Herstellung und Charakterisierung tetraetherlipidhaltiger Lipoplexe und Lipopolyplexe als neuartige Vehikel für die orale Gentherapie

Die Gentherapie bietet ein großes Potential für die Behandlung von schweren chronischen Erkrankungen wie z.B. Krebs oder Erbkrankheiten. Hierbei werden nicht nur die Symptome der Krankheiten behandelt, sondern man versucht defekte Gene zu ersetzen. Mit sogenannten Genvektoren (viral oder nicht-viral...

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Bibliographic Details
Main Author: Engelhardt, Konrad
Contributors: Bakowsky, Udo (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2017
Online Access:PDF Full Text
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Table of Contents: Gene therapy offers great potential for the treatment of severe chronic diseases such as cancer or hereditary diseases. It not only treats the symptoms of the disease, but also attempts to replace defective genes. Genes are introduced into human cells using so-called gene vectors (viral or non-viral). The structural and functional requirements for a suitable gene vector are manifold. It must not only have a high transfection efficiency, but also a low cell toxicity and high patient compliance. A gene therapeutical medicine for the oral application, which has a high stability against the acidic pH value in the stomach, would meet these criteria. A promising approach to produce gene vectors that remain stable under acidic pH conditions is the use of tetraether lipids from the archaea. Archaea form one of the three domains of cellular organisms, along with bacteria and eukaryotes. They are adapted to extreme environmental conditions, e. g. they grow preferably at +80 °C (hyperthermophil), in highly concentrated salt solutions (halophilic) or at low pH-values (acidophil). The reason for this extraordinary stability is that the cell membrane of the archaea is composed of tetraether lipids. In contrast to eukaryotic membrane lipids, tetraether lipids do not contain ester but ether bonds. Furthermore, tetraether lipids span the entire cell membrane and do not arrange themselves into a bilayer membrane. In the present work, tetraether lipids were extracted from the freeze-dried bio masses of archaea using soxhlet extraction. A subsequent purification was performed on a silica column. The lipid fractions extracted were limited to TEL (raw lipids), PLFE (polar lipid fraction E), hGDNT (hydrolyzed glycerol-di-alkyl-nonitol tetraether lipid) and hGDGT (hydrolyzed glycerol-di-glycerol tetraether lipid). In addition, a novel tetraether lipid (MI-0907) with a positively charged head groups was synthesized. All five tetraether lipids were obtained as dark brown or pale yellow wax-like masses with yields of 1.9 - 46.89 % (referring to the initial mass). For the preparation of liposomes, tetraether lipids and conventional lipids like DPPC (1,2-di-palmitoyl-sn-glycero-3-phosphocholine), CH (cholesterol) or DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) were mixed in different molar ratios in an organic solvent. Evaporation of the solvent produced a thin lipid film, which then was hydrated with an aqueous buffer solution resulting in a liposomal suspension. The liposomes prepared in this way showed diameters of 101 - 351 nm and PDI values of iv 0.2 - 0.4. Liposomes with favourable parameters regarding size and PDI-value provided the formulations hGDNT/DPPC/DOTAP (20/55/25 mol/mol/mol), MI-0907/DPPC/CH (20/55/25 mol/mol/mol) and hGDNT/DPPC/CH (20/55/25 mol/mol/mol). They were incubated in buffer solutions in further experiments with pH-values ranging from 2 - 9 and were evaluated with regard to the change of the diameter according to a scoring system (--- to +++). The liposomal formulations showed minimal changes in diameter and PDI values. The complexation of the positively charged formulations with pDNA led to lipoplexes, which also showed high stability at pH values of 2 - 9. The transfection efficiencies of the formulations were approximately 30 - 40 % below the reference substances like 25kDa-bPEI or DOTAP. In order to improve the transfection efficiencies, liposomes, polymers and pDNA composites, so-called lipopolyplexes, were prepared. The above mentioned formulations were used as liposomes, which had a high pH stability. The transfection efficiency of lipopolyplexes was up to 50 % above the reference substances. In addition, lipopolyplexes in toxicity tests such as LDH assay showed a reduced toxicity of about 75 % compared to simple polyplexes. The special stability of the tetraether lipid-containing lipopolyplexes was determined in a heparin assay, whereby the stability effect was higher than for simple polyplexes. The morphological characteristics of tetraether lipid-containing lipoplexes could be investigated by atomic force microscopy. It was found that pDNA is wrapped around liposomes and the structure is multilamellar, i. e. several lipid layers are arranged on top of each other and form a typical "onion-like" structure of the complexes. Lipopolyplexes, which have diameters of 138 nm to 156 nm, have an additional lipid layer of approx. 4 nm thickness. The successful extraction and purification of tetraether lipids from archaea is an important basis for the preparation of gene vectors. Quality-determining criteria of the lipoplexes and lipopolyplexes obtained, such as reduced toxicity, stability in the acidic pH environment and increased transfection efficiency suggest that gene vectors based on archaeal tetraether lipids should be suitable for oral gene therapy.