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Even, Marie-Paule (2015): Twin-screw extruded lipid implants for vaccine delivery. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



In recent years there has been a considerable focus on the development of subunit vaccines, preferred over traditional vaccines for reasons of safety and purity. However, subunit vaccines are less immunogenic than attenuated vaccines and need therefor multiple administrations in combination with immunostimulatory adjuvants, in order to induce immunity. The sustained release of a vaccine together with the release of an adjuvant is a potential alternative to giving multiple doses. The aim of this thesis was to manufacture lipid implants for vaccine delivery by twin-screw (tsc) extrusion and evaluate the potency of these lipid systems to stimulate an immune response in vivo. To accomplish this, lipid implants consisting of cholesterol, soybean lecithin, and Dynasan 114 (D114) were prepared. Different formulations were evaluated for their extrudability before adding the model antigen ovalbumin (OVA) and the adjuvant Quil-A (QA) to the formulation. Investigating the release behaviour of OVA and QA showed that mainly cholesterol influences the release behaviour of OVA, increasing the fraction of cholesterol slows down the release of OVA. To further slow down the release of OVA from the implants, they were cured at different temperature resulting in an even longer OVA release. Furthermore, the addition of QA to the implants influenced the release behaviour of OVA and vice versa. The investigation of the implant polymorphism after the extrusion process as well as during storage showed good stability. To combine the advantage of particulate delivery and sustained release, preformed liposomes were incorporated into the implants prior to extrusion. For the analysis of the immune response, two sets of animal experiments in mice were performed, one evaluating the kinetics of the release of the model antigen in vivo, a second one to evaluate the immune response in vivo. Evaluation of these data indicated a correlation between the in vitro and in vivo release behaviour of OVA. Furthermore, immune responses similar to those induced by two booster injections, consisting of OVA and alum could be achieved using implant formulations containing QA. These results further emphasized the importance of adjuvant in the formulation. The incorporation of preformed liposomes into the implants on the other hand did not lead to an improved outcome. In a second part of this work, an in vivo tumour study was prepared, using the TRP2 peptide as active ingredient. Due to the use of this expensive peptide, a transfer to a different extruder was necessary. The influence that a change of the production device has on the implants characteristics was investigated. Once the formulation was adapted to the new extruder, implants containing TRP2 and QA were produced. The in vitro release of TRP2 proved to be very slow, much different from the OVA release. Furthermore, the preparation of vesicular phospholipid gels (VPGs) as an alternative lipid delivery system for TRP2 was investigated. The TRP2 release from the VPGs was also slow and incomplete. Both formulations were used in an in vivo tumour growth study. Mice were injected with B16F10luc2 melanoma cells, 6 days later formulations were administered. VPGs showed adverse reactions in the mouse and are therefore not s suitable delivery system. TRP2 implants showed a slow delay in the start of tumour growth, but were not more potent that TRP2 in PBS injections given to the mice. The very slow in vitro release data of TRP2 brought up the question about interactions between the lipid implants and the peptide influencing the release. Choosing peptides of different size and hydropathy, an investigation of their release behaviour and interaction with the implants was conducted. In conclusion, lipid implants were well tolerated and offer a great potential as sustained release delivery system for vaccines. They allow releasing the active component and the adjuvant together, enabling to achieve a strong immune response.