized ex vivo using autoradiography. Nevertheless, also ACPP activation in the circulation resulted in retention of the activated probe in the surrounding tissues, especially in the liver. Consequently, a strong background signal was observed. Chapter 4 focuses on the development of long circulating MMP-2/9 sensitive ACPPs to achieve an extended exposure time to the target proteases. Incorporation of two different albumin ligands i.e. palmitic acid (Palm) and deoxycholic acid (DOCA), in the ACPPs resulted in a strong increase in circulation time of these albumin-binding ACPPs compared to the ACPP without albumin ligand. In vivo biodistribution studies in a mouse model of myocardial infarction pointed towards local activation of a DOCA-conjugated ACPP in areas of cardiac remodelling. Despite the increased circulation time of this probe, the infarct-to-remote ratios and absolute probe uptake in infarcted areas of the heart was comparable to dACPP. In view of the findings discussed in Chapters 2 and 3, we hypothesized that ACPPs sensitive for tissue-specific biomarkers should exhibit reduced activation in the vasculature and background probe uptake of the activated ACPP in all tissues. Consequently, this should improve the signal-to-background ratios of these probes. Therefore, Chapter 5 and Chapter 6 are dedicated to the development of radiolabeled ACPPs activatable by membrane-type matrix metalloproteinase-1 (MMP-14), and the transmembrane protein angiotensin converting enzyme (ACE), respectively. In Chapter 5, the design and synthesis of MMP-14 sensitive ACPPs (ACPP-14) is addressed. MMP-14, like MMP-2 and -9, plays an important role in adverse cardiac remodeling. The most effective ACPP-14 probe was selected by employing MMP-14 sensitivity and enzyme specificity assays. This probe showed efficient cellular uptake upon activation. In a pilot in vivo biodistribution study, the level of in vivo background activation in the vasculature was decreased compared to MMP-2/9 ACPP (Chapters 2 and 3), while an increased uptake in infarcted heart tissue was observed compared to remote heart tissue, warranting further research into the in vivo biodistribution of this probe. Chapter 6 presents the development of an ACPP sensitive for the carboxy exopeptidase angiotensin converting enzyme (ACE). Upregulated heart-associated ACE activity has been related to adverse cardiac remodeling in nearly all cardiovascular diseases. Using molecular modeling approaches, various ACE ACPPs were designed to fit into the catalytic pocket of ACE. These probes were subsequently synthesized, but unfortunately showed no in vitro sensitivity towards ACE. Chapter 7 deals with the design of an ACPP that responds to hydrogen peroxide (H2O2). Production of reactive oxygen species like H2O2 typically occurs during elevated oxidative stress and contributes to the pathogenesis of several diseases, including cardiac ischemia-reperfusion injury. In this Chapter, we propose to extend the application of the ACPP imaging concept to the detection and imaging of H2O2 after cardiac ischemia-reperfusion injury. The suggested H2O2 ACPP uses a H2O2 self-immolative linker moiety to which the cell penetrating polycationic peptide and the polyanionic peptide are conjugated. H2O2-triggering initiates self-immolation of the linker, and thereby releases the polycationic cell penetrating peptide in H2O2-producing tissues. This H2O2 ACPP probe is currently in development. Finally, Chapter 8 concludes with a general discussion on the preceding chapters, followed by some future perspectives of activatable cell penetrating peptide imaging probe

Verspilde levens. Een verkennend onderzoek naar het ervaren van erkenning en miskenning door West-Afrikaanse ongedocumenteerde migranten in Nederland

Dit verkennende empirische onderzoek beschrijft op welke wijze West-Afrikaanse ongedocumenteerde migranten erkenning en miskenning ervaren in Nederland. De erkenningstheorie van Axel Honneth (1995) is toegepast als erkenningstheoretisch kader en zijn theorie is vergeleken met de empirische onderzoeksresultaten. De resultaten van dit onderzoek beschrijven levendig de ervaringen van negen West-Afrikaanse ongedocumenteerde migranten, die met name gevonden zijn door het toepassen van de snowball method. De respondenten ervaren miskenning door geen gelijke rechten te hebben, geen hulp van en verbondenheid met andere mensen te ervaren en de ervaring gezien en behandeld te worden als een minderwaardig mens. De beschreven miskenningservaringen benadrukken de noodzaak tot een meer menswaardig vreemdelingenbeleid. Erkenning wordt ervaren door de aandacht en hulp van vrienden, hulpverleners en God. Tevens ervaren zij erkenning door onderdeel te zijn van een geloofsgemeenschap en ervaren de werkende respondenten sociale waardering. Binnen dit onderzoek wordt inclusie, door het toebehoren aan een gemeenschap, gezien als een zeer waardevolle toevoeging aan Honneths erkenningstheorie. Voor deze vorm van erkenning zijn gelijke rechten namelijk niet noodzakelijk. Tot slot duidt dit onderzoek aan dat Honneths verschillende vormen van erkenning zich hiërarchisch tot elkaar verhouden. Gelijke rechten blijken een belangrijke voorwaarde voor andere vormen van erkenning. Dit impliceert dat mensen zonder rechten amper erkenning als mens kunnen ervaren, waardoor zij als het ware uit de mensheid getild worden