Dokument

Zusammenfassung:
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) werden dabei Gene in menschliche Zellen eingeschleust. Ein Genvektor muss nicht nur eine hohe Transfektionseffizienz besitzen, sondern auch eine geringe Toxizität und hohe Patientencompliance aufweisen. Ein Gentherapeutikum zur oralen Anwendung, das eine hohe Stabilität gegenüber dem sauren pH-Wert im Magen hat, würde diese Kriterien am besten erfüllen. Ein vielversprechender Ansatz, um Genvektoren herzustellen, die in sauren pH-Werten stabil bleiben, stellt die Nutzung von Tetraetherlipiden der Archaeen dar. Archaeen bilden neben den Bakterien und Eukaryoten eine der drei Domänen zellulärer Lebewesen. Sie sind an extreme Milieubedingungen angepasst, z.B. wachsen sie bevorzugt bei +80 °C (hyperthermophil), in stark konzentrierten Salzlösungen (halophil) oder bei niedrigen pH-Werten (acidophil). Der Grund für diese außergewöhnliche Stabilität liegt darin, dass die Zellmembran der Archaeen aus Tetraetherlipiden aufgebaut ist. Im Gegensatz zu eukaryotischen Membranlipiden besitzen Tetraetherlipide keine Ester- sondern Etherbindungen. Des Weiteren durchspannen Tetraetherlipide die Membran vollständig und ordnen sich nicht zu einer Doppelmembran an. In der vorliegenden Arbeit wurden Tetraetherlipide aus den gefriergetrockneten Biomassen von Archaeen mittels Soxhlet-Extraktion extrahiert. Eine darauffolgende Aufreinigung fand an einer Kieselgelsäule statt. Die extrahierten Lipidfraktionen beschränkten sich auf TEL (Rohlipide), PLFE (polare Lipidfraktion E), hGDNT (hydrolysiertes Glycerol-Di-Alkyl-Nonitol-Tetraetherlipid) und hGDGT (hydrolysiertes Glycerol-Di-Glycerol-Tetraetherlipid). Zudem wurde ein neuartiges Tetraetherlipid (MI-0907) mit positiv geladener Kopfgruppe synthetisiert. Alle fünf Tetraetherlipide erhielt man als dunkelbraune bzw. hellgelbe waxartige Massen mit Ausbeuten von 1,9 - 46,89 % (bezogen auf die Ausgangsmasse). Für die Herstellung von Liposomen wurden Tetraetherlipide und konventionelle Lipide wie z.B. DPPC (1,2-Di-palmitoyl-sn-glycero-3-phosphocholin), CH (Cholesterol) oder DOTAP (1,2-Dioleoyl-3-Trimethylammoniumpropan) in verschiedenen molaren Verhältnissen in einem organischen Lösungsmittel miteinander gemischt. Durch Verdampfung des ii Lösungsmittels bildete sich ein dünner Lipidfilm, der durch Zugabe einer wässrigen Pufferlösung in eine liposomale Suspension überführt wurde. Die so hergestellten Liposomen zeigten Durchmesser von 101 - 351 nm und PDI-Werte von 0,2 - 0,4. Liposomen mit günstigen Parametern bzgl. Größe und PDI-Wert stellten die Formulierungen hGDNT/DPPC/DOTAP (20/55/25 mol/mol/mol), MI-0907/DPPC/CH (20/55/25 mol/mol/mol) und hGDNT/DPPC/CH (20/55/25 mol/mol/mol) dar. Sie wurden in weiteren Experimenten in Pufferlösungen mit pH-Werten von 2 - 9 inkubiert und bzgl. der Änderung des Durchmessers nach einem Scoring System (--- bis +++) bewertet. Die oben genannten Formulierungen zeigten minimale Veränderungen bzgl. der Durchmesser und PDI-Werte. Die Komplexierung der positiv geladenen Formulierungen mit pDNA führte zu Lipoplexen, die ebenfalls eine hohe Stabilität bei pH-Werten von 2 - 9 zeigten. Die Transfektionseffizienzen der Formulierungen lagen ca. 30 - 40 % unter den Referenzsubstanzen (25kDa-bPEI oder DOTAP). Zur Verbesserung der Transfektionseffizienzen, bildete man Komposite aus Liposomen, Polymer und pDNA, sogenannte Lipopolyplexe. Als Liposomen wurden die oben genannten Formulierungen eingesetzt. Die Transfektionseffizienz lag bei Lipopolyplexen bis zu 50 % über den Referenzsubstanzen. Zudem wiesen Lipopolyplexe in Toxizitätsversuchen wie z.B. LDH-Assay eine um ca. 75 % reduzierte Toxizität im Vergleich zu einfachen Polyplexen auf. Die besondere Stabilität der tetraetherlipidhaltigen Lipopolyplexe wurde in einem Heparin-Assay ermittelt, wobei der Stabilitätseffekt deutlich höher war als für einfache Polyplexe. Die morphologischen Besonderheiten von Lipoplexen konnte mittels Rasterkraftmikroskopie untersucht werden. Hierbei war zu erkennen, dass sich pDNA um Liposomen windet und der Aufbau multilamellar erfolgt, d.h. mehrere Lipidschichten sind übereinander angeordnet und bilden einen „zwiebelartigen“ Aufbau der Komplexe. Bei Lipopolyplexen, die Durchmesser von 138 nm bis 156 nm annehmen, ist ein Polyplexkern von einer ca. 4 nm dicken Lipidschicht ummantelt. Die erfolgreiche Extraktion und Aufreinigung von Tetraetherlipiden aus Archaeen, ist für die Herstellung von Genvektoren eine wichtige Grundlage. Qualitätsbestimmende Kriterien der gewonnenen Lipoplexe und Lipopolyplexe wie reduzierte Toxizität, Stabilität im sauren pH-Milieu und erhöhte Transfektionseffizienz legen nahe, dass sich Genvektoren auf der Basis von Tetraetherlipiden der Archaeen für die orale Gentherapie eignen sollten.

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