Hypericin inclusion complexes encapsulated in liposomes for antimicrobial photodynamic therapy.

The naturally occurring anthraquinone derivative hypericin is a highly potent photosensitiser. Several in vitro studies show high phototoxicity of the pigment towards gram-positive bacteria. Nevertheless, the highly lipophilic nature and poor bioavailability prevent its application in daily clinical practice thus leading to a limited therapeutic value of hypericin. Liposomal encapsulation could help overcome these limitations and would make hypericin available for daily clinical practice. The use of liposomes as carriers for hypericin in antimicrobial photodynamic therapy (aPDT) is quite new. The aim of this work was to improve the photodynamic efficiency of the previously mentioned carriers by entrapping hypericin in the aqueous compartment of the liposomes. Therefore, a water-soluble inclusion complex of hypericin and (2-hydroxypropyl)-beta-cyclodextrin (Hyp-HPßCD) was prepared. After encapsulation of the inclusion complex into DSPC and DSPC/DPPC/DSPE-PEG liposomes with the dehydration-rehydration vesicle (DRV) method, the formulations were physicochemical characterised. The photodynamic efficiency towards the gram-positive model strain Staphylococcus saprophyticus subsp. bovis. was tested on planktonic cells as well as on biofilms. DSPC liposomes achieved a 4.1log reduction and the DSPC/DPPC/DSPE-PEG liposomes a 2.6log reduction in growth of planktonic bacteria, while Hyp-HPßCD showed total eradication. Even bacterial cells growing in a biofilm could be treated effectively in vitro.

Synergistic effects of ultrasound and photodynamic therapy leading to biofilm eradication on polyurethane catheter surfaces modified with hypericin nanoformulations.

Catheter related infections are causing one third of all blood stream infections. The mortality of those infections is very high and the gold standard for catheter related blood stream infections (CR-BSI) is still the removal of the catheter and systemic antibiotic therapy. There already exist some approaches to prevent the biofilm formation on catheter material, which are far from ideal. A new strategy to prevent bacterial colonization on catheter surfaces is the application of photodynamic therapy (PDT). Therefor the surface has to be modified with substances that can be activated by light, leading to the production of cell toxic reactive oxygen species (ROS). Only small concentrations of the so called photosensitizer (PS) are necessary, avoiding side effects in human therapy. Furthermore, there is no resistance development in PDT. In this study polyurethane (PUR) surfaces were coated with hypericin nanoformulations, leading to 4.3 log10 reduction in bacterial growth in vitro. The effect could be enhanced by the application of ultrasound. The combination of PDT with ultrasound therapy led to a synergistic effect resulting in a 6.8 log10 reduction of viable counts. This minimal invasive method requires only an optical fibre inserted in the catheter lumen and an ultrasound device. Thus the implementation in daily clinical practice is very simple.

Photodynamic therapy - hypericin tetraether liposome conjugates and their antitumor and antiangiogenic activity.

Photodynamic therapy (PDT) is an established noninvasive tumor treatment. The hydrophobic natural occurring pigment hypericin shows a lot of attractive properties for the application in PDT. Hence, the administration to biological systems or patients requires the formulation in drug carriers enabling sufficient bioavailability. Therefore, free hypericin was encapsulated by the thin film hydration method or a hypericin-hydroxypropyl-ß-cyclodextrin inclusion complex (Hyp-HPßCD) was incorporated by dehydration-rehydration vesicle method in either conventional or ultra-stable tetraether lipid (TEL) liposomes. The hydrodynamic diameter of the prepared nanoformulations ranged between 127 and 212 nm. These results were confirmed by atomic force microscopy. All liposomes showed a good stability under physiological conditions. TEL liposomes which tend to build more rigid bilayers, generate higher encapsulation efficiencies than their conventional counterparts. Furthermore, the suitability for intravenous application was confirmed by hemocompatibility studies resulting in a hemolytic potential less than 20% and a coagulation time less than 50 sec. The uptake of liposomal hypericin into human ovarian carcinoma cells (SK-OV-3) was confirmed using confocal microscopy and further characterized by pathway studies. It was demonstrated that the lipid composition and intraliposomal hypericin localization influenced the anti-vascular effect in the chorioallantoic membrane (CAM). While hypericin TEL liposomes exhibit substantial destruction of the microvasculature drug-in-cyclodextrin TEL liposomes showed no effect. Nevertheless, both formulations yielded severe photocytotoxicity in SK-OV-3 cells in a therapeutic dosage range. Conclusively, hypericin TEL liposomes would be perfectly suited for anti-vascular targeting while Hyp-HPßCD TEL liposomes could deliver the photosensitizer to the tumor site in a more protected manner.