Dermatophytosis is a fungal infection that affects the skin, hair, and nails, impacting approximately 25% of the global population. Nannizzia gypsea is a geophilic fungus that can cause infections in humans and animals. Several studies have been conducted regarding its virulence, or ability to cause disease. This species may produce keratinolytic enzymes and form biofilms, which can increase resistance to treatment. Thus, this study focuses on investigating the biofilm formation of N. gypsea isolated from canine dermatophytosis using an ex vivo hair model, its biofilm extracellular matrix macromolecular contents, and the expression of genes involved in the colonization of keratinized surfaces. The biofilm was analyzed for metabolic activity using the XTT reduction assay, crystal violet staining to measure biofilm biomass, scanning electron microscopy (SEM), and the presence of polysaccharides, proteins, and extracellular DNA in the biofilm extracellular matrix. The virulence genes subtilisin 7, fungalysin (extracellular metalloproteinase), and efflux pump (Multidrug and Toxin Extrusion Protein 2) were evaluated by qPCR, comparing the planktonic and biofilm phenotypes. N. gypsea formed a robust biofilm, which matured after 5 days. Scanning electron microscopy (SEM) revealed the presence of an extensive extracellular matrix. In the hair model, the characteristic ectothrix parasitism of the species is observable. The gene expression analysis revealed a higher expression of all evaluated genes in the biofilm form compared to the planktonic form. Thus, N. gypsea exhibits a biofilm characterized by a robust extracellular matrix and high gene expression of factors related to pathogenesis and resistance.

Background/Objectives: Fungal infections caused by Candida species remain a significant clinical challenge, exacerbated by limitations in current antifungal therapies, including toxicity and poor bioavailability. This study aimed to develop and evaluate voriconazole-loaded zein–pectin–hyaluronic acid nanoparticles (ZPHA-VRC NPs) as a novel drug delivery system to enhance efficacy and reduce toxicity. Alternative in vitro and in vivo models were utilized to assess the safety and therapeutic potential of the nanoparticles. Methods: ZPHA-VRC NPs were prepared using a nanoprecipitation method and characterized for particle size, polydispersity index, zeta potential, and encapsulation efficiency. Antifungal activity was assessed via MIC assays against Candida albicans, C. krusei, and C. parapsilosis. Cytotoxicity was evaluated on Vero cells, while in vivo toxicity and efficacy were assessed using Galleria mellonella and Caenorhabditis elegans models. The therapeutic efficacy was further evaluated in an infected Caenorhabditis elegans model using survival and health scores. Results: ZPHA-VRC nanoparticles exhibited favorable physicochemical properties, including a particle size of approximately 192 nm, a polydispersity index of 0.079, a zeta potential of −24 mV, and an encapsulation efficiency of 34%. The nanoparticles retained antifungal activity comparable to free voriconazole while significantly reducing cytotoxicity. In vivo studies using G. mellonella and C. elegans demonstrated that ZPHA-VRC NPs markedly improved survival rates, reduced fungal burden, and enhanced health scores in infected models, outperforming the free drug. Additionally, the nanoparticles exhibited a superior safety profile, minimizing systemic toxicity while maintaining therapeutic efficacy. Conclusions: ZPHA-VRC NPs offer a safer and more effective delivery system for VRC, addressing the limitations of conventional formulations. The integration of alternative efficacy and safety models highlights their value in preclinical research.

BACKGROUND: Hypoxia-inducible factor 1a (HIF-1alpha) induces transcription of glycolytic genes, including carbonic anhydrase IX (CA IX) and glucose transporter-1 (GLUT-1), and angiogenic proteins like vascular endothelial growth factor (VEGF). HIF-1alpha expression is increased in human glioblastoma multiforme (GBM) compared to low-grade tumors. We have previously shown that HIF-1alpha inhibition decreases tumor growth in a flank glioma model. This study examines methods of anti-HIF-1 siRNA delivery utilizing nanoparticle systems in an intracranial model and the effects on tumor angiogenesis and growth. METHODS: We use siRNA directed against HIF-1alpha in an intracranial luciferase reporter glioma model with the ability to measure HIF-1 activity and tumor growth in vivo. These molecules are delivered with a variety of nanoparticle delivery molecules we call multifunctional carrier (MFC) and chemically modified Accell siRNAs. Tumor growth was monitored by luciferase activity over time and compared between various nanoparticle carrier molecules. We also examined local osmotic pump delivery and systemic therapy. At the end of treatment, tumors were removed and examined for expression of downstream-regulated molecules well as measures of angiogenesis and cellular proliferation. RESULTS: We demonstrated reduced tumor growth and HIF-1 activity in tumors treated with anti-HIF-1alpha siRNA delivered by our MFC nanoparticles compared with other carriers and negative control siRNA injections. No neurotoxicities were found. We also found reductions of HIF-1alpha, VEGF, and GLUT-1 expression, as well as decreased MIB-1 index and decreased vascular density. Difficulties and future strategies of local delivery with osmotic pumps and systemic delivery using targeting moieties are discussed. CONCLUSIONS: Our preliminary results suggest that intracranially injected siRNA against HIF-1alpha complexed to a newly developed carrier might be used for treatment of malignant brain tumors. This treatment strategy needs to be further developed with more specific tumor targeting and possibly with systemic delivery strategies.

Paracoccidioidomycosis (PCM) is a chronic endemic mycosis in Latin America, predominantly caused by Paracoccidioides brasiliensis (Pb18) and Paracoccidioides lutzii (Pl01). Diagnosing PCM is challenging due to species-specific antigenic differences, therefore new biomarkers for accurate and rapid detection are needed. This study explores multiple tolerization subtractive immunization (MTSI) to generate monoclonal antibodies against rare or weakly expressed epitopes of Pb18 and Pl01, potentially improving PCM diagnosis. These strains were cultured to obtain cell-free antigens (CFA). MTSI involved immunizing BALB/c mice with CFA from Pb18 as a tolerogen and Pl01 as an immunogen, using Freund’s adjuvant and cyclophosphamide to induce immune tolerance. The immune response was monitored via Enzyme-linked immunosorbent assay (ELISA) and Western blotting. Hybridomas were generated by fusing splenocytes from immunized mice with myeloma cells, after which clonal selection was conducted based on reactivity to Pl01 antigens. The study explores the presence of various proteins, including gp43 and Hsp60, in the protein profile of CFAs. Additionally, polyclonal antibody reactivity to Pb18 antigens was significantly reduced, suggesting that MTSI effectively promoted immunological tolerance. Followig the screening of hybridomas, clones with good reactivity to Pl01 and less reactive to Pb18 were selected. The monoclonal clones C1 and E6 exhibited potential specificity for Pl01 antigens. The effective generation of P. lutzii -specific antibodies by MTSI demonstrates this technology’s promise for the development of accurate PCM diagnostic instruments. These antibodies have the potential to enhance patient outcomes and reduce the incidence of false-negative diagnoses, which could lead to better disease management.