Messenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology... Show moreMessenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology for mRNA delivery and are the basis for the approved mRNA vaccines. In many of these lipid formulations, polyethylene glycol (PEG)-functionalized lipid provides steric stabilization and thus plays a key role in improving the stability both ex vivo and in vivo. However, immune responses towards PEGylated lipids may compromise the use of those lipids in some applications (e.g., induction of antigen specific tolerance), or within sensitive tissues (e.g., central nervous system (CNS)). With respect to this issue, polysarcosine (pSar)-based lipopolymers were investigated as an alternative to PEG-lipid in mRNA lipoplexes for controlled intracerebral protein expression in this study. Four polysarcosine-lipids with defined sarcosine average molecular weight (Mn = 2 k, 5 k) and anchor diacyl chain length (m = 14, 18) were synthesized, and incorporated into cationic liposomes. We found that the content, pSar chain length and carbon tail lengths of pSar-lipids govern the transfection efficiency and biodistribution. Increasing carbon diacyl chain length of pSar-lipid led up to 4- and 6-fold lower protein expression in vitro. When the length of either pSar chain or lipid carbon tail increased, the transfection efficiency decreased while the circulation time was prolonged. mRNA lipoplexes containing 2.5% C14-pSar2k resulted in the highest mRNA translation in the brain of zebrafish embryos through intraventricular injection, while C18-pSar2k-liposomes showed a comparable circulation with DSPE-PEG2k-liposomes after systemic administration. To conclude, pSar-lipid enable efficient mRNA delivery, and can substitute PEG-lipids in lipid formulations for controlled protein expression within the CNS. Show less
In this thesis we aimed to expand our knowledge on the pathophysiological aspects of the metabolic syndrome in transgenic mice. The metabolic syndrome involves multiple aspects and has a major... Show moreIn this thesis we aimed to expand our knowledge on the pathophysiological aspects of the metabolic syndrome in transgenic mice. The metabolic syndrome involves multiple aspects and has a major impact on cardiovascular diseases. In the first part of thesis the role of PAI-1 in the development of insulin resistance will addressed. This part will also focus on the mechanism of plasma PAI-1 clearance. In the second part of this thesis, the roles of LRP in atherosclerosis and LPL activity in lipid metabolism are addressed. In this thesis we showed the PAI-1 catabolism is facilitated by a RAP-sensitive mechanism other than LRP, LDLR and VLDLR. The increased plasma PAI-1 levels observed in insulin resistance and obesity is not explained by impaired clearance of PAI-1. The increased plasma PAI-1 levels might be an epiphenomenon of the chronic inflammatory state of insulin resistance or obesity. Furthermore, alternative pathways other than the traditional lipoprotein receptors are involved in the regulation of plasma cholesterol and triglyceride levels. The development of atherosclerosis is multi-factorial in which the balance between the antiand pro-inflammatory processes plays a central role. Macrophage LRP might be one of the features that control this balance. Inflammation not only promotes to the development of atherosclerosis, but might also be involved in the processes that restore the damaged vascular wall. Show less
