Liposomale Formulierungen von Hypericin zur Anwendung in der photodynamischen Therapie

The purpose of this doctoral thesis was the development of liposomal hypericin formulations and the investigation of their photodynamic activity against bacteria and tumour cells. Since hypericin is very lipophilic and forms aggregates in aqueous solutions, clinical application is a challenge and requires the development of an aqueous hypericin formulation. For this purpose, two different strategies to encapsulate hypericin into liposomes viz. hypericin incorporated within the liposomal membrane prepared by thin film hydration method and its cyclodextrine complex encapsulated inside the aqueous milieu of the liposome prepared by dehydration-rehydration method were compared. Chapter 1 of the thesis introduces the reader to the background and state of art in PDT and liposomal technology. This is followed by Chapter 2 which deals with the summary and explanation of the methods employed. Chapter 3 involves the characterisation of the liposomes. Hydrodynamic diameter, PDI, zeta potential, morphology, encapsulation efficiency and the stability during storage or in the presence of serum were determined with photon correlation spectroscopy (PCS), laser doppler anemometry (LDA), atomic force microscopy (AFM) and size exclusion chromatography (SEC) respectively. All formulations characterised showed a hydrodynamic diameter ranging between 127 and 212 nm and a PDI between 0.21 and 0.32. The zeta potential was especially low in case of dehydration-rehydration vesicles (DRV). This leads to the hypothesis that the Hyp-HPβCD complex also adheres on the liposomal surface. The AFM micrographs confirmed the typical morphology of liposomes showing spherically shaped vesicles. Liposomes composed of DPPC/TEL encapsulated more hypericin than their DSPC counterparts. The stability studies showed that the formulations were stable in cell culture medium or 60% FCS. The photodynamic activity of hypericin liposomes on Staphylococcus saprophyticus subsp. bovis and E. coli DH5α is summarised in Chapter 4 of the thesis. Additionally, CLSM micrographs and binding assay served as qualitative and quantitative analysis of hypericin delivery in gram positive bacteria. E. coli DH5α was not susceptible to hypericin, Hyp-HPβCD or hypericin liposome mediated photodynamic antimicrobial chemotherapy (PACT). Concerning the PACT of gram-positive bacteria, the DRV vesicles were more effective than conventional liposomes prepared using the thin-film hydration method. The strongest effect could be seen using DSPC/Hyp-HPβCD liposomes, which led to a bacterial reduction of 4.1 log10¬. In order to build a bridge between in vitro and in vivo, the best formulation was tested in the chick embryo model. Interestingly, the results, which are presented in Chapter 5, were quite opposite to the in vitro studies. The DSPC/Hyp-HPβCD liposomes led to a decrease of the bacterial load of 1.2 log10 and thus showed a stronger antibacterial effect than the complex. Chapter 6 evaluates the photodynamic activity of hypericin liposomes on biofilms of Staphylococcus saprophyticus subsp. bovis. Additionally, Hyp-HPβCD and/or DSPC/Hyp HPβCD modified implant material was examined in regard to its antibacterial efficacy. The surface coating containing only Hyp-HPβCD led to a reduction of 4.3 log10 of biofilm bacteria. This effect could be increased to a 6.8 log10 reduction by applying ultrasound. Chapter 7 investigates the antitumor characteristics of hypericin liposomes. Therefore, DRV liposomes consisting either of DPPC/TEL or DSPC were compared to vesicles of the same composition prepared using the thin film hydration method. The photodynamic in vitro activity on SK OV-3 cells was concentration dependent in case of all formulations. At a low irradiation fluence of 2.1 J/cm², the conventional liposomes were more phototoxic than the DRV liposomes. By increasing the irradiation fluence up to 12.4 J/cm², the phototoxicity of all formulations approached a similar level. Moreover, the photodynamic activity of the liposomes was dependent on the incubation time with an ideal incubation period of 4 h. Qualitative evidence for the uptake of liposomes into SK-OV-3 could be acquired from the CLSM micrographs. Endocytosis pathway studies led to the assumption that the DRV liposomes were mainly taken up by clathrin mediated endocytosis. Furthermore, the haemolysis assay and the aPTT time indicate that the liposomes were haemocompatible and thus suitable for intravenous injection. The photodynamic effect of the formulations on the microvasculature of the chorioallantoic membrane (CAM) is shown in Chapter 8. DRV liposomes exhibited only a moderate to no antivascular effect, while the conventional liposomes caused a substantial photodestruction of the microvasculature. These results indicate that the conventional liposomes are suitable for antivascular targeting by delivering hypericin to the endothelial cells. The DRV vesicles on the contrary seem to yield the Photosensitiser from light by a very stable encapsulation. Thus we assume that these vesicles can be further developed for direct tumour targeting.