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|Apolipoprotein E-Peptid-modifizierte Wirkstoffträgersysteme|
|Main title||Apolipoprotein E-Peptid-modifizierte Wirkstoffträgersysteme|
|Subtitle||Untersuchungen zur Aufnahme in Endothelzellen und Makrophagen|
|Title variations||Apolipoprotein E-peptide-modified carriersystems|
|Subtitle for translated title||investigation of the uptake in endothelial cells and macrophages|
Place of birth: Berlin
|1. Referee||Prof. Dr. Schäfer-Korting|
|Further Referee(s)||Prof. Dr. Bienert|
|Keywords||apolipoprotein E; endocytosis; artherosclerosis; endothelial cells; macrophages|
|Classification (DDC)||572 Biochemistry|
|Summary||Apolipoprotein E-derived peptides with alternative palmitoylation-pattern
The objective of the study was to investigate the influence of the palmitoylation-pattern on the stability of the adsorptive peptide-liposome-complexes and the secondary structure of the Apo E-derived peptide upon binding to the liposomal bilayer.
All dipalmitoylated Apo E-peptides bind efficiently to liposomes and mediate their internalisation into brain endothelial cells. The N-terminal at position 1 dipalmitoylated P2A2-1 is ideally suited for stable adsorptive binding to POPC-liposomes. P2A2-1 has a low redistribution-rate when bound to liposomes, a high liposome-induced helicity and a moderate membrane-disturbing activity.
No influence of the peptide-helicity on the uptake into endothelial cells was observed. The uptake-efficiency correlated with the stability of the peptide-liposome-complexes.
So far mostly covalent strategies are described for the functionalisation of liposomes. The adsorptive binding of peptides to liposomes using palmitoylic residues is an elegant method, that is easily applied. In comparison to covalent coupling of peptides to liposomes the chemical modification of the peptide is shifted to the peptide-synthesis. The stability of adsorptive peptide-liposome-complexes is comparable to covalent complexes, as the two spatial closely positioned palmitoylic residues are structurally similar to a covalently coupled lipid.
Uptake-studies with endothelial cells of the blood-vessels
Endothelial cells of the blood vessels constitute the first contact area of intravenously applied drug formulations. To assess the transmembrane transport properties of cells lining blood capillaries and large vessels, we compared the uptake of different ApoE-peptide-based carrier-systems into brain capillary endothelial cells (b.End3) and aortic endothelial cells (BAEC).
The fluorescence-properties of carboxyfluorescein are influenced considerably by the carrier-system. The deviating fluorescence-intensities hamper the quantitative comparison of the uptake-efficiency between different ApoE-peptide-based carrier-systems. Therefore, the uptake-studies had to be limited to the characterisation of the uptake-mechanism of each carrier-system individually.
The results indicate complex uptake-patterns for ApoE-peptide-micelles and ApoE-peptide-tagged liposomes. Stereo-specific as well as –unspecific components seem to be involved. Stereo-specific components could be the LDLr or LRP1. For both receptors interactions with ApoE-derived peptides were previously shown (Dyer et al. 1995; Croy et al. 2004). However, the polysaccharide-chains of the HSPG-network can form stereo-specific interactions to a minor degree, too (Brewer et al. 2002).
The endocytotic internalisation of small ApoE-peptide-micelles with a high peptide-density on their surface is mediated by clathrin and is independent of HSPG. In contrast, liposomes tagged with ApoE-peptides are internalised by a clathrin- and caveolin-independent endocytosys.
The LDLr is involved in the uptake of both, ApoE-peptide-micelles and ApoE-peptide-tagged liposomes. Considering the differing endocytotic mechanisms by which these particles are internalised, this seems to be unlikely. However, because of the high sequence-homology in the LDLr-family, different receptors might be targeted. These results show, that the uptake-mechanism is not only determined by the uptake-mediating ligand, but is also influenced by the carrier-type. Important factors are the size of the carrier as well as the peptide-density on the particle-surface.
The efficient internalisation of ApoE-peptide-micelles into brain-capillary endothelial cells but low uptake into endothelial cells of large vessels may provide a basis for the development of more site-specific carrier systems. Further research needs to be done in this field. However, endosomes formed after clathrin-mediated endocytosis eventually fuse with lysosomes where degradation of the internalised material occurs (Maxfield und McGraw 2004). Hence, the advantage of site specificity is likely opposed by a rather unfavorable intracellular fate of the peptide micelles.
Application of ApoE-derived peptides for the diagnosis of atherosclerosis
Atherosclerosis and atherosclerosis-induced secondary disorders are the leading cause of death in the industrialized countries (Naghavi et al. 2003). Therefore, the importance of the development of accurate and sensitive diagnostic methods for this disease can not be underestimated. Due to the ability of MRI to visualize soft tissue with high resolution and contrast it is highly suitable for the non-invasive and timely diagnosis of atherosclerosis.
The application of specific contrast-agents will permit the characterisation of plaque composition. ApoE-peptide-tagged PEG-lipid-micelles target macrophages in the atherosclerotic plaques. The advantages are an easy synthesis, a long shelf-life and a high contrast-enhancement of atherosclerotic plaques in MRI. The signal-enhancement is higher than that of anti-MSR-A antibody-tagged lipid-micelles (Amirbekian et al. 2007).
Compared to standard Gd3+-based contrast-agents (e.g. Gadovist, Magnevist), signal-enhancement of atherosclerotic plaques in MRI by the targeting of macrophages constitutes an pronounced improvement. The stability of atherosclerotic plaques is correlated with its macrophage-density (Virmani et al. 2000) and thus the probability of plaque-rupture can be estimated by MRI.
The successful targeting of early atherosclerotic plaques provides the chance for an effective therapy. The application of statines, e.g. paclitaxel, as used in drug-eluting-stents, may prevent the development of advanced plaques (Schömig et al. 2007).
ApoE-peptide-tagged PEG-lipid-micelles containing Gd3+ are promising new contrast-agents that are excellently suited for the improved MRI based diagnosis of atherosclerosis.
Dataobject from FUDISS_thesis_000000014792
|Number of pages||VI, 116 S.|
|FU Department||Department of Biology, Chemistry and Pharmacy|
|Year of publication||2009|
|Document type||Doctoral thesis|
|Date of defense||2009-12-01|
|Created at||2009-12-21 : 08:53:33|
|Last changed||2010-02-19 : 12:06:11|