Abstract

In this work, combined interdisciplinary research in the fields of Pharmaceutical Technology, (Bio)Materials Science, and Biology is presented to understand and promote lyotropic liquid crystalline dispersions as novel vaccine carriers. The work was initiated with a detailed development of lyotropic mesophases based on phytantriol. The aim was to gain knowledge on the phase behavior of the system and also on the structural impact of additives. Therefore, formulations with increasing degree of complexity were sequentially characterized. The study started establishing a phase diagram, in which bulk phases of phytantriol in excess of highly purified water were characterized, upon temperature increase, applying polarization microscopy and small angle X-ray scattering (SAXS). Subsequently, pursuing the development of a system with immunostimulatory properties, the impact of mannide monooleate on phytantriol-based mesophases was investigated. Mannide monooleate is an emulsifier widely used in adjuvant formulations and found to promote immune responses in emulsion form. Thus, progressively higher amounts of mannide monooleate were incorporated into the system and the structural modifications were monitored upon temperature increase. The phase diagram allowed for a deep understanding of the mesophase behavior and enabled the selection of a feasible phytantriol/mannide monooleate ratio for the subsequent investigations. In this way, a stable formulation, featuring inverse hexagonal structure was identified. Next, to enable dispersion of the characterized bulk and to produce a nanoparticulate system, different steric stabilizers were evaluated. Here, the selection criteria were based on the dispersion efficiency at low concentrations and on the impact on the internal structure of the particles. The last part of the formulation development was concerning the application and customization of the formed hexosomes. Thus, the particle loading with the model antigens ovalbumin and lysozyme was studied, as well as strategies to introduce new features to the particulate system in a controlled way, preserving the inverse hexagonal structure. Here, negatively and positively charged hexosomes could be prepared through the incorporation of charged phospholipids into the formulation. Additionally, a formulation based on phytantriol and mannide monooleate, which typically formed an inverse hexagonal structure was shown to self-assemble with bicontinuous cubic double diamond structure upon addition of octyl-β-D-glucopyranoside. The aim of the second part of this work was the verification of one of the most important hypothesis with respect to the biological performance of non-lamellar lyotropic liquid crystalline dispersions. More specifically, it is known that non-lamellar structures are involved in fusogenic processes of the cell membrane. For this reason, it was speculated that particles internally structured with non-lamellar phases, such as hexosomes, could have fusogenic properties. Thus, to verify this hypothesis and elucidate the cellular uptake mechanism of hexosomes, we investigated the interactions of these particles with cells and with models of the cell membrane. For this purpose, the established hexosomes, based on phytantriol/mannide monooleate, were fluorescently labeled and their toxicity in cultures of HeLa cells was determined. Series of uptake experiments were carried out after suppression of the major endocytic pathways using highly specific approaches (e.g. single and double knockdown of regulatory proteins), and also inhibition strategies of broader effect (e.g. temperature reduction to 4°C, hypotonic treatment, cytochalasin D treatment). Additionally, experiments using models of the cell membrane (e.g. phospholipid monolayers and bilayers) were performed. All the analyses were carried out with hexosomes and liposomes in parallel to enable the comparison between particles featuring non-lamellar and lamellar structures, respectively. The results revealed a steeper toxicity curve and faster internalization kinetics for hexosomes in comparison to liposomes. Interestingly, against the expectations, indications of fusogenic properties were not observed. However, strong evidences that hexosomes have an alternative cell entry pathway that bypasses standard endocytosis were identified. In contrast, liposomes appeared to enter the cells through well-known endocytic pathways, such as caveolae-mediated and clathrin- mediated endocytosis. The final part of this work consisted of the evaluation of the biological performance of hexosomes within the vaccination context. The experiments were performed in vitro and in vivo, in parallel with the already established and promising cationic liposomal formulation CAF04, which is based on dimethyldioctadecylammonium (DDA) and on a synthetic analogue of monomycoloyl glycerol (MMG-1). Since MMG-1 and the positive charge of DDA are considered the core of the success of this formulation, differently charged hexosomes containing MMG-1 were developed and included in the study. The development process was driven by the lessons learned in the first phase of the project with phytantriol/mannide monooleate systems. In this way, to create particles internally structured with inverse hexagonal phase, MMG-1 was combined to phytantriol. After the establishment of the formulations, the immunogenicity of plain particles (without antigen) was investigated in vitro in cultures of dendritic cells derived from human peripheral blood mononuclear cells. Through the evaluation of the expression of several surface markers, it was found that, while CAF04 liposomes induced the upregulation of the homing receptor CCR7, all hexosomal formulations tested did not provoke any sign of adjuvanticity. Considering that the cultures of dendritic cells represent a dramatic simplification of the immune system, further experiments were performed in vivo. Thus, mice were immunized with the formulations loaded with Chlamydia trachomatis major outer membrane protein (MOMP) antigen. The immunological output was analyzed through several parameters (e.g. T-cell activation, cytokine expression and release, polyfunctionality, specific serum IgG) and all of them revealed dramatic differences between the immunizations performed with the unadjuvanted antigen and antigen adjuvanted with either liposomes or hexosomes. In comparison to the unadjuvanted systems, liposomes and hexosomes were both able to increase the magnitude of the immune responses. However, the immune responses elicited by CAF04 liposomes and by hexosomes were very distinct, the first eliciting mainly cellular mediated responses and the second humoral responses. Throughout this work, it was shown that stable and robust lipid-based lyotropic liquid formulations, that allow customization without impairment of the internal structure, can be rationally designed. In addition, keeping track of the structural modifications induced by each additive (separately) was shown to be a key strategy to enable further optimization in advanced stages of the formulation development. Regarding the performance of hexosomes in the biological environment, we have obtained accurate results demonstrating, in vitro and in vivo, drastic differences in comparison to liposomes. This intriguing outcome was observed for different formulations and in completely independent experiments. Overall, the insights presented in this thesis show hexosomes as promising delivery systems with uncommon properties and an applicability potential that goes beyond the vaccination context.