Use of selenium nanoparticles (SNPs) stabilized with polyvinyl alcohol (PVA) and chitosan with antifungal action against Cryptococcus neoformans.

Description

1 / 24 Use of selenium nanoparticles (SeNPs) stabilized with polyvinyl alcohol (PVA) and chitosan with antifungal action against Cryptococcus neoformans. Field of invention

[001] The present invention belongs to the technical field of Biotechnology, Chemistry, Materials, Pharmacology and Medicine. More specifically, the invention relates to the development of selenium nanoparticles (SeNPs) stabilized with polyvinyl alcohol (PVA) and chitosan (QUI), intended for antifungal therapy, with the aim of offering an innovative and effective alternative to the treatment of fungal infections caused by C. neoformans.

[002] Furthermore, the invention finds application in the field of developing new drugs aimed at combating antifungal resistance and reducing adverse effects associated with conventional treatments, employing Fluconazole (FLU) and Amphotericin B (ANFB), for example. The invention also covers the technical field for the development of pharmaceutical formulations including the integration of nanomaterials in therapies targeting fungal infections. Fundamentals of the invention

[003] Fungal infections represent a group of diseases caused by fungi, ranging from superficial manifestations, such as dermatophytosis, to serious systemic infections, such as cryptococcosis and invasive candidiasis, which mainly affect immunocompromised individuals. In addition, fungal infections represent a significant cause of morbidity and mortality globally, with recent estimates pointing to approximately 3.75 million deaths annually, higher than in previous years. This shows that these infections cause a mortality rate six times higher than malaria and almost three times higher than tuberculosis. It is estimated that approximately 68% of these deaths (2.55 million) are a direct result of fungal infections, while the remainder are related to underlying diseases such as leukemia and HIV / AIDS.These numbers reveal not only the seriousness of the problem, but also the underreporting associated with these conditions, reinforcing the pressing need to develop more effective and accessible antifungal therapies capable of reducing this impact on public health (IKUTA, KS; Petition 870250071264, dated 08 / 13 / 2025, p. 6 / 29 2 / 24 MESTROVIC, T.; NAGHAVI, M. Global incidence and mortality of serious fungal diseases. The Lancet. Infectious diseases, v. 24, n. 5, p. e268, 2024).

[004] In Brazil, the situation is especially worrying, with increasing reports of the high incidence of serious fungal infections, such as invasive candidiasis and cryptococcosis, in hospitalized patients and immunocompromised populations, including those with HIV / AIDS and individuals undergoing transplants. Furthermore, these infections not only prolong hospital stays but also significantly increase healthcare costs due to the need for expensive antifungal medications and intensive supportive treatments. Additionally, mortality rates associated with these diseases frequently exceed 40%, depending on the causative agent and the patient's clinical condition, demonstrating the significant impact of these illnesses on public health and the country's economy. Moreover, fungal infections represent the third or fourth leading cause of hospital-acquired infections in Brazil (SUEHARA, MB; SILVA, MCP).Prevalence of anemophilous fungi in Brazil and its correlation with respiratory diseases and fungal infections. Ciência & Saúde Coletiva, v. 28, p. 3289-3300, 2023; TONON, PHC et al. Profile of fungal infections in the post-pandemic period in a hospital in Campos Gerais. Pesquisa, Sociedade e Desenvolvimento, v. 13, n. 9, p. e9113946929e9113946929, 2024; HAMBURGER, FG; GALES, AC; COLOMBO, AL Systematic Review of Candidemia in Brazil: Unlocking Historical Trends and Challenges in Conducting Surveys in Middle-Income Countries. Mycopathologia, v. 189, n. 4, p. 60, 2024; MAGALHÃES, VCR et al. Clinical factors associated with systemic sporotrichosis in Brazil. Mycoses, v. 67, no. 1, p. e13656, 2024; FONSECA, SNS Overview of invasive fungal infections in children in South America-the threat of resistant Candida species and the role of climate change in the new geographic distribution of endemic systemic mycosis. Current Opinion in Pediatrics, vol. 36, no. 2, p. 136-143, 2024).

[005] In October 2022, the World Health Organization (WHO) released the first list of fungi that pose a significant threat to global public health. This list classifies fungal pathogens into three priority categories: critical, high, and Petition 870250071264, dated 08 / 13 / 2025, p. 7 / 29 3 / 24 average. The fungus C. neoformans is included in the critical priority category, standing out as one of the most concerning fungi due to its impact on human health and increasing resistance to available treatments (World Health Organization. WHO. Fungal priority pathogens list to guide research, development, and public health action. 2022. Available at: (https: / / www.who.int / publications / i / item / 9789240060241. Accessed on: January 11, 2025).

[006] In general, the species C. neoformans is an encapsulated basidiomycete fungus belonging to the genus Cryptococcus, widely disseminated in the environment, especially in pigeon droppings, from where the main form of transmission to humans originates, through inhalation of its spores. This pathogen is responsible for serious fungal infections, notably cryptococcosis, a systemic disease that, when not treated properly, can progress to meningoencephalitis, characterized by inflammation of the meninges and brain. The fungus was identified at the end of the 19th century by microbiologist Charles Nicolle and, over the decades, has become a cause for concern for immunocompromised patients, being one of the main causes of fungal meningitis among this population. The etiology of cryptococcosis is closely related to the ability of C.Cryptococcus neoformans invades the central nervous system, where its polysaccharide capsule plays an essential role in protecting against the host's immune system, contributing to the progression of the infection (CASADEVALL, A.; PERFECT, JR. Cryptococcus neoformans. Washington, D.C.: ASM Press, 1998; BLACK, B. et al. Glutathione-mediated redox regulation in Cryptococcus neoformans impacts virulence. Nature Microbiology, v. 9, n. 8, p. 2084-2098, 2024; AL-HUTHAIFI, AM et al.

[007] It is estimated that infection caused by C. neoformans is responsible for approximately 112,000 deaths annually worldwide, of which about 19% are directly associated with HIV. These numbers, alarming in themselves, reflect the severity of the disease, whose mortality rate can reach 100% in the absence of adequate treatment. These data reflect not only the high burden of morbidity Petition 870250071264, dated 08 / 13 / 2025, page 8 / 29 4 / 24 of this infection, but also its intrinsic relationship with immunosuppressive diseases, such as HIV, which further aggravate the clinical picture of vulnerable patients. The infection, which can manifest as either pneumonia or meningitis, becomes even more concerning when we observe that cryptococcal meningitis is responsible for approximately 15% of deaths related to acquired immunodeficiency syndrome (AIDS) (World Health Organization. WHO fungal priority pathogens list to guide research, development and public health action. World Health Organization, 2022; IYER, KR et al. Treatment strategies for cryptococcal infection: challenges, advances and future outlook. Nature Reviews Microbiology, v. 19, n. 7, p. 454-466, 2021; FRANCIS, VI et al. Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia. Mbio, v. 15, n. 4, p. e03078-23, 2024; RATEMO, SN; DENNING, DW).Burden of fungal infections in Kenya. Mycology, vol. 14, no. 2, p. 142-154, 2023; ALHUTHAIFI, AM et al. Mechanisms and virulence factors of Cryptococcus neoformans dissemination to the central nervous system. Journal of Fungi, vol. 10, no. 8, p. 586, 2024).

[008] The global burden of fungal infections is alarming, with approximately 223,000 individuals co-infected with multiple myeloma (CM) and HIV, resulting in about 180,000 deaths annually. In the United States, the mortality rate associated with these fungal infections ranges from 15-20%. Furthermore, it is estimated that about 3 million people have destructive fungal lung disease, a condition that is frequently misdiagnosed as tuberculosis. When not treated properly, this chronic fungal lung infection can have a mortality rate of up to 80% within five years (GAFFI. Global Action Fund for Fungal Infections. Available at https: / / www.gaffi.org / . Accessed on 11 / 01 / 2025).

[009] Recent economic studies highlight the significant financial impact of fungal diseases in the United States. In 2017, it is estimated that fungal diseases cost the country's healthcare system more than US$7.2 billion. This includes US$4.5 billion from hospitalizations and US$2.6 billion from outpatient visits. In terms of outpatient visits, more than half were related to dermatophyte infections, which generated a total cost of US$1.6 billion. Petition 870250071264, dated 08 / 13 / 2025, page 9 / 29 5 / 24 These data reinforce the economic impact of fungal diseases and underscore the urgency of more effective and affordable therapeutic strategies that can alleviate clinical and financial impacts (BENEDICT, K. et al. Estimation of direct healthcare costs of fungal diseases in the United States. Clinical Infectious Diseases, v. 68, n. 11, p. 1791-1797, 2019).

[0010] In Latin America, the situation is equally worrying, with the region ranking third in terms of the absolute number of cases, with an estimated 5,300 fungal infections annually. The etiological agent C. neoformans is responsible for more than 90% of cryptococcosis cases, particularly among people living with HIV / AIDS. Brazil and Colombia have the highest incidences of cryptococcosis, with rates ranging from 1,000 to 2,500 cases annually, followed by Argentina and Mexico, which have incidences of 501 to 1,000 cases annually. In terms of lethality, cryptococcal meningitis has a mortality rate ranging from 30 to 60% in Latin America, values ​​substantially higher when compared to those of European countries, Canada and Japan, highlighting the severity of this disease in the region and the urgent need for effective and accessible therapeutic approaches (FIRACATIVE, C; LIZARAZO, J; ILLNAIT-ZARAGOZÍ, MT; CASTANEDA, E.The status of cryptococcosis in Latin America. Mem. Inst. Oswaldo Cruz, Rio de Janeiro, vol. 113, n 7, 2018; Freire, CP Clinical and cost analysis of the treatment of patients co-infected with cryptococcal meningitis and HIV in a hospital in northeastern Brazil. 2021. Dissertation (Master's) Federal University of Ceará, Faculty of Medicine, Postgraduate Program in Public Health, Fortaleza, 2021).

[0011] Conventional treatment of C. neoformans infections involves the use of antifungals that act to eradicate the infection and prevent recurrences, but most classes of antifungal agents have limitations in terms of high cost and toxicity. Most infections are preferentially treated with ANFB and FLU, which represent the standard therapeutic options for managing the infection (MENDONÇA, AMS et al. Ethyl Acetate Fraction of Punica granatum and Its GalloylHHDP-Glucose Compound, Alone or in Combination with Fluconazole, Have Antifungal and Antivirulence Properties against Candida spp. Antibiotics, v. 11, n. 2, p. 265, 2022; Petition 870250071264, dated 08 / 13 / 2025, page 10 / 29 6 / 24 AHMADY, L. et al. Antifungal drug resistance in Candida: a special emphasis on amphotericin B. APMIS, v. 132, no. 5, p. 291-316, 2024).

[0012] Amphotericin B (ANFB) is a polyene antifungal widely used in the treatment of serious fungal infections, including cryptococcal meningitis and other conditions caused by C. neoformans. Its mechanism of action is based on direct interaction with ergosterol, an essential sterol present in the fungal cell membrane. Amphotericin B has a selective affinity for ergosterol, forming complexes that organize themselves into transmembrane pores. These pores drastically increase membrane permeability, leading to the loss of intracellular ions, such as potassium and magnesium, as well as essential macromolecules. This osmotic imbalance culminates in fungal cell death, characterizing its fungicidal action. However, this interaction with sterols is not completely specific, allowing ANFB to also bind to cholesterol present in human cells. This lack of selectivity is the main cause of its systemic toxicity.Nephrotoxicity, considered the most serious adverse effect, occurs in approximately 80% of patients treated with the conventional formulation, resulting in acute kidney injury and, in severe cases, permanent renal failure. Furthermore, infusion reactions, such as fever, chills, hypotension, and bronchospasm, are frequently reported, further reducing its tolerability. Studies indicate that the total cost of treatment with ANFB can be substantially higher compared to other therapeutic options. For example, a pharmacoeconomic analysis conducted in the United States in 2015 revealed that treatment with ANFB combined with FLU had a total cost of approximately US$31,000, while the combination of liposomal ANFB and flucytosine had a cost of approximately US$75,000 (MISTRO, S. et al. Cost-effectiveness of caspofungin versus liposomal amphotericin B in the treatment of systemic fungal infections: a systematic review of economic analyses).Expert review of pharmacoeconomics & outcomes research, v. 16, n. 4, p. 465-473, 2016;. AKINOSOGLOU, K. et al. Amphotericin B in the Era of New Antifungals: Where Will It Stand? Journal of Fungi, v. 10, n. 4, p. 278, 2024; CAVASSIN, F. et al. Acute InfusionRelated Side Effects of Amphotericin B Lipid Complex (ABLC) in Oncohematological Patients: Real-World Data from Brazilian Reference Centers. Infectious Diseases and Petição 870250071264, de 13 / 08 / 2025, pág. 11 / 29 7 / 24 Therapy, p. 1-16, 2024; DASH, S. K.; BENIVAL, D.; JINDAL, A. B. Formulation Strategies to Overcome Amphotericin B Induced Toxicity. Molecular Pharmaceutics, v. 21, n. 11, p. 5392-5412, 2024).

[0013] On the other hand, FLU, an antifungal from the triazole class, is frequently used in the treatment of cryptococcosis, especially in less severe cases or for maintenance treatment after the initial phase with ANFB. The mechanism of action is based on the inhibition of the cytochrome P450-dependent enzyme lanosterol 14-α-demethylase, which plays an essential role in the biosynthesis of ergosterol, a critical structural component of the fungal cell membrane. FLU interacts directly with the heme group of the enzyme, blocking its catalytic activity. As a result, lanosterol demethylation is interrupted, leading to the accumulation of methylated sterols in the cell membrane, which compromises its integrity and functionality.Furthermore, FLU has fungistatic action, inhibiting growth but not killing yeast cells, which can lead to the development of resistance. This characteristic can also lead to persistent infections in severe cases or in immunocompromised patients. Despite being effective and having a more favorable safety profile than ANFB, FLU has limitations, such as acquired resistance in C. neoformans strains, which can compromise its effectiveness, especially in prolonged or recurrent infections. In addition, resistance to FLU is a growing concern, especially in long-term or recurrent fungal infections. Mutations in the ERG11 gene, which encodes lanosterol 14-adesmethylase, are frequently associated with resistance, leading to overexpression of the enzyme and reduced FLU efficacy. Toxicity associated with FLU has also been documented, although less frequently compared to ANFB.Adverse reactions include hepatotoxicity, gastrointestinal disturbances, and, in rare cases, acute neurological symptoms such as seizures and encephalopathy. Such adverse events may limit the use of FLU in patients with pre-existing hepatic impairment or neurological impairment (LU, H. et al. Candida albicans targets that potentially synergize with fluconazole. Critical reviews in microbiology, v. 47, n. 3, p. 323-337, 2021;). MENDONÇA, AMS et al. Ethyl Acetate Fraction of Punica granatum and Its GalloylHHDP-Glucose Compound, Alone or in Combination with Fluconazole, Have Antifungal Petition 870250071264, dated 08 / 13 / 2025, page 12 / 29 8 / 24 and Antivirulence Properties against Candida spp. Antibiotics, v. 11, n. 2, p. 265, 2022; BHASKARAN, N. A. et al. Development of cream to enhance the antifungal activity and reduce the side effects of fluconazole for the treatment of Candida albicans. Tenside Surfactants Detergents, v. 59, n. 3, p. 231-239, 2022; YANG, Yan-Li et al. Adverse effects associated with currently commonly used antifungal agents: A network metaanalysis and systematic review. Frontiers in Pharmacology, v. 12, p. 697330, 2021; EÇKUT, N. et al. Acute fluconazole toxicity: a case presenting with protean manifestations including systemic and neurologic symptoms. Postgraduate Medicine, v. 133, n. 2, p. 250-252, 2021).

[0014] According to a study by the Brazilian Association of Tropical Medicine (ABMT), the cost of treating serious fungal infections, such as cryptococcosis, can exceed R$ 10,000 per patient in Brazil, considering the costs of medication, hospitalization, and constant monitoring. ANFB, which is administered intravenously, requires specialized hospital infrastructure and monitoring of side effects, which significantly increases costs, which are even higher when drug resistance occurs. Therefore, treatment may need to be prolonged and combined with other drugs, increasing complexity and total cost. Furthermore, in low- and middle-income countries, such as Brazil, the production and acquisition of these medications represent a major financial challenge (FREIRE, CP Clinical and cost analysis of the treatment of patients co-infected with cryptococcal meningitis and HIV in a Hospital in Northeast Brazil. 2021. 65 p.Dissertation (Master's in Public Health) - Faculty of Medicine, Federal University of Ceará, Fortaleza, 2021.

[0015] Conventional treatment, although widely used, still faces challenges, such as toxicity associated with ANFB and the development of resistance to FLU. Furthermore, the duration of treatment can be prolonged, leading to high costs and increasing the burden on public health systems. These factors highlight the urgent need for new antifungal therapies with fewer adverse effects and the ability to combat resistant strains, aiming to improve clinical outcomes and reduce the impact of fungal infections globally. These costs Petition 870250071264, dated 08 / 13 / 2025, page 13 / 29 Elevated 9 / 24 rates have a substantial impact on public finances, especially considering the high burden of infectious diseases in Brazil. The need for prolonged treatments and the use of high-cost medications result in significant expenses for hospitalizations, medications, and clinical monitoring. Furthermore, antifungal resistance, although not widely documented in Brazil, can lead to therapeutic failures, increasing the need for additional interventions and prolonging the treatment period (KIM, JH; CHENG, LW; LAND, KM Advances in antifungal development: Discovery of new drugs and drug repurposing. Pharmaceuticals, v. 15, n. 7, p. 787, 2022; AKINOSOGLOU, K. et al. Amphotericin B in the Era of New Antifungals: Where Will It Stand? Journal of Fungi, v. 10, n. 4, p. 278, 2024; HOENIGL, M. et al. Novel antifungals and treatment approaches to tackle resistance and improve outcomes of invasive fungal disease. Clinical Microbiology Reviews, p.e00074-23, 2024).

[0016] The limited availability of targeted antifungal drugs (AFAs) has contributed to increased mortality and morbidity associated with fungal infections. In this context, there are fewer AFAs compared to antibiotics, and the development of these drugs has slowed since the 1990s. Available AFA classes include echinocandins, azoles, flucytosine, and polyenes, with azoles being the most widely used due to their ability to interact with non-specific enzymes present in cytochrome P450; however, they can lead to the emergence of resistance and toxic effects. Polyenes, such as amphotericin B, have serious side effects, such as nephrotoxicity. Although 5-fluorocytosine is effective, it can be hepatotoxic and lead to bone marrow suppression, as well as induce resistance when used alone.Combined therapy with 5-fluorocytosine and amphotericin B is common in severe cases, but the therapeutic results of conventional AFAs are still unsatisfactory due to toxicity, fungal resistance, and limitations in bioavailability and tissue penetration (MOHD-ASSAAD, N; MCDONALD, BA; CROLL, D. Multilocus resistance evolution to azole fungicides in fungal plant pathogen populations. Molecular ecology, v. 25, n. 24, p. 6124-6142, 2016; WALL, G.; LOPEZ-RIBOT, JL Current antimycotics, new prospects, and future approaches to antifungal therapy. Antibiotics, v. 9, n. 8, p. 445, 2020; MADKHALI, OA A comprehensive review on potential applications of metallic nanoparticles as antifungal). Petition 870250071264, dated 08 / 13 / 2025, page 14 / 29 10 / 24 therapies to combat human fungal diseases. Saudi Pharmaceutical Journal, p. 101733, 2023).

[0017] Patent BR2022 / 0018517 describes a process for the treatment of fungal infections, including those caused by C. neoformans, which involves an induction phase with the administration of amphotericin B (cAMB) and 5-Flucitosis or an azole compound via the mucosa, followed by a consolidation phase. Although the proposed process is innovative and cAMB formulations are also disclosed, the patent has limitations, such as the reliance on mucosal administration, which may be less effective or difficult to apply in certain patients, as well as the risk of adverse effects due to the combined use of multiple drugs.

[0018] In recent decades, new inventions have been developed for application against various microorganisms, including C. neoformans, as shown in Table 1. However, the technologies available for the treatment of fungal infections caused by C. neoformans and others are still limited, due to multi-step synthetic routes and low yield, toxic effects, the need for elaborate purification procedures, or activity inferior to commercial products (positive controls). Petition 870250071264, dated 08 / 13 / 2025, page 15 / 29 11 / 24 Table 1. General characterization of patented inventions relating to fungicidal action against Cryptococcus sp. Patent No. Category Derivative Main Target Specific Properties Applications Similarities BR102021003550 Synthetic organic and biological compound Biosurfactant of Bacillus subtilis, Sodium dodecyl sulfate (SDS), Triton X100 Candida albicans, Candida glabrata and Cryptococcus sp, S. aureus and Enterococcus faecalis, Klebsiella pneumoniae, Shigella sp. and Pseudomonas aeruginosa Surface tension reducing agent, interfacial tension and high emulsifying capacity (91%) Treatment of fungal and bacterial infections, surface cleaning Use of biosurfactants and synthetic surfactants. BR102020023890 Synthetic organic compound But-3-in-1-IL 2,3-dideoxy2-enopyranoside-4-ulose Cryptococcus sp (MIC: 16 pg mL·1) Inhibition of biofilms on surfaces (organic and inorganic) Treatment of biofilms and fungal infections Synthetic compounds targeting biofilms, against a variety of microorganisms.BR102020023770 Synthetic organic compound Benzyl 2,3-dideoxy-2-enopyranoside-4-ulose Cryptococcus spp. (MIC: 32 pg mL·1) Inhibition of biofilms on surfaces (organic and inorganic) Treatment of biofilms and fungal infections Synthetic compounds targeting biofilms, against a variety of microorganisms. BR102022001851 Synthetic organic compound Amphotericin B formulations Cryptococcus spp (dose = 1 g per day) Mucosal administration Treatment of cryptococcal meningitis Treatment with amphotericin B formulations as an alternative to intravenous administration BR102020013971 Synthetic organic compound N-Propyl 2,3-dideoxy-2-enopyranoside-4-ulose Cryptococcus sp (MIC: 16 pg mL⁻¹) Inhibition of biofilms on surfaces (organic and inorganic) Treatment of biofilms and fungal infections Synthetic compounds targeting biofilms, against a variety of microorganisms. BR102020004201 Synthetic organic compound 1,3,4-Oxadiazoles Candida spp.(MIC: 32 pg mL⁻¹), Cryptococcus spp. (MIC: 16 pg mL⁻¹), Paracoccidioides spp. (MIC: 2 pg mL⁻¹) Antimicrobial activity, high chemical reactivity and high solubility Treatment of human infections Formulation of a drug, additive, agent or formulation for the treatment and / or prevention of infections caused by fungi. BR102019027141 Synthetic organic compound Halofantrine, artesunate and amodiaquine Cryptococcus sp. and Candida sp (MIC >512 pg mL⁻¹) Antimicrobial activity and low cost of synthetic route Treatment of fungal infections, preferably caused by Cryptococcus sp. and Candida sp The use of a composition containing halofantrine, artesunate and amodiaquine BR102019019689 Synthetic organic compound 2-amino-thiophenes Cryptococcus sp. (MIC: 8 pg mL·1) Low toxicity treatment of human infections 2-aminosubstituted thiophene derivatives show promise as molecules in the development of antifungal compounds. Petition 870250071264, dated 08 / 13 / 2025, p. 16 / 29 12 / 24 BR102019017783 Synthetic organic compound Benzyl 2,3-Dideoxy-2-Enopyranoside-4-Ulose Cryptococcus sp. (MIC: 8 pg mL·1) Inhibition of fungal biofilms on organic and non-organic surfaces Treatment of biofilms, fungal infections Synthetic compounds with a specific focus on biofilms, acting against a variety of microorganisms. BR102018009020 Synthetic organic compound 1,3,4-oxydiazoles: 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furan-211)-1,3,4-oxadiazol-2-yl)benzamide Candida spp. (MIC: 128 pg mL⁻¹) Cryptococcus spp. (MIC; 32 pg mL⁻¹) Target of action in the thioredoxin reductase enzyme Treatment of human infections Formulation of a drug, additive, agent or formulation for the treatment of systemic fungal infections, such as Candidiasis, Cryptococcosis and Paracoccidioidomycosis. BR102017010830 Synthetic organic compound 2-(5-nitro-thiophene) thiosemicarbazone Cryptococcus spp.(MIC: 16 pg mL⁻¹) Low synthesis cost. Treatment of microbial infections. Thiosemicarbazone compounds containing a thiophene nucleus may become a future alternative for the treatment of fungal infections. BR102014029027 Synthetic organic compound 2-aminothiophene Cryptococcus sp. (MIC: 0.32-83.3 pg mL⁻¹, depending on the formulation) Low toxicity and antimicrobial activity. Use in the treatment of microbial infections. Nanoformulations based on 6CN₁O as an alternative for the treatment of the fungal infection cryptococcosis. BR102013033880 Synthetic organic compound Thiazole heterocyclic compounds Cryptococcus sp. and Candida sp (MIC: 0.5-250 pM, depending on the formulation) Easily synthesized from commercially available starting materials and with good yields. Used in the treatment of microbial infections. Thiazole heterocyclics as new antimicrobial agents. BR102019025251 Natural product (essential oil) Leaves of Croton argyrophyloides Mull.Arg (sacatinga) Cryptococcus sp. and Candida sp. (MIC: 2 to 64 pg mL·1) Fungicidal effect, low toxicity and natural product. Use in the treatment of microbial infections. Use of natural products as antimicrobials. BR102017016889 Amino acids and derivatives N-methyl-C-4-phenylglycine hydrochloride, N-methyl-C-4-methylphenylglycine hydrochloride, N-methyl-C-4-methoxyphenylglycine hydrochloride, N-methyl-C-4-isopropylphenylglycine hydrochloride Cryptococcus sp.and Candida sp (MIC: 128-512 pg mL·1) Low synthesis cost and low toxicity Use in the treatment of microbial infections Use of amino acids as antimicrobial agents BR102019001518 Synthetic peptide Defensin from Manihot esculenta Staphylococcus aureus (MIC: 16 pg mL·1), Acinetobacter baumannii (MIC: 64 pg mL·1) Candida albicans (MIC: 128 pg mL·1) Cryptococcus neoformans (MIC: 64 pg mL·1) Less susceptible to microbial resistance processes Synthetic peptide for use against fungal and bactericidal infections Use of synthetic peptide as antimicrobial agents. Petition 870250071264, dated 08 / 13 / 2025, page 17 / 29 13 / 24 BR102019001520 Synthetic peptide Defensin (PDef-Caj I) plant species Cajanus cejan Staphylococcus aureus (MIC: 16 pg mL⁻¹) Pseudomonas aeruginosa (MIC: 512 pg mL⁻¹) Acinetobacter baumannii (MIC: 256 pg mL⁻¹) Candida albicans (MIC: 64 pg mL⁻¹) Candida parapsilosis (MIC: 128 pg mL⁻¹) Cryptococcus neoformans (MIC: 64 pg mL⁻¹) Less susceptible to microbial resistance processes and bacteriostatic mode of action Synthetic peptide for use against fungal and bactericidal infections Use of synthetic peptide as antimicrobial agents BR102017024728 Synthetic peptide ToAP2 (NDBP, non-disulfide-bridged peptide) Cryptococcus spp.and Candida albicans (MIC: 1.6-200 pM) Low in vitro toxicity to mammalian cells, act on microbial membranes and are less susceptible to resistance development Inhibit the growth of two pathogenic fungi Use of synthetic peptide as antimicrobial agents BR102013029245 Fruit and vegetable extract Antibiotic from sugarcane pulp extract (Saccharum otticinarum) Candida, Cryptococcus, Trichosporon, E. coli, Staphylococcus aureus Biofilm degradation and reduction, microbicide Fungal and bacterial infections and biofilm treatment Use of natural extract, action on biofilms and wide variety of microorganisms treated BR102013018492 Fruit and vegetable extract Propolis gel Candida, Cryptococcus, Staphylococcus, E. coli Good adhesion, biocompatibility and easy application Premature implants Natural extract with application in dental implants.BR102013022601 Lipid derivative Phosphocholine oil (C18:1-PC) Candida, Cryptococcus, Aspergillus, Fusarium, Trichophyton (IC50 value ranging from 0.5-20 pM) Combats systemic fungal infections and acts on various strains Treatment of mycoses and systemic fungal infections Use of phospholipids, antifungal action on a wide variety of pathogens Current patent invention Synthetic compounds Selenium nanoparticles (stabilized with PVA or Chitosan) Cryptococcus neoformans (MIC: 0.78-1.56 pg mL·1) Biodegradable, non-toxic and low cost Fungal infections Use of selenium nanoparticles (stabilized with PVA or Chitosan) for the treatment of fungal infections. Petition 870250071264, dated 08 / 13 / 2025, page 18 / 29 14 / 24

[0019] The medicinal use of nanoparticles (NPs) with different compositions emerges as a promising alternative for the development of new pharmaceutical formulations capable of effectively combating fungal infections. NPs are widely applied in the treatment of fungal and bacterial infections, as they offer a lower probability of microorganisms developing resistance. Several types of NPs demonstrate significant antifungal properties, showing great potential for use in the treatment of infections of microbial origin (LIMA, R.; DEL, FS; BALCÃO, VM Prospects for the use of new technologies to combat multidrug-resistant bacteria. Frontiers in pharmacology, v. 10, p.692, 2019; WU, Z. et al. Microbial resistance to nanotechnologies: An important but understudied consideration using antimicrobial nanotechnologies in orthopaedic implants. Bioactive Materials, v. 16, p. 249-270, 2022).

[0020] The growing interest in NPs is due to their characteristics, such as shape, size, and large surface area, which are selective, stable, and biocompatible. These properties allow NPs to be administered in a targeted manner to specific areas of the body, through systemic administration, with low toxicity and high safety. Among the different options for NPs for the treatment of fungal infections, selenium nanoparticles stand out (XIA, Zhi-Kuan et al. The antifungal effect of silver nanoparticles on Trichosporon asahii. Journal of microbiology, immunology and infection, v. 49, n. 2, p. 182-188, 2016; NAYAK, D. et al. Green synthesis mediated by bark extract of silver nanoparticles: evaluation of antimicrobial activity and antiproliferative response against osteosarcoma. Materials Science and Engineering: C, v. 58, p. 44-52, 2016; MADKHALI, OAA comprehensive review on potential applications of metallic nanoparticles as antifungal therapies to combat human fungal diseases. Saudi Pharmaceutical Journal, p. 101733, 2023; RANA, D. et al. Nanomedicines for the Treatment of Systemic Candidiasis. In: Nanomedicines for the Prevention and Treatment of Infectious Diseases. Cham: Springer International Publishing, p. 95-124, 2023).

[0021] NPSe exhibit a promising biocompatibility and safety profile. Hemocompatibility tests indicated that concentrations up to 500 pg mL-1 are Petition 870250071264, dated 08 / 13 / 2025, page 19 / 29 15 / 24 safe for use, without harming the integrity of blood cells. The cytotoxicity study revealed an IC50 of 113.73 μg mL-1, suggesting low cellular toxicity. These results reinforce the potential of NPSe as a safe and effective alternative for therapeutic applications, without associated toxic risks (HASHEM, AH et al. Pomegranate peel extract stabilized selenium nanoparticles synthesis: promising antimicrobial potential, antioxidant activity, biocompatibility, and hemocompatibility. Applied Biochemistry and Biotechnology, v. 195, n. 10, p. 5753-5776, 2023).

[0022] Nesse contexto, tem sido produzidas NPSe estabilizadas por diferentes agentes estabilizantes, como Ulva lactuca (46,03 - 83,80 nm) e com alginato (38,94 nm) com o objetivo de avaliar sua atividade antifúngica contra C. neoformans (MAKHLOF, M. E. M et al. In vitro Assessment of Ulva lactuca Mediated Selenium Nanoparticles (USeNPs) through Elevating the Action of Ketoconazole Antibiotic against Pathogenic Yeast Species and Wound Healing Capacity through Inhibition of Cyclooxygenase (Cox-1) Activity. Egyptian Journal of Botany, v. 63, n. 3, p. 1155-1171, 2023; SINDI, H. A. et al. Alginate Extracted from Azotobacter chroococcum Loaded in Selenium Nanoparticles: Insight on Characterization, Antifungal and Anticancer Activities. Polymers, v. 16, n. 14, p. 2065, 2024).

[0023] Depending on the stabilizing agent and the synthesis process, the size of the nanoparticles varies, thus influencing biological activity. Therefore, such alterations can directly impact antifungal efficacy, as different stabilizers and sizes are associated with how NPSe interact with fungal cells, and with the absorption and distribution process in tissues. Thus, the development of nanoparticles with suitable characteristics can result in an innovative antifungal, competitive in the pharmaceutical market, effective in combating fungal infections and safe for users (MADKHALI, OA A comprehensive review on potential applications of metallic nanoparticles as antifungal therapies to combat human fungal diseases. Saudi Pharmaceutical Journal, p. 101733, 2023). Petition 870250071264, dated 08 / 13 / 2025, page 20 / 29 16 / 24

[0024] However, to ensure wide applicability and effectiveness, especially in medical treatments, it is necessary to use materials that are preferably low cost, biodegradable, non-toxic and provide high yield. In this sense, the use of chitosan (QUI) and polyvinyl alcohol (PVA) as stabilizers for NPSe stands out due to improved stability and controlled release in NPSe (BOROUMAND, S. et al. Selenium nanoparticles: synthesis, characterization and study of their cytotoxicity, antioxidant and antibacterial activity. Materials Research Express, v. 6, n. 8, p. 0850d8, 2019; IBRAHIM, MA et al. Selenium loaded sodium alginate / polyvinyl alcohol nanocomposite film as wound dressing: Synthesis and cytotoxic activities of selenium nanoparticles incorporated nano-chitosan, v.2, p. 1421-1437, 2024).

[0025] Chitosan is a natural polysaccharide found in the cell wall of some organisms, such as certain fungi. Commercially, it can be obtained economically through the N-deacetylation of chitin, exhibiting characteristics such as intra- and intermolecular hydrogen bonds, which influence its properties regarding solubility, viscosity, and crystallinity. This biomaterial is widely used in various areas, including pharmaceuticals, food, textiles, agriculture, and environmental remediation processes. Its versatility of application is due to its biodegradability, biocompatibility, low toxicity, reduced cost, and wide availability (WANG, J.; ZHUANG, S. Chitosan-based materials: Preparation, modification and application. Journal of Cleaner Production, v. 355, p. 131825, 2022; QU, B.; LUO, Y. Chitosan-based hydrogel beads: Preparations, modifications and applications in food and agriculture sectors - A review).International Journal of Biological Macromolecules, vol. 152, p. 437-448, 2020).

[0026] Polyvinyl alcohol (PVA) is a semi-crystalline synthetic polymer composed of various functional groups that confer polarity to the macromolecule, allowing the formation of hydrogen bonds and facilitating the stabilization of different systems. Due to its hydrophilic character and high density of functional groups, PVA is Petition 870250071264, dated 08 / 13 / 2025, page 21 / 29 17 / 24 stands out as an efficient stabilizer, capable of interacting with various systems. Furthermore, PVA exhibits properties such as low toxicity, high water solubility, film-forming capacity, and biocompatibility with different systems, being considered safe for the environment (AZIZ, SB et al. Impedance study and behavior of a solid-state double-layer capacitor of proton-conducting polymers (H+) based on CS:PS electrolytes. Ionics, v. 26, n. 9, p. 4635-4649, 2020; AZIZ, SB et al. The study of the degree of crystalline, electrical equivalent circuit, and dielectric properties of polyvinyl alcohol (PVA)-based biopolymer electrolytes. Polymers, v. 12, n. 10, p. 2184, 2020; HASHEMI-FIROUZI, N. et al. The effects of polyvinyl alcohol-coated selenium nanoparticles on memory impairment in rats. Metabolic Brain Disease, v. 37, n. 8, p. 3011-3021, 2022; JING, Qiang et al.pH responsive fabrication of PVAstabilized selenium nano formulation encapsulated with luteolin to reduce diabetic ureteral injury by decreasing NLRP3 inflammasome via Nrf2 / ARE signaling. Regenerative Therapy, v. 27, p. 434-444, 2024).

[0027] Patent BR2024 / 023561 consisted of the use of selenium nanoparticles (NPSe) stabilized with polyvinyl alcohol (PVA) and chitosan (QUI) as urease inhibitors in soils, which showed promise for the development of fertilizers with increased efficiency due to urease inhibition. Therefore, it is possible to infer that one of the fungicidal mechanisms of action of NPSe_PVA and NPSe_QUI against the pathogen C. neoformans is through urease inhibition, since this microorganism is a ureolytic fungus. Thus, the use of QUI and PVA in the stabilization of selenium nanoparticles in the present invention aims to overcome the limitations of conventional antifungal treatments, providing a more effective and sustainable solution for combating C. neoformans, with significant benefits for health and the environment.Thus, the present invention proposes an innovative approach by employing polyvinyl alcohol (PVA, NPSe_PVA) and chitosan (QUI, NPSe_QUI) stabilized NPSe as an antifungal agent against C. neoformans (ureolytic fungus). Brief description of the drawings Petition 870250071264, dated 08 / 13 / 2025, pp. 22-29 18 / 24

[0028] The attached figures, incorporated into this descriptive report, illustrate various aspects and, together with the description, serve to explain the principles of the invention. Figure 1. Characterization of NPSe by Transmission Electron Microscopy varying the scale of the images: (1) NPSe_PVA (1 μm); (2) NPSe_PVA (200 nm); (3) NPSe_PVA (100 nm); (4) NPSe_QUI (1 μm); (5) NPSe_QUI (200 nm) and (6) NPSe_QUI (100 nm). Figure 2. Evaluation of the antifungal activity of NPSe_PVA (A) and NPSe_QUI (B) at different concentrations against the growth of C. neoformans after 48 h. Concentrations evaluated: 1 (25 μg mL-1), 2 (12.5 μg mL-1), 3 (6.25 μg mL-1), 4 (3.12 μg mL-1) and 5 (1.56 μg mL-1). R1 ​​and R2 are real replicates of the system and CT represents the positive control. Description of the invention

[0029] The present invention relates to the development of selenium nanoparticles (SeNPs) stabilized with polyvinyl alcohol (PVA) and chitosan (QUI), designed to act as antifungal agents against C. neoformans. This technological solution aims to offer an effective, biocompatible and economically viable alternative for the treatment of fungal infections, particularly in immunocompromised patients.

[0030] The invention innovatively addresses the technical problem of resistance to conventional antifungal treatments by proposing aqueous dispersions containing nanoparticles (NPSe_QUI or NPSe_PVA), characterized by a nanomaterial with antifungal properties, colloidal stability, and potential for clinical application. The technical advantages of this invention include reducing the risk of fungal resistance, a simple, scalable, and low-cost synthesis method, and significantly expanding therapeutic and preventive applications, providing a promising solution for the treatment of resistant fungal infections. Examples of how the invention can be implemented

[0031] For the synthesis of chitosan-stabilized selenium nanoparticles (SeNPs), 1 mL of a 24 mmol L-1 sodium selenite solution is added to an Erlenmeyer flask containing 4 mL of a 0.5% (w / v) chitosan solution, 3 mL of 230 mmol L-1 ascorbic acid and 2 mL of 2.4 mol L-1 acetic acid, under constant stirring. Petition 870250071264, dated 08 / 13 / 2025, pp. 23 / 29 19 / 24 The reagents used are of analytical grade (PA) and do not require prior purification steps before use.

[0032] To synthesize PVA-functionalized NPSe, a 50 mmol L-1 sodium selenite solution was mixed with a 20 mg mL-1 PVA solution in a 1:1 (v / v) ratio. Subsequently, under continuous stirring, a 100 mmol L-1 ascorbic acid solution was added, maintaining a final ratio of 3:20 (v / v).

[0033] To obtain NPSe_PVA and NPSe_QUI in suspension, the NPSe were subjected to continuous agitation at 25°C for 2 hours and 30 minutes, respectively, ensuring complete reduction of selenite by ascorbic acid and, consequently, the formation of elemental selenium.

[0034] After synthesis, the solutions containing the NPSe were subjected to dialysis using 12 kDa cellulose membranes (Sigma-Aldrich, HE, Germany) for a period of 8 hours, in order to remove excess reagents such as ascorbic acid and acetic acid. The membranes were previously hydrated in ultrapure water for 15 minutes before receiving the NPSe suspensions. After proper sealing, the membranes were placed in beakers containing 2 liters of ultrapure water, with the dialysis water being changed every 4 hours. Furthermore, pH measurements were performed before and after synthesis, ensuring complete removal of acids and adjusting the pH of the nanomaterial suspension to approximately 6.0 (ZIDAN, Nahla S. et al. Microwave synthesis of Chitosan-stabilized selenium nanoparticles: Intrinsic oxidant scavenging capabilities, hemocompatibility, anticancer, and antibacterial potency. Journal of Molecular Structure, v. 1295, p. 136715, 2024).

[0035] The characterization of the NPSe was performed using various techniques, including transmission electron microscopy (TEM), energy-dispersive X-ray spectrometry (EDX), dynamic light scattering (DLS), Fourier transform infrared (FTIR) molecular absorption spectroscopy, and UV-Vis absorption spectroscopy. Through TEM, it was observed that the NPSe_PVA had an average size of 76 ± 12 nm, while the NPSe_QUI had an average size of 82 ± 20 nm, both with spherical morphology. High-resolution transmission electron microscopy (HR-TEM) also revealed that the nanoparticles had average dimensions of 76 ± 12 nm for the NPSe_PVA and 82 ± 20 nm for the NPSe_QUI. The presence Petition 870250071264, dated 08 / 13 / 2025, pages 24 / 29 The 20 / 24 selenium content in the NPSe was confirmed by EDX and X-ray photoelectron spectroscopy (XPS) techniques. FTIR spectra exhibited characteristic profiles of the stabilizers PVA and chitosan, confirming their presence on the nanoparticle surfaces. Furthermore, UV-Vis absorption spectra showed maximum absorption peaks for NPSe_PVA and NPSe_QUI at 258 nm and 260 nm, respectively.

[0036] The antifungal activity of the compounds NPSe_QUI and NPSE_PVA was evaluated using the minimum inhibitory concentration (MIC) test, according to the Reference Method for Broth Dilution Tests for Determination of Yeast Sensitivity to Antifungal Therapy described in the Clinical and Laboratory Standards Institute (2017), using a sterile 96-well plate. The C. neoformans ATCC 208821 strain was previously cultured in solid YPD medium (2% (w / w) agar, 2% (w / w) glucose, 1% (w / w) peptone and 0.5% (w / w) yeast extract) for 48 ha at 25-27°C (CLINICAL AND LABORATORY STANDARDS INSTITUTE. M27: Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. Wayne, PA: CLSI, 2017).

[0037] For inoculum preparation, pure yeast colonies were diluted in saline solution (0.85% (w / w) NaCl) and adjusted to OD530 (optical density at 530 nm) between 0.12 and 0.15. After adjustment, the inoculum was added to Sabouraud broth. The volume of inoculum added to Sabouraud broth was calculated following a 1:20 dilution followed by a 1:50 dilution. Subsequently, 100 μL of the previously diluted inoculum was added to all wells of columns 1 to 11 in the microplate. Then, 100 μL of NPSe_QUI and NPSE_PVA (initial concentration of 200 μg mL-1) were added to the wells of column 1, in duplicate, reaching a final concentration of 100 μg mL-1. To perform the microdilution, a multichannel pipette was used, homogenizing and removing 100 μL from the wells of column 1 and dispensing these 100 μL into the wells of column 2. This step was performed up to the wells of column 10, completing the microdilution.Wells in column 11 contained only inoculum as a positive control for yeast growth, while wells in column 12 contained only sterile Sabouraud broth as a negative control for growth. After the microdilution step, the plates were... Petition 870250071264, dated 08 / 13 / 2025, pages 25 / 29 21 / 24 were incubated for 48 ha at 35°C in a refrigerated incubator. Plate measurements were taken by recording the OD530 values ​​for wells without growth and wells with reduced growth.

[0038] To evaluate the antifungal activity of the tested systems, a 2 μL aliquot was taken from the wells that did not show growth and placed in plates containing YPD medium. The plates were incubated for 48 ha at 35°C. The measurement (OD530) was performed, noting the absence of yeast growth as fungicidal activity and the presence of yeast growth as fungistatic activity. Additionally, the commercial antifungals amphotericin B and fluconazole were tested as positive controls, with initial concentrations of 8 and 32 μg mL-1, respectively.

[0039] The antifungal activity of NPSe_QUI and NPSe_PVA was evaluated against C. neoformans, and the results were compared with conventional antifungal agents, Amphotericin B (ANFB) and Fluconazole (FLU), as well as with isolated stabilizers (PVA and QUI). The minimum inhibitory concentration (MIC) was used as a parameter to determine the efficacy of the tested samples, as well as the relative activity index (RAI), calculated from the ratio between the MIC values ​​of the antifungals ANFB and FLU in relation to the evaluated systems (Table 2). In this evaluation, pure PVA did not show activity in any of the dilutions evaluated up to a concentration of 100 pg mL-1 Table 2. Comparison of the antifungal activity (MIC) of stabilized NPSe and commercial antifungals against C. neoformans. Where Iar = relative activity index. MIC System (Mg mL-1) Iar-flu (comparison with FLU) Iar-anfb (comparison with ANFB) NPSe_PVA 1.56 10.26 2.56 NPSe_QUI 0.78 20.51 5.13 QUI 6.25 2.56 0.64 ANFB 4 4 1 Petition 870250071264, dated 08 / 13 / 2025, pages 26 / 29 22 / 24 FLU 16 1 0.25

[0040] NPSe_PVA and NPSe_QUI showed superior antifungal activity compared to the commercial antifungals FLU and ANFB evaluated in assays against C. neoformans. In this context, the present invention proposes a safe and more effective alternative for the treatment of fungal infections, considering that NPSe_PVA was 3 to 10 times more efficient, while NPSe_QUI was 5 to 20 times more efficient compared to FLU and ANFB in in vitro studies (Table 2).

[0041] Among the antifungals tested, NPSe_QUI showed the lowest minimum inhibitory concentration (MIC = 0.78 pg mL-1), being more active than NPSe_PVA (MIC = 1.56 pg mL-1). This superior performance of NPSe_QUI can be attributed to the combined action between NPSe and QUI, since the isolated biopolymer demonstrated antifungal activity (MIC = 6.25 pg mL-1). The incorporation of selenium into chitosan, in the form of NPs, potentiated its activity.

[0042] On the other hand, the activity of NPSe_PVA is attributed exclusively to NPSe, since pure PVA did not show any inhibitory effect on the fungus (up to 100 pg mL-1). This reinforces the importance of selenium as an active component in nanoparticles stabilized with PVA. These results highlight the relevance of NPSe_QUI and NPSe_PVA as promising alternatives to usual antifungals, offering a more active approach to the treatment of fungal infections caused by C. neoformans.

[0042] Therefore, the fungicidal activity of NPSe_QUI and NPSe_PVA was determined after determining the minimum inhibitory concentration (MIC) using the subculture test. After exposing C. neoformans ATCC 208821 cells to the nanoparticles, an aliquot of this mixture was transferred to plates containing solid YPD medium in order to observe the absence or presence of fungal growth.

[0043] The results demonstrated that NPSe_PVA and NPSe_QUI exhibited clear fungicidal activity at concentrations of 1.56 and 0.78 pg mL-1, respectively (Figure 2). The fungicidal action could be confirmed by the absence of growth of C. neoformans ATCC 208821 cells in solid medium, indicating that these nanoparticles not only inhibited fungal growth but also caused damage to its cells. Petition 870250071264, dated 08 / 13 / 2025, pages 27 / 29 23 / 24

[0044] The classification as a fungicidal mode of action is an important differentiating factor compared to commercial antifungals such as FLU, which has a predominantly fungistatic action. Fungistatic antifungals only inhibit fungal growth in the medium without eliminating the cells; thus, there is a possibility that the residual fungal population may become more susceptible to developing resistance over time. This limitation is particularly concerning in the treatment of chronic or recurrent infections, such as those caused by C. neoformans, where selective pressure may favor the selection of resistant strains.

[0045] In contrast, the fungicidal capacity of NPSe_PVA and NPSe_QUI offers an effective and promising solution, reducing the chance of viable cell survival and, consequently, the possibility of resistance development. Furthermore, the fungicidal activity observed even at low concentrations highlights the potential of these nanoparticles as an innovative alternative to conventional antifungals.

[0046] It is worth noting that experimental antimicrobial activity assays were performed for bacteria such as P. mirabilis, S. aureus, and E. coli, and the fungus C. albicans using concentrations from 0 to 100 pg mL-1. However, NPSe_QUI and NPSe_PVA did not show antimicrobial activity against P. mirabilis and E. coli. Nevertheless, they showed a MIC value of 50 pg mL-1 only for the fungus C. albicans, a value higher than that of conventional antifungals (ANFB and FLU, Table 2). This selectivity can be attributed to structural and biochemical differences between the microorganisms. In addition, C. neoformans has a polysaccharide capsule rich in glucuronoxylomannan, which can interact specifically with the nanoparticles, favoring internalization.

[0047] In addition, C. neoformans exhibits urease production as a crucial virulence factor, suggesting that one of the antifungal mechanisms of action of NPSe_QUI and NPSe_PVA is the inhibition of this enzyme, as reported in patent BR2024 / 023561, which demonstrated that these nanoparticles possess antiureolytic activity. Additionally, the NPSe were evaluated against other microorganisms (bacteria and fungi), with and without ureolytic activity. Petition 870250071264, dated 08 / 13 / 2025, pages 28 / 29 24 / 24

[0048] In contrast, C. albicans and S. aureus, which do not produce urease as a virulence factor or related to metabolic processes, were not effectively active by NPSe_QUI and NPSe_PVA (MIC of 50g mL-1 only for C. albicans). In these microorganisms, pathogenicity is mainly related to biofilm formation, the production of hydrolytic enzymes (such as proteases and phospholipases), and the transition between morphological forms (yeast and hyphae) (NIEWERTH, M.; KORTING, HC Phospholipases of Candida albicans. Mycoses, v. 44, n. 9-10, p. 361-367, 2001; KASHYAP, B. et al. Candida albicans Induces Oral Microbial Dysbiosis and Promotes Oral Diseases. Microorganisms, v. 12, n. 11, p. 2138, 2024; SCHOLZ, R. et al. Epigenetic control of microglial immune responses. Immunological Reviews, 2024). On the other hand, Gram-negative bacteria such as P. mirabilis (ureolytic bacteria) and E.Escherichia coli (non-ureolytic bacteria) have a complex cell wall, including an outer membrane rich in lipopolysaccharides, which functions as a protective barrier against antimicrobial agents (CALDARELLI, M. et al. Gut-Brain Axis: Focus on Sex Differences in Neuroinflammation. International Journal of Molecular Sciences, v. 25, n. 10, p. 5377, 2024; KADHUM, WN; ZAIDAN, IA The synergistic effects of chitosan alginate nanoparticles loaded with doxycycline antibiotic against multidrug-resistant Proteus mirabilis, Escherichia coli and Enterococcus faecalis). Therefore, this outer membrane may also contain efflux proteins, which eliminate foreign compounds, further hindering the action of NPSe_QUI and NPSe_PVA, which were not active against these bacteria. Petition 870250071264, dated 08 / 13 / 2025, p. 29 / 29

Claims

CLAIMS 1. Use of selenium nanoparticles (SeNPs) stabilized with polyvinyl alcohol (PVA), characterized by their antifungal action against C. neoformans.

2. Use of selenium nanoparticles (SeNPs) stabilized with chitosan (QUI), characterized by their antifungal action against C. neoformans. Petition 870250013062, dated 02 / 18 / 2025, page 9 / 35

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