Oral delivery of brain-targeted miltefosine-loaded alginate nanoparticles functionalized with polysorbate 80 for the treatment of cryptococcal meningitis.

OBJECTIVES: To develop alginate nanoparticles functionalized with polysorbate 80 (P80) as miltefosine carriers for brain targeting in the oral treatment of cryptococcal meningitis. METHODS: Miltefosine-loaded alginate nanoparticles functionalized or not with P80 were produced by an emulsification/external gelation method and the physicochemical characteristics were determined. The haemolytic activity and cytotoxic and antifungal effects of nanoparticles were assessed in an in vitro model of the blood-brain barrier (BBB). A murine model of disseminated cryptococcosis was used for testing the efficacy of oral treatment with the nanoparticles. In addition, serum biomarkers were measured for toxicity evaluation and the nanoparticle biodistribution was analysed. RESULTS: P80-functionalized nanoparticles had a mean size of ∼300 nm, a polydispersity index of ∼0.4 and zeta potential around -50 mV, and they promoted a sustained drug release. Both nanoparticles were effective in decreasing the infection process across the BBB model and reduced drug cytotoxicity and haemolysis. In in vivo cryptococcosis, the oral treatment with two doses of P80 nanoparticles reduced the fungal burden in the brain and lungs, while the non-functionalized nanoparticles reduced fungal amount only in the lungs, and the free miltefosine was not effective. In addition, the P80-functionalization improved the nanoparticle distribution in several organs, especially in the brain. Finally, treatment with nanoparticles did not cause any toxicity in animals. CONCLUSIONS: These results support the potential use of P80-functionalized alginate nanoparticles as miltefosine carriers for non-toxic and effective alternative oral treatment, enabling BBB translocation and reduction of fungal infection in the brain.

Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris.

Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia's low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract.

Macropinocytosis as a potential mechanism driving neurotropism of Cryptococcus neoformans.

Cryptococcus neoformans can invade the central nervous system by crossing the blood-brain barrier via a transcellular mechanism that relies on multiple host factors. In this narrative, we review the evidence that a direct interplay between C. neoformans and brain endothelial cells forms the basis for invasion and transmigration across the brain endothelium. Adherence and internalization of C. neoformans is dependent on transmembrane proteins, including a hyaluronic acid receptor and an ephrin receptor tyrosine kinase. We consider the role of EphA2 in facilitating the invasion of the central nervous system by C. neoformans and highlight experimental evidence supporting macropinocytosis as a potential mechanism of internalization and transcytosis. How macropinocytosis might be conclusively demonstrated in the context of C. neoformans is also discussed.