Can polymeric particles potentially deliver antifungals inside phago(lyso)somes containing intracellular persistent conidia of Aspergillus fumigatus?

Author:

Julien Alex, Katherine González, Gauri Gangapurwala, Antje Vollrath, Zoltán Cseresnyés, Christine Weber, Justyna A. Czaplewska, Stephanie Hoeppener, Carl-Magnus Svensson, Thomas Orasch, Thorsten Heinekamp, Carlos Guerrero-Sánchez, Marc Thilo Figge, Ulrich S. Schubert, Axel A. Brakhage

Abstract:

Objectives: In immunocompromised patients, conidia of Aspergillus fumigatus can avoid their elimination inside of alveolar macrophages by interfering with the maturation of phagolysosomes. Currently used antifungals in clinics often prove to be inefficient to treat intracellular persisting pathogens because the drugs have to cross two membranes, i.e., the cytoplasmic and the phagosomal membrane. The utilization of drug delivery systems bears the potential to alter the internalization route of antifungals and thus, reach intracellular persistent microorganisms inside phagosomes or phagolysosomes more efficiently compared to the pristine drug. The aim of this study was to elucidated whether polymeric particles (PP) can target intracellular persistent pathogens.

Methods: We formulated dye-labeled PPs via single emulsion technique to generate PP sizes large enough for their phagocytosis by macrophages. The covalent conjugation of dye molecules to the polymer was confirmed by size exclusion chromatography. PPs were characterized by means of dynamic light scattering and scanning electron microscopy. The uptake of PPs into RAW 264.7 macrophages was analyzed by imaging flow cytometry. Studies with regards to the intracellular localization of PPs was executed via immunofluorescent staining of Rab7 and Lamp1 (confocal laser scanning microscopy) and transmission electron microscopy.

Results: RAW 264.7 macrophages internalized PPs efficiently. Co-localization of PPs and conidia in the same phagolysosome was observed after PPs were added to prior infected macrophages. The fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was confirmed. The number of phagolysosomes containing both conidia and PPs was increased at elevated PP concentrations or after utilization of the fusion enhancer Vacuolin-1.

Conclusion: Our study suggests the fusion of conidia- and PP- containing phagolysosomes as the underlaying mechanism for PPs reaching intracellular A. fumigatus. Methods for increasing fusion events were established in order to exploit the investigated delivery route of PPs. These results represent the requisite for the development of advanced delivery systems reaching intracellular persistent pathogens.

Abstract Number: 84

Conference Year: 2024

Conference abstracts, posters & presentations

Showing 10 posts of 17325 posts found.

  • Title

    Author

    Year

    Number

    Poster