Effect of ethanolic extract propolis-loaded niosomes on phospholipase and biofilm productions of cryptococcus neoformans

Abstract

Pulmonary cryptococcosis is initiated by inhalation of the small infective particles of Cryptococcus neoformans from environmental sources. The respiratory defense mechanism does not prevent these propagules reaching the distal lung. C. neoformansproduces several virulence factors, most notably the polysaccharide capsule, melanin and urease. Among these virulence factors of C. neoformans, secretory phospholipase and biofilms are also the potential role in fungal pathogenesis. Phospholipase B1 (PLB1) facilitates adherence to lung epithelium and destabilization of cell membranes, while biofilm production protects themselves from host’s immune cells and anti-mycotic agents. For these reasons, causing significant the dissemination of C. neoformans in multiple organ systems and invasion of the blood-brain barrier (BBB) leading to increase morbidity and mortality. Despite the treatment of systemic mycosis, the high toxicity, reduced spectrum of activity and resistance have still emergence. Therefore, targeting virulence factors associated with PLB1 and biofilm of cryptococci is an attractive therapeutic approach for pulmonary cryptococcosis. This study aimed to formulate the ethanolic extract propolis-loaded niosomes (Nio-EEP) using thin film hydration method and evaluate the biological activities on PLB1 production and biofilm formation of C. neoformans. The physicochemical characterization of niosomal vesicles had a mean diameter of approximately 270 nm of spherical shape, zeta-potential of -10.54±1.37 mV, 88.13±0.01 entrapment efficiency. Nio-EEP can release the EEP in a sustained release manner and has been consistent physicochemical properties of Nio-EEP for a mont 4C. Nio-EEP exhibited less cytotoxicity on A549 and NR8383 cells. Cellular uptake of Nio-EEP by C. neoformans was also observed. Its biological activity was further evaluated on some cryptococcal virulence factors. The results demonstrated no difference in phospholipase activity by Egg Yolk Agar (EYA) assay between Nio-EEP treated C. neoformans and control group. However, a significant decrease in the mRNA expression level of PLB1 was also determined. Interestingly, biofilm formation and expression level of biofilm-related genes (UGD1 and UXS1) were significantly reduced. The pre-treatment with Nio-EEP prior to yeast infection reduced intracellular replication of C. neoformans in NR8383 macrophages for 47% by cultivation. In conclusion, Nio-EEP not only mediates as an anti-virulence agent to inhibit the production of PLB1 and biofilm for the preventing fungal colonization of the lung epithelium cells, but also enhance killing of phagocytosed cryptococci. Anti-virulence agents might be potential therapeutic strategies in prophylaxis and treatment of pulmonary cryptococcosis in the future.