Nanocontainer targeting macrophages as antimicrobial therapy

Invasive fungal infections are responsible for more than 1.5 million deaths every year. Almost half of these cases are due to chronic or invasive mold infections of immunocompromised patients, predominantly caused by Aspergillus fumigatus. The ubiquitous conidia of this fungus are inhaled by humans and face alveolar macrophages belonging to the first line of immune defense. After uptake of conidia by these immune cells, the conidia-containing cellular compartment (phagosome) fuses with degrading cell organelles (lysosomes) to form mature phagolysosomes. Like several other pathogens , A. fumigatus is able to avoid phagolysosomal killing, but the exact mechanism behind this remains obscure. The treatment of A. fumigatus infections is still problematic, especially due to the development of resistances against available antifungal drugs. On the other hand, newly discovered antifungal drugs are often highly toxic, display limited bioavailability and/or limited efficacy against certain species. Therefore, there is a strong need for the development of new antifungal applications and a better understanding of the immune evasion techniques of A. fumigatus. The dihydroxynaphthalene(DHN)-melanin layer of the conidia was shown to scavenge reactive oxygen species (ROS) and being an important virulence factor of A. fumigatus. ROS are important for the immune defense, as patients displaying defects in the ROS producing machinery are highly susceptible for infections. However, a direct role of ROS in killing the conidia of this fungus within macrophages became unlikely. Therefore, one part of this project deals with the investigation on the role of ROS as signaling molecules for phagosome maturation. Macrophages defective in the ROS machinery will be generated and intracellular processing of conidia with and without the DHN-melanin layer will be analyzed by sophisticated microscopical techniques. The importance of ROS for phagosome maturation will be followed by analyzing distinct parameters like formation of lipid-raft microdomains. Another part of this project deals with the development of new formulations for antifungal drugs for targeted therapy. The new compound jagaricin, displaying high antifungal activity but also high toxicity, will be incorporated in nanocontainers of different sizes and surface properties, to allow targeted therapy with this antifungal drug to macrophages and thereby decrease the toxicity. Finally, also the molecular mechanism allowing the uptake of nanoparticles by fungi will be elucidated. The findings of this project will expand the knowledge on phagosome maturation, the immune evasion of A. fumigatus and enable new application strategies of pharmacological problematic antifungal drugs, which is much needed for the development of new antifungal compounds. The strategy developed here will be most likely also applicable for the treatment of other pathogens surviving phagolysosomal killing.

Every year, around 1.5 million people die due to invasive fungal disease caused, for example, by the opportunistic mold pathogen Aspergillus fumigatus. Everybody inhales hundreds of spores of this mold every day, which can be easily taken up and degraded in macrophages of the lung of healthy individuals. By contrast, in patients with a suppressed immune system these spores can survive in specific cell organelles, called phagosomes, and thereby cause life threatening infections. Furthermore, there is only a limited number of antifungal drugs available to treat invasive fungal infections and the situation gets even worse due to increasing resistances against approved drugs in fungal pathogens. Therefore, a better understanding of the immune evasion mechanisms of A. fumigatus as well as the development of novel antifungal therapeutics is urgently needed. The aim of this project was the investigation on the role of reactive oxygen species (ROS) on the maturation of spore-containing phagosomes as well as the use of polymeric nanoparticles to treat A. fumigatus infections. The investigation on the role of ROS gained interesting insights into the persistence of A. fumigatus conidia in macrophages and the inhibition of the fusion of phagosomes with degradative cell organelles. Nevertheless, this aim needs further investigations on the exact mechanism and can therefore not be concluded so far. The investigations regarding nanoparticles showed that they can be used to reach spores persisting within macrophages, which is one disadvantage of conventional therapy. In addition, it could be proven that nanoparticles can be used for the efficient delivery of antifungal compounds into pathogenic fungi and that this delivery is much more efficient than the uptake of the pristine drug. The pharmacological properties like solubility or toxicity of these antifungal nanoparticles are dominated by the used polymer and not by the antifungal drug itself, which potentially allows the use of novel substances for treatment, which display pharmacological issues like high toxicity as pristine substance.