**3. Drug delivery systems loaded with itraconazole**

Itraconazole (**Figure 3**) is an antifungal drug from the azole group, widely used in the treatment of aspergillosis, cryptococcosis, candidiasis, blastomycosis, and mild histoplasmosis. Its action mechanism encompasses the disruption of ergosterol synthesis to avoid the formation of the fungal cell membrane [37, 38].

### **3.1 Polymeric nanoparticles of itraconazole**

Polymeric nanoparticles are capable of not only safely carrying drugs for specific target organs but also of effectively permeating cellular membranes [39].

A striking example of a novel nano-based delivery system is the encapsulation of itraconazole in nanosphere polymeric nanoparticles (NP) based on poly-(lactic-coglycolic acid) (PLGA) and functionalized with F4/80 antibodies and mannose. The optimized NP was made up of PLGA 75:25 and a mix of surfactants (Kolliphor P188 and vitamin E-TPGS) at pH 5. It showed optimal drug-loading capacity (6.6%), high encapsulation efficiency (80%), and fitted well with the Fickian diffusion model [23]. A schematic representation of this system is outlined in **Figure 4**.

This study has demonstrated increased J774 macrophage uptake *in vitro* and more efficacy in eliminating the *H. capsulatum* on murine macrophages compared with bare NP. Moreover, these NP did not affect the viability in macrophages

#### **Figure 3.**

*Chemical structure of itraconazole (C35H38CI2N8O4). Drawn using ChemDraw Professional 22.0 from PerkinElmer Informatics, Inc.*

**Figure 4.**

*Schematic representation of the functionalized PLGA NPs loaded with itraconazole and their interaction with F4/80 antibodies receptor in macrophages. Created with BioRender.com.*

at different concentrations, which proves they are not cytotoxic. A successful antibody-NP surface binding was achieved while keeping its stability and avoiding aggregation. The 200 nm size is adequate to prevent rapid elimination by the endothelial reticulum system. *H. capsulatum* induces IFN-γ expression in the macrophages and the functionalized NPs developed were able to reduce the expression of this cytokine, emphasizing the role of this antibody as a binding molecule with immunomodulatory properties. In addition, the treatment with these nanoformulations also significantly reduced IL-6 and IL-10 expression compared to free itraconazole. All in all, this research supports the idea that the encapsulation of itraconazole into NP allows a controlled and targeted drug release into macrophages, along with enhanced efficacy and efficiency in battling the fungal intracellular infection [8, 23, 25, 40].

In line with this research was the report of a new polymeric drug delivery system for targeted brain delivery. The authors achieved a stable linkage between RVG29 peptide (a brain-targeting ligand) and itraconazole-loaded albumin nanoparticles by means of the biotin-binding crosslinker streptavidin. This conjugation simplified the intracellular delivery of NPs and enhanced drug distribution in mice brain [41]. Albeit the non-specificity to *H. capsulatum* of either *in vitro* or *in vivo* conducted studies*,* there is undeniable potential to be exploited herein. In fact, CNS histoplasmosis, despite being rare, is of difficult diagnosis and quickly escalates to disseminated infection with a lower chance of recovery [12].
