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Background: Like many other cancer patients, most pancreatic carcinoma patients suffer from severe weight loss. As shown in numerous studies with fish oil (FO) supplementation, a minimum daily intake of 1.5 g n-3-fatty acids (n-3-FA) contributes to weight stabilization and improvement of quality of life (QoL) of cancer patients. Given n-3-FA not as triglycerides (FO), but mainly bound to marine phospholipids (MPL), weight stabilization and improvement of QoL has already been seen at much lower doses of n-3-FA (0,3 g), and MPL were much better tolerated. The objective of this double-blind randomized controlled trial was to compare low dose MPL and FO formulations, which had the same n-3-FA amount and composition, on weight and appetite stabilization, global health enhancement (QoL), and plasma FA-profiles in patients suffering from pancreatic cancer. Methods: Sixty pancreatic cancer patients were included into the study and randomized to take either FO- or MPL supplementation. Patients were treated with 0.3 g of n-3-fatty acids per day over six weeks. Since the n-3-FA content of FO is usually higher than that of MPL, FO was diluted with 40% of medium chain triglycerides (MCT) to achieve the same capsule size in both intervention groups and therefore assure blinding. Routine blood parameters, lipid profiles, body weight, and appetite were measured before and after intervention. Patient compliance was assessed through a patient diary. Quality of life and nutritional habits were assessed with validated questionnaires (EORTC-QLQ-C30, PAN26). Thirty one patients finalized the study protocol and were analyzed (per-protocol-analysis). Results: Intervention with low dose n-3-FAs, either as FO or MPL supplementation, resulted in similar and promising weight and appetite stabilization in pancreatic cancer patients. MPL capsules were slightly better tolerated and showed fewer side effects, when compared to FO supplementation. Conclusion: The similar effects between both interventions were unexpected but reliable, since the MPL and FO formulations caused identical increases of n-3-FAs in plasma lipids of included patients after supplementation. The effects of FO with very low n-3-FA content might be explained by the addition of MCT. The results of this study suggest the need for further investigations of marine phospholipids for the improvement of QoL of cancer patients, optionally in combination with MCT.

Conventional liposome preparation methods bear many limitations, such as poor entrapment efficiencies for hydrophilic drugs, batch size limitations, and limited options for aseptic manufacturing. Liposome preparation by dual centrifugation (DC) is able to overcome most of these limitations. DC differs from normal centrifugation by an additional rotation of the samples during the centrifugation process. Thus, the direction of the centrifugal forces changes continuously in the sample vials. The consequential powerful sample movements inside the vials result in powerful homogenization of the sample. Since this “in-vial” homogenization is optimal for viscous samples, semisolid “vesicular phospholipid gels” (VPGs) are preferred intermediates in the liposome manufacturing by DC. The DC method easily enables aseptic preparation and is gentler as compared to other methods, such as high-pressure homogenization. The method allows very small samples to be prepared, and VPG batches down to 1–5 mg scale have been prepared successfully. VPGs have several applications; they are attractive as depot formulations, or as stable storage intermediates, and can be easily transferred into conventional liposomal formulations by simple dilution. Here, we aim to present the novel DC-liposome technique; the concept, advantages, and limitations; and provide an overview of the experiences of liposome preparation by DC so far.

Drugs are encapsulated in nano-sized liposomes to refine their pharmacokinetic, pharmacodynamic or physico-chemical behavior upon administration. Immunoliposomes utilize targeting ligands conjugated to their surface to specifically target the encapsulated drug to the disease affected tissue. The conjugation reaction combining the carrier and ligand is a critical manufacturing step since it is a possible source of product heterogeneities. In this work, Sortase A transpeptidase was investigated as chemoenzymatic tool providing a site specific anchoring of ligands on liposomes. Pentaglycine motifs serving as Sortase-A acceptor motifs were integrated into various liposomal formulations. Liposome manufacturing via a solvent injection process and subsequent tangential flow filtration was characterized regarding liposome size, polydispersity and lipid composition. Sortase-A catalyzed the conjugation of LPETG-modified antibodies to the liposomal surface. The reaction kinetics and efficiency were examined under aspects of liposomal surface properties, temperature, enzyme and substrate concentration. Additionally, Sortase-A depletion procedures from the reaction bulk and alternative manufacturing strategies (post-insertion) were characterized. Conjugation of CD11b¬ specific antibodies led to a targeting of liposomes to human and murine myeloid cells in vitro. For the first time, sortagged liposomes specifically delivered model fluorophores to murine splenic myeloid cells in vivo. Additionally, epidermal growth factor receptor targeted liposomes loaded with doxorubicin exhibited a specifically higher cytotoxicity on a breast cancer cell line compared to their non targeted counterparts. In this thesis, it became obvious that Sortase A transpeptidation is a suitable technology to anchor targeting ligands on liposomes. The obtained results contribute to the elucidation of site-selective conjugation strategies utilized for the design of targeted nanotherapeutics. Arzneistoffe werden in nanoskalierte Liposomen verkapselt, um pharmakokinetische, pharmakodynamische oder physikochemische Eigenschaften des Wirkstoffs zu verbessern. Immunoliposomen verwenden Targeting-Liganden auf ihrer Oberfläche, um den verkapselten Stoff zielgerichtet zu krankheitsbefallenen Geweben zu transportieren. Die Konjugationsreaktion, die Ligand und Liposom vereint, ist ein kritischer Herstellungsschritt, da sie eine mögliche Ursache für Produktheterogenität darstellt. In dieser Arbeit wurde daher eine chemoenzymatische Transpeptidierung mittels Sortase A zum gerichteten Verankern von Liganden auf Liposomen untersucht. Dazu wurden verschiedene Liposomentypen mit Pentaglycin-Motiven versehen, die als Akzeptor für die Sortase-A Reaktion dienten. Diese Liposomen wurde erfolgreich mittels Sortase-A mit LPETG-tragenden Antikörpern modifiziert, wobei die Reaktion unter den Aspekten der liposomalen Oberfläche, Temperatur, Enzym- und Substratkonzentration sowie dem Aufreinigungsprozess charakterisiert wurde. Die Konjugation von CD11b spezifischen Antikörpern auf Liposomen führte zu einer erfolgreichen Targetierung von humanen und murinen myeloischen Zellen in vitro. Mittels Sortase A hergestellte Immunoliposomen wurden erstmalig auch in einem Tierexperiment getestet, in dem die spezifische Färbung von myeloischen Zellen in der Milz gezeigt werden konnte. Weiterhin zeigten mit Doxorubicin beladene und gegen den epidermalen Wachstumsfaktor-Rezeptor gerichtete Immunoliposomen eine spezifisch höhere Toxizität auf einer Brustkrebs-Zelllinie als nicht-targetierte Formulierungen. Die vorliegende Arbeit demonstriert die Eignung der Sortase A katalysierten Transpeptidierung als Methode zum Verankern von Liganden auf Liposomen aus technologischer wie auch biologischer Perspektive. Die Resultate tragen daher zur Erforschung von selektiven Konjugationsstrategien für das Design von zielgerichteten Nanotherapeutika bei.