Striking Back against Fungal Infections: The Utilization of Nanosystems for Antifungal Strategies

Fungal infections have become a major health concern, given that invasive infections by Candida, Cryptococcus, and Aspergillus species have led to millions of mortalities. Conventional antifungal drugs including polyenes, echinocandins, azoles, allylamins, and antimetabolites have been used for decades, but their limitations include off-target toxicity, drug-resistance, poor water solubility, low bioavailability, and weak tissue penetration, which cannot be ignored. These drawbacks have led to the emergence of novel antifungal therapies. In this review, we discuss the nanosystems that are currently utilized for drug delivery and the application of antifungal therapies.


Fungal Infection
Fungal infections are a major threat to humans and number in the billions, leading to more than 1.5 million deaths annually [1][2][3]. Candida, Aspergillus, and Cryptococcus, are the major pathogenic fungi in humans, causing 90% of the deaths in both immunocompetent and immunocompromised individuals [2,3]. Other pathogenic fungi, including Pneumocystis, Coccidioides, and Histoplasma, can also cause severe tissue damage and even death ( Table 1).
The type of fungal infection is highly dependent on the fungal species and the immune status of the hosts [4]. For instance, superficial infections in humans are among the most common fungal infections, occurring in over 1 billion people. Over 135 million women are diagnosed with Candida mucosal infections [5]. However, the most devastating fungal infections are invasive. These are initiated by the inhalation or inoculation of fungal spores or by an imbalance of the commensal fungi of the host. Invasive candidiasis, aspergillosis, and cryptococcosis occur in the bloodstream and deep-seated organs as a result of fungi from the genera Candida, Aspergillus, and Cryptococcus, respectively. Additionally, fungal infection also results in or enhances severe host allergic responses, including asthma, cystic fibrosis, or chronic nasal sinus symptoms [6][7][8][9][10].
Candida spp. make up the commensal fungi residing within human superficial, mucosal, or intestinal tract regions, and their colonization and growth are highly restricted when the host is immunocompetent. Studies demonstrate that over 15 distinct Candida species are pathogenic fungi, of which five species cause the most invasive infections in humans: Candida albicans, Candida krusei, Candida glabrat, Candida tropicalis, and Candida parapsilosis [11][12][13][14]. Recent studies show that Candida auris has emerged globally as a multidrug-resistant fungal pathogen that leads to significant patient mortality [15][16][17][18]. In addition to Candida, Cryptococcus, and Aspergillus lead to severe lung infections and can lead to fatal infections, including pneumonia and meningocephalitis. they have developed sophisticated strategies, such as thermo-tolerance, pH-tolerance, and resistance to phagocytosis from host immune cells, which facilitate fungal growth and persistence within environmental niches and vertebrates [39][40][41][42][43]. These strategies endow cryptococci with growth advantages, including severe virulence. Cryptococcal infection begins with the inhalation of cryptococci spores into the lungs and can cause pneumonia in immunosuppressed patients. However, these fungal cells establish an asymptomatic latent infection in immunocompetent hosts, where the colonizing fungal cells can disseminate to other tissues, especially the central nervous system, which occurs through uncharacterized mechanisms [44,45]. Once the brain has been colonized, cryptococcosis leads to a devastating infection of the meninges and lethal meningoencephalitis [46].
Aspergillus is a saprophytic fungus found in soil and comprises at least 200 species. Aspergillus spp. is common and isolated from cultures of the respiratory tracts of asymptomatic patients who lack evidence of invasive or allergic disease [47][48][49]. Moreover, aspergillosis is highly associated with chronic obstructive pulmonary disease [50]. Aspergillus spp. is much more common than other fungi, and approximately 37% of healthy adults carry these fungi [51]. However, they are not always associated with infection. Nevertheless, colonization by Aspergillus spp. is associated with increased risk of invasive infections across a wide range of immunocompromised individuals [52]. To date, Aspergillus pathogens include A. fumigatus, A. flavus, A. niger, A. terrus, A. clavatus, and A. nidulans, the first of which is the most common.
Distinct from Cryptococcus, however, Aspergillus can produce small asexual spores, called conidia, which are 2-3 µm in diameter. The dispersal of A. fumigatus produces conidia that are hydrophobic and can spread and sustain for a much longer time in the air, making it a highly virulent fungus [53][54][55]. In healthy individuals, epithelial cells in the airway and alveolar macrophages efficiently eliminate inhaled conidia, but resident conidia can evade mucocilliary clearance and lodge in the alveoli deep in the lungs, germinating into short hyphae in less than 6 h [56]. Aside from host immune status, Aspergillus colonization is related to certain genetic factors of the host. Impaired expression of the transcriptional factor ZNF77 in bronchial epithelia can result in the destruction of epithelial cell integrity, and upregulation of extracellular matrix proteins has been shown to facilitate conidial adhesion [57].

Conventional Treatments for Candidiasis, Cryptococcosis, and Aspergillosis
Researchers have developed several antifungal treatments to contend with the increasing challenge of fungal infections. However, the significant side effects and development of drug resistance in fungi have not been adequately addressed [58][59][60][61].
In addition, yeast cells construct galactosaminoglycans and protein-based outer layers, and galactomannan is an additional element in the final layer of filamentous cells. Moreover, fungal cells utilize ergosterol rather than cholesterol to manufacture cell membranes [66,67]. These elements of fungal cell walls and membranes distinguish invasive fungi from host mammalian cells and act as therapeutic targets for antifungal drugs (Figure 1).
Common clinical antifungal drugs have distinct molecular targets and can be divided into 5 categories ( Figure 1, Table 2): (i) azoles, (ii) polyenes, (iii) echinocandins, (iv) allylamines, and (v) antimetabolites. Azoles are chemically synthesized small molecules that primarily block the ergosterol synthesis pathway by inhibiting lanosterol 14α-demethylase (Erg11), which is essential for fungal cell membrane formation. They have been used as antifungal drugs since the 1970s, when they were found to impart a broad spectrum of activity against various fungal species. , is a fluorinated pyrimidine analog with fungicidal activity via interfering the pyrimidine metabolism, RNA/DNA and protein synthesis. First, 5-FC is taken up by fungal cells via a cytosine permease (encoded by gene FCY2) and is converted to 5-fluorouracil , and is then transformed by UMP pyrophosphorylase into 5-fluorourdine monophosphate (5-FUMP). Then, 5-FUMP is incorporated into RNAs to inhibit the protein synthesis. Additionally, ribonucleotide reductase enables the conversion of 5-FUMP into 5-fluorodeoxyuridine monophosphate (5-FdUMP), a potent inhibitor of thymidylate synthase that inhibits fungal DNA synthesis and nuclear division. Azoles are inhibitors for cytochrome P450-dependent enzyme lanosterol 14α-demethylase (CYP51) encoded by the ERG11 gene, and thus block the conversion of lanosterol to ergosterol. Allylamines block ergosterol biosynthesis via inhibiting squalene epoxidase (ERG1) that lead to squalene accumulation and increased permeability may cause the disruption of cellular organization. Echinocandins act as noncompetitive inhibitors of β-(1, 3)-D-glucan synthase enzyme complex and leads to disruption of the cell wall structure, resulting in osmotic instability and fungal cell death. Polyenes specifically bind to the lipid bilayer and form a complex with the ergosterol producing pores that leads to the disruption of the cell membrane, leakage of the cytoplasmic, contents and oxidative damage in fungal cells. Amphotericin B (AmB) binds ergosterol and forms an extra-membranous fungicidal sterol sponge destabilizing membrane function.
Common clinical antifungal drugs have distinct molecular targets and can be divided into 5 categories ( Figure 1, Table 2): (i) azoles, (ii) polyenes, (iii) echinocandins, (iv) allylamines, and (v) antimetabolites. Azoles are chemically synthesized small molecules that primarily block the ergosterol synthesis pathway by inhibiting lanosterol 14α-demethylase (Erg11), which is essential for fungal cell membrane formation. They have been used as antifungal drugs since the 1970s, when they were found to impart a broad spectrum of activity against various fungal species. Schematic representation of the conventional antifungal agents, their targets, and actions. Antimetabolite, 5-Fluorocytosine (5-FC), is a fluorinated pyrimidine analog with fungicidal activity via interfering the pyrimidine metabolism, RNA/DNA and protein synthesis. First, 5-FC is taken up by fungal cells via a cytosine permease (encoded by gene FCY2) and is converted to 5-fluorouracil (5-FU), and is then transformed by UMP pyrophosphorylase into 5-fluorourdine monophosphate (5-FUMP). Then, 5-FUMP is incorporated into RNAs to inhibit the protein synthesis. Additionally, ribonucleotide reductase enables the conversion of 5-FUMP into 5-fluorodeoxyuridine monophosphate (5-FdUMP), a potent inhibitor of thymidylate synthase that inhibits fungal DNA synthesis and nuclear division. Azoles are inhibitors for cytochrome P450-dependent enzyme lanosterol 14α-demethylase (CYP51) encoded by the ERG11 gene, and thus block the conversion of lanosterol to ergosterol. Allylamines block ergosterol biosynthesis via inhibiting squalene epoxidase (ERG1) that lead to squalene accumulation and increased permeability may cause the disruption of cellular organization. Echinocandins act as noncompetitive inhibitors of β-(1, 3)-D-glucan synthase enzyme complex and leads to disruption of the cell wall structure, resulting in osmotic instability and fungal cell death. Polyenes specifically bind to the lipid bilayer and form a complex with the ergosterol producing pores that leads to the disruption of the cell membrane, leakage of the cytoplasmic, contents and oxidative damage in fungal cells. Amphotericin B (AmB) binds ergosterol and forms an extra-membranous fungicidal sterol sponge destabilizing membrane function.  Inhibits the amino acid and glucose transportation, leads to ergosterol-specific and reversible inhibition of membrane transport proteins without altering the cell membrane permeability [85] Topical No severe side effects have been reported Rare cases reported mild irritation, redness, foreign body sensation, stinging, burning sensation, and tearing [86] Echinocandins Anidulafungin (AFG) Candida spp. [87,88] Acts as the noncompetitive inhibitor of β-1, 3-D-glucan synthase, which leads to the inhibition of the synthesis of glucan. Thus, it compromises the fungal cell wall stability and synthesis.  Polyenes were isolated from Streptomyces spp., where they have functions in the bacterial defense mechanism. This class of drug mainly sequesters ergosterol and disrupts the fungal cell membrane via pore formation, resulting in leakage of cytoplasmic contents and fungal cell death [95,96]. The most potent, amphotericin B (AmB), is the most common polyene used for invasive fungal infections by forming an extra-membranous fungicidal sterol sponge that destabilizes membrane function [97]. In contrast with other kinds of polyenes, natamycin (NAT) inhibits fungal growth by reversibly inhibiting the amino acid and membrane transport proteins without altering the cell membrane permeability [85].

Intravenous
Enchinocandins target β-1, 3-glucan synthase and negatively impact fungal cell wall integrity. These antifungal agents have good safety profiles, but have poor oral bioavailability, due to the lipid side chains. They have efficient therapeutic applications against both the planktonic cells of Candida and their biofilm formation. Additionally, this antifungal agent has been used to treat aspergillosis [98,99].

Unsatisfactory Properties of Currently Used Antifungal Drugs
The five classes of conventional antifungal drugs have been determined to have great efficiency for treating both superficial and invasive fungal infection. Nevertheless, their side effects and unpleasant properties highly restrict their applications. As the most commonly used antifungal drugs in clinical practice, the major concerns of using azoles are their interactions with drugs that act as substrates for cytochrome P450, leading to off-target toxicity and fungal resistance to azoles [101,102]. Polyenes target fungal ergosterol, which is structurally similar to mammalian cholesterol. As a result, AmB displays devastating nephrotoxicity and infusion-related reactions [103,104]. As a result, its dosage is highly restricted, and it is usually replaced by an azole drug (voriconazole). Rather than invasive fungal infections, allylamines are normally used for treating superficial fungal infection, such as onychomycosis, which occurs in the fingernails or toenails [105].
As a highly effective antifungal agent, antimetabolite 5-FC is severely hepatoxic and results in bone-marrow depression [106][107][108]. Additionally, monotherapy with 5-FC triggers significant fungal resistance. Its primary clinical use is in combination with AmB for severe cases of candidiasis and cryptococcosis [109,110]. Although several effective antifungal agents have been prescribed for decades, their therapeutic outcomes remain unsatisfactory. Aside from these traditional antifungal agents being highly toxic, fungi tend to become resistant to them. Moreover, these antifungal agents display distinct efficiencies in tissue penetration and oral bioavailability.
In general, fluconazole, 5-FC, and voriconazole are small molecules and display better tissue penetration than the larger, more lipophilic agents (itraconazole) and amphipathic agents (AmB and echinocandins). Additionally, AmB and echinocandins exhibit delayed drug metabolism and accumulate in tissues [111]. Current strategies for improvement include developing analogs of these compounds, evaluating current drugs for their potential antifungal effects, finding new targets for antifungal drugs, and determining new fungal antigens as vaccine candidates [112,113].
Another possible strategy is using nanotechnology to modify or encapsulate currently used antifungal agents to improve their efficacy. To date, several nanomaterials have been investigated and presented as innovative antifungal agents, which include biodegradable polymeric and co-polymeric-based structures, metallic nanoparticles, metallic nanocompos-ites, and lipid-based nanosystems [114][115][116]. Additionally, the size range of nanoparticles endows them with the ability to deliver current antifungal agents by various routes of administration, such as oral, nasal, and intraocular routes [117].

Nanotechnology-Based Therapies for Fungal Infections
Since nano theory was firstly hypothesized by Richard Feynman in 1959, it has become a broad arena for integrating various areas of knowledge, such as biology, chemistry, physics, and engineering. Nanoscience has been shown to have great potential in the treatment of pathologies [118]. Moreover, nano-sized carriers enable the delivery of multiple drugs or imaging agents in the treatment of cancer or infections and in pathologic diagnostics [119,120]. The advantages of using nano-sized carriers include prolonged drug release, resistance to metabolic degradation, augmented therapeutic effects, and even avoidance of drug resistance mechanisms [119]. Metallic nanoparticles, mesoporous silica nanoparticles, polymeric nanoparticles, and lipid-based nanosystems are possible solutions to the challenges faced in the treatment of fungal infections. As the threat of invasive and superficial fungal infections continuously increases, hundreds of studies have led to a variety of synthesized and fabricated nanosystems for the optimization of antifungal therapy.

Metallic Nanoparticles
Metal nanoparticles are 1 to 100 nm in size and offer advantages of chemical stability, potential antifungal effects, low toxicity, and low pathogen resistance [121][122][123][124]. They can inhibit fungal cell membrane synthesis and certain fungal protein syntheses, as well as facilitate the production of fungal reactive oxygen species [125][126][127][128]. Gold, silver, zinc, and iron oxide nanoparticles are the most studied for antifungal drug delivery [121]. Several related studies are listed Table 3.
Nano-sized gold materials have been shown to have anti-candida effects with low toxicity [129,130]. Normally, gold nanoparticles are conjugated with effective agents to improve their antifungal effects. For example, indolicidin, a host defense peptide, was conjugated with gold nanoparticles to treat fluconazole-resistant clinical isolates of C. albicans. The indolicidin-gold nanoparticles did not show cytotoxicity for the fibroblast cells and erythrocytes and they significantly reduced the expression levels of the ERG11 gene in C. albicans [130].
Other methods of obtaining antifungal nanoparticles include the SnCl 2 and NaBH 4 based synthesis methods, which provide nanoparticles average sizes of 15 nm and 7 nm, respectively. Interestingly, the smaller size of gold nanoparticles displayed better antifungal activity and greater biocidal action against Candida isolates than 15 nm gold nanoparticles by restricting the transmembrane H + efflux [131]. In another study, triangular gold nanoparticles were synthesized and conjugated with specific peptide ligands that inhibit secreted aspartyl proteinase 2 (Sap2) in C. albicans. Both non-conjugated and peptide gold nanoparticles showed high antifungal activity for 30 clinical isolates of C. albicans, although the peptide-conjugated nanoparticles had the highest uptake efficiency [129].
Silver nanoparticles have been shown to have great potential for antifungal growth and avoiding resistance in microorganisms [132]. As with gold, silver nanoparticles are easily modified and synthesized and display stable physicochemical characteristics [133]. Monotherapy with silver nanoparticles has been evaluated in various studies in vitro, where the growth and survival of C. albicans and C. tropicalis were significantly hampered. Moreover, they show great potential against fluconazole-resistant isolates of C. tropicalis in clinical settings. The antifungal efficiency of silver nanoparticles can be optimized when used in conjugation with AmB and fluconazole [134][135][136].
Silver and gold nanoparticles have also been biosynthesized to fight fungi-induced dermal infections. Interestingly, the growth of Candida, Microsporum, and Trichophyton dermatophyte isolates was inhibited by silver particles, but C. neoformans was susceptible to both gold and silver nanoparticles. Both of these heavy-metal-based nanoparticles were shown to lack cytotoxicity to human keratinocytes [137]. Despite its ability to impart anti-fungal activity, an overload of silver is toxic to mammalian cells, so the toxicity and use of silver nanoparticles needs further evaluation.
Aside from directly inhibiting the growth of fungal pathogens, a low dosage of silver nanoparticles has been demonstrated to have great potential for inhibiting mycotoxin biosynthesis [138]. Mycotoxin contamination has affected over 25% of the world's crops and leads to losses of around 1 billion metric tons of foods and food products annually according to the Food and Agriculture Organization of the United States. F. chlamydosporum and P. chrysogenum were used to produce biogenic silver nanoparticles, which inhibited the fungal growth of A. flavus and completely prevented its aflatoxin production [139]. A. terreus and P. expansum were also used to produce silver nanoparticles, which inhibited A. orchraceus and its mycotoxin production [140]. The uptake of these silver nanoparticles is believed to be localized to the endosomes. They are thought to significantly influence the fungal cells' oxidative stress response and secondary metabolism, as well as to increase transcripts of the superoxide dismutase, which is associated with aflatoxin inhibition [138].
Zinc-containing metallic nanoparticles are also commonly studied. Zinc oxide nanoparticles are considered the most promising of these for drug release and low toxicity [141][142][143]. As with silver nanoparticles, zinc nanoparticles show significant anti-candida effects both as a monotherapy [144,145] and in combination with antifungal drugs such as fluconazole [146]. Thus far, the in vitro antifungal activities of zinc nanoparticles have been evaluated with various strains of C. albicans, C. krusei, C. aprapsilosis, and C. tropicalis [116,144,147]. However, the in vivo studies remain unconvincing; as a result, zinc nanoparticles are currently not indicated for the treatment of a specific candidiasis. Biomedical applications of iron oxide nanoparticles have also been widely investigated due to several attractive characteristics, including magnetism, biocompatibility, and stability [148,149]. Although this type of nanoparticle is mainly used in tissue imaging to assist the diagnosis, several studies indicate its great potential in treating antifungal infection. For example, Candida species are able to form a drug-resistant biofilm in medical apparatuses and instruments, such as catheters. Thus, Chifiriuc et al. synthesized oleic acid and CHCl 3 fabricated iron oxide nanoparticles (Fe 3 O 4 /oleic acid: CHCl 3 ) as a delivery system to carry essential oil from Rosmarinus officinalis and cover the catheter pieces. According to confocal laser scanning microscopy, they found that the essential oil and pulsed iron oxide nanoparticles significantly inhibited the fungal adherence of C. albicans and C. tropicalis. Furthermore, the same research group investigated these nanoparticles for their anti-bacterial capabilities by inhibiting the biofilm formation of Enterococcus faecalis [150,151].
Aside from anti-fungal effects, metallic nanoparticles have been used in fungal diagnoses [152]. The two common causes of human cryptococcosis, C. neoformans and C. gatti, have distinct pathogenic properties, so they require different therapeutic strategies. Detecting Cryptococcus in clinical specimens is time-consuming, and diagnosis is difficult. Artificial positively charged silver nanoparticles have been evaluated to directly distinguish between C. neoformans and C. gattii in clinical specimens using surface-enhanced Raman scattering and spectral analysis. These nanoparticles resulted in better signals than the standard substrate of negatively charged silver nanoparticles in that they selfassembled on the surface of the cryptococcal cell walls via electrostatic aggregation. This novel method based on silver nanoparticles was 100% accurate in distinguishing between the two Cryptococcus species. Biogenic silver nanoparticles Amphotericin B (AmB) C. albicans, C. tropicalis Anti-fungal growth Amphotericin B-conjugated silver nanoparticles with more activity in inhibiting C. albicans and C. tropicalis as compared to AmB only [135] Silver/silver chloride nanopartilces Latex of Azadirachta indica Sensitive and resistant strains of C. tropicalis

Inhibited fungal growth and biofilm formation
Latex fabricated silver/silver chloride nanoparticles inhibited fungal growth and biofilm formation [136] Polyvinylpyrrolidone (PVP)-capped quantum-sized silver nanoparticles (SNPs) Polyvinylpyrrolidone and silver C. albicans Anti-fungal growth The MIC determined that PVP-capped SNP displayed antifungal effects in 70 ng/mL, which was lower than AmB (500 ng/mL), fluconazole (500 ng/mL), and ketoconazole (8 µg/mL) [137] Biogenic gold and silver nanoparticles The high astaxanthin content yeast, Phaffia rhodozyma, is utilized for microbe-assisted nanoparticle synthesis  Rosmarinus officinalis essential oil C. albicans and C. tropicalis Biofilm formation in the medical apparatus and instruments The essential oil pulsed iron oxide nanoparticles significantly inhibit fungal adherence of C. albicans and C. tropicalis. Thus, they inhibit the biofilm formation in the medical instruments [150] Gold and silver nanoparticles Heparin C. albicans, C. krusei, C. parapsilosis Anti-fungal growth

Mesoporous Silica Nanoparticles
Mesoporous silica nanoparticles (MSNs) were firstly developed in the early 1990s and have been shown to have potential applications in areas of biomedicine, such as drug delivery, disease diagnosis, medical imaging, and tissue regeneration [155][156][157]. MSNs are fascinating in drug delivery because they are biocompatible, they are highly chemically and thermally stable, and they provide a large surface area for carrying drug payloads. Moreover, bioactive components can be assembled on the nanoparticle surface and guide the drug-loaded system directly to a specific location [158]. Thus, MSNs have been developed as novel drug delivery systems in the treatments of various diseases [158][159][160]. Table 4 shows a summary of several successful studies that use this nanomaterial in drug delivery systems for treating fungal infections. The anti-microbial effect of Levofloxacin was enhanced by functionalized mesoporous silica nanoparticles with lactic acid [163] pH-sensitive gated MSNs were synthesized for carrying tebuconazole. The assembled nanosystem significantly inhibited yeast growth and could possibly provide an optimal treatment for vaginal mycoses [161]. Furthermore, econazole incorporated into an MSN system has been demonstrated to have antifungal activity against C. albicans [162]. Aside from treatment strategies, MSN systems have also been used to study the functionality of macrophages in fungal infections. Portoles et al. developed mesoporous SiO 2 -CaO nanospheres that were labeled with fluorescein isothiocyanate (FITC-NanoMBGs), and the red fluorescence signal from this fluorescent-labeled MSN system was used to measure the ability of C. albicans to carry out phagocytosis. This provides a critical method for evaluating the macrophage-fungus interface and offers a perspective for understanding host immunity responses during fungal infections [164].
The controlled release of a drug is considered one of the most attractive improvements in anti-fungi therapeutic strategies. Polylactic acid was coated with functionalized MSNs, and this nanosystem was conjugated with levofloxacin to form an MSN-based drug delivery system. In vitro antimicrobial tests showed that this MSN-based nanosystem had significant anti-microbial properties against Staphylococcus aureus, E. coli, and C. albicans. However, this nanosystem was not effective in preventing the growth of A. niger. Nevertheless, this nanosystem was shown to have acceptable cytotoxicity to normal human fibroblasts [163].
MSNs have also been used in combination with metallic nanoparticles, which enhance the fungicidal effects. In 2016, a laboratory from Spain developed a nanosystem that utilized electrospun cellulose acetate to contain silver and copper nanoparticles and supported it with sepiolite and mesoporous silica. This resulted in significantly fungistatic membranes against Aspergillus niger [165]. The controlled release of metal was facilitated by the MSNs. Thus, the antifungal effects were optimized by the synergic use of metallic nanoparticles and MSNs.
Although the cytotoxicity of MSN-based antifungal treatments is acceptable [161,163], overloads of antifungal drugs or heavy metals lead to severe side effects. Thus, the use of naturally isolated antimicrobial agents might be the optimum antifungal therapy. Klouček's research group loaded an essential oil component, eugenol, into MSNs and capped them with various saccharide gates, such as starch, maltodextrin, maltose, and glucose. In vitro experiments showed that the eugenol-loaded MSN system inhibited the growth of A. niger and that the release of eugenol could occur through the degradation of the anchored saccharides by exogenous enzymes.

Polymeric Nanoparticles
Polymeric nanoparticles (PNs) were first developed in the 1970s, and range in size from 5 to 1000 nm. Active ingredients such as antifungal drugs can be loaded by either entrapping them inside the particle or adsorbing them onto polymeric cores. Polymeric nanoparticles are divided into two classes, based on their morphological properties: nanocapsules and nanospheres [166]. A nanocapsule is covered by a polymeric wall and has oil or water in the core, whereas a nanosphere is made up of a continuous polymer network that can retain a drug inside or absorb it onto the surface [166][167][168]. Thus, nanocapsules and nanospheres can be considered reservoir systems and matrix systems, respectively. Several production methods have been used, including polyelectrolytic complexation, ionotropic gelation, and emulsification by evaporation, solvent diffusion, solvent evaporation, or nanoprecipitation [169][170][171][172].
The efficiency of drug dispersion into PNs is determined by several factors, including the physicochemical characteristics of the drug, nanoparticle surface characteristics, the nature of the polymer, and the amount of drug to be loaded. Thus, natural oils and polymeric surface surfactants are used to improve the drug payload in PNs [173,174]. The advantages of PN-based treatments include enhanced therapeutic efficiency and penetration, reduced toxicity, and the delivery of drug action to a target site [175]. This nanosystem has been investigated as an effective therapeutic strategy for treating fungal infections, typically with drugs that are already in use (Table 5).

C. neoformans induced meningoencephalitis in mouse model
Cryptococcal meningoencephalitis The intravenous administration of AmB-PBCA-NP enabled crossing the blood brain barrier and reduced the colony-forming unit counts of cryptococcal meningoencephalitis mouse model. [184] Poly (lactic-co-glycolic acid) nanoparticles with fungal chitosan-binding peptide Itraconazole C. neoformans Cryptococcal pneumonia Polymeric nanoparticle carried cryptococcal chitosan-binding peptide specific recognized and bind to fungal capsules, and thus precisely delivered the itraconazole to clear the C. neoformans from mice lungs [185] To improve the therapeutic effects of antifungal agents, PNs have been widely used in the treatment of candidiasis, including oral candidiasis [176,177,186] and vaginal candidiasis [178][179][180][181][182]. In the former, chitosan was incorporated into PNs, resulting in inhibition of the biofilm adhesion and formation of Candida. Chitosan-PNs significantly inhibited Candida biofilm formation and decreased the number of colony forming units of Candida spp., in contrast with NaOCl, which was used as a positive control [177].
In other cases, PNs were used to optimize antifungal therapies by taking advantage of the large contact surface of the vagina, on which local or systemic fungal infections are obtained. Because it has a broad spectrum of action, itraconazole is a common treatment for vaginal fungal infections. In an attempt to optimize itraconazole treatment, the drug was loaded onto both nanocapsules and nanospheres, which improved the encapsulation efficiency. In a mouse model of vaginal Candida infection, itraconazole-loaded nanocapsules significantly reduced fungal infection, whereas loaded nanospheres did not. Histological analysis indicated that mice treated with the loaded nanospheres had augmented levels of IL-1β and TNF-α and typical tissue inflammation. This showed that the nanocapsules were a more ideal means of improving vulvovaginal candidiasis and a host of immune responses [178].
Another common antifungal drug used for vaginal candidiasis is AmB. In one recent study, nanoprecipitation was used to load AmB onto Eudragit RL100 nanoparticles coated with hyaluronic acid. The Eudragit RL100 nanoparticles facilitated 81% AmB release that lasted for 96 h, although the vaginal fungal burden was eliminated within 24 h. Thus, this PN system provided an optimized drug release platform that significantly promotes antifungal effects. Moreover, this study suggested that the innovative PNs facilitate AmB penetration into the vaginal epithelium via CD44 receptors, rendering this treatment a great improvement over conventional PNs and thus representing a novel vaginal means of optimizing vulvovaginal candidiasis treatment [181].
Polymeric nanoparticles have also displayed low toxicity in murine fibroblasts [180], human keratinocytes [187], kidney epithelial cells, and murine macrophages [183]. Consequently, they can provide nanosystems for optimized treatment of candidiasis. Cryptococcus infections have similarly been treated using PNs. In one case, AmB was loaded onto polybutylcyanoacrylate nanoparticles, which have a great ability to cross the blood-brain barrier (BBB), resulting in an effective treatment for cryptococcal meningoencephalitis. The loaded nanoparticles significantly increased the survival rate of BALB/c mice with cryptococcal infections, and the number of colony-forming units was much lower in the brain tissues of mice treated with nanoparticles compared to those treated with AmB alone. Thus, PNs have great potential for delivering antifungal drugs across the BBB as treatments for encranial infections [184].
The pulmonary region is another infection site for cryptococcosis, where the pulmonary mucus layer is the first point of contact with C. neoformans. In order to eliminate Cryptococcal related pulmonary infection, Li and Liao's team developed a sophisticated drug carrier based on chitosan-conjugated poly (lactic-co-glycolic acid, PLGA) nanoparticles [185]. Chitosan is not only part of the ectocellular structure of Cryptococcus neoformans, but also a typical biomaterial for improving drug oral absorption. Thus, they firstly utilized the phage display library to select a chitosan-binding peptide (12-mer peptide: ADGVG-DAESRTR, CP) that covalently conjugates to PLGA to form CP-PLGA NPs. This was followed by incubation with free chitosan in vitro to form chitosan covered nanoparticles, which were referred to as C-CP-PLGA NPs.
The outer and noncovalent chitosan is responsible for enhancing the CP-PLGA NPs' permeability across the oral absorption barrier. Furthermore, bound chitosan enhances the adhesion to the mucosal layer. The CP-PLGA NPs can be released into circulation to research an infection site. After loading the itraconazole (ITZ) into PLGA, both C-PLGA/ITZ and C-CP-PLGA/ITZ showed remarkable clearing capability for cryptococcal lung infections and increased the survival rate of mice. However, the mortality of C-CP-PLGA/ITZ treated cryptococcsis mice was significantly lower than that of C-PLGA/ITZ treated mice [185].

Lipid Based Nanoparticles
Lipid-based nanosystems are also excellent drug delivery tools for treating fungal infections, reaching therapeutic targets, and providing effective delivery systems (Figure 2).
Liposomes were firstly developed as double-layer lipid systems for drug delivery in 1965. Phospholipids are the primary chemical component of the liposomal structure and offer both hydrophobic and hydrophilic characteristics. Artificial liposomal structures are primarily cholesterol, which provides advantages such as promising stability, moderation of the fluidity of the lipid bilayer, controlled drug release, and stabilization of the vesicles [188][189][190]. These constituents give liposomal structures the ability to incorporate drugs of various polarities: hydrophilic substances are encapsulated in the aqueous region, but hydrophobic substances are held inside the liposome [191].
Liposome nanoparticles are easily fabricated and modified to improve the therapeutic effects of antifungal drugs. The advantages of liposomal nanoparticles also include good biocompatibility and biodegradability. Additionally, they have considerable therapeutic potential and have desirable pharmacokinetics and pharmacodynamics, providing sustained drug release and reduced toxicity [192]. As a result, liposomes are widely utilized as primary delivery vehicles for pharmaceuticals [193][194][195]. Table 6 showed several studies that utilized liposome nanoparticles for treating fungal infections.

Lipid Based Nanoparticles
Lipid-based nanosystems are also excellent drug delivery tools for treating fungal infections, reaching therapeutic targets, and providing effective delivery systems (Figure 2).    Lipid-based nanoparticles (LNP) Voriconazole C. glabrata, A. flavus Ophthalmic candidiasis and aspergillosis LNP improved the poor solubility of voriconaole and enhanced its antifungal effects [211] Solid lipid nanoparticles (SLN) Amphotericin B (AmB) C. albicans Oral candidiasis SLN improved AmB oral bioavailability and alleviated its toxicity, and it enhanced its antifungal effects both in vivo and in vitro [212] Solid lipid nanoparticles (SLN) Terbinafine hydrochloride (TH) C. albicans Skin candidiasis SLN improved the stability and antifungal effects of TH [213] Solid lipid nanoparticles (SLN) Clotrimazole (CLZ) and alphalipolic acid (ALA) C. albicans Anti-fungal growth SLN prolonged drug release without any burst effect and facilitated antifungal effects in animal model [214] Solid lipid nanoparticles (SLN) Natamycin C. albicans, A. fumigatus Fungal keratitis SLN sustained drug release and increased corneal penetration. SLN-natamycin increased antifungal activity without cytotoxic effects on corneal tissues [215] Nanostructured lipid carriers (NLC) Voriconazole C. albicans Anti-candidal hyphae formation NLC sustained drug release, inhibited hyphae formation, and improved antifungal activities in vitro [216] Nanostructured lipid carriers (NLC) Fluconazole Fluconazole-resistant strains of C. albicans, C. glabrata, C. parapsilosis Anti-fungal growth NLC sustained drug release and improved fungicidal activities [217]  Lipid nanocapsules (LNC) Fluconazole Fluconazole-resistant strains of C. albicans, C. glabrata, C. krusei, C. tropicalis Anti-fungal growth NLC provided the high drug carrying and controlled drug release system, enhanced antifungal activity to fluconazole-resistant Candida spp. [223] Lipid nanocapsules (LNC) Amiodarone (AMD) and/or fluconazole (FLU) C. neoformans Anti-fungal growth LNC displayed better antifungal effects, whether it was encapsulated with AMD, FLU, or AMD+FLU, than single usage of these two drugs in cryptococcal infected mice [224] Fungal infections are treated using AmB, the "gold standard" among polyene-based antifungal drugs. Because it is relatively hydrophobic, conventional administration relies on intravenous administration. However, it is significantly nephrotoxic and is associated with infusion-related reactions (up to 53% of patients experience renal dysfunction) [225]. The AmB-liposome conjugates AmBisome ® (a liposomal amphotericin) and Abelcet (an AmB-lipid complex; The Liposome Co., Princeton, NJ, USA) were developed as alternatives to AmB deoxycholate injections. Both are successful commercial nanoformulations of this antifungal drug. Liposomal AmB agents were developed to overcome the severe side effects and nephrotoxicity of AmB [226], and AmB lipid complexes have been developed over decades. Amphotec, an AmB colloidal dispersion system, was also developed. It offers lower renal toxicity and lower mortality according to a case study of 174 patients with invasive aspergillosis [196]. In another case study of 556 patients with various invasive fungal infections, Albecet showed anti-aspergillosis effects with moderately compromised nephrotoxicity compared to AmB [197]. Albecet appears to be better at treating filamentous fungal infections (Aspergillus) than yeast infections (Candida, Cryptococcus).
AmBisome ® provides much greater antifungal effects against Candida [227]. Aside from being a liposome-based treatment for Candida, this nanosystem has been used to optimize Cryptococcus treatment strategies, although the literature is limited. Fu et al. firstly selected a fluorescent polypyridyl ruthenium complex, RC-7, with significantly lower minimum inhibitory and minimum fungicidal concentrations than fluconazole. It was loaded into brain-targeting nano-liposome particles to form RDP-liposome (RDP is a peptide derived from rabies virus glycoproteins). As a result, the fluconazole loaded RC-7 liposome nanoparticles could be guided through the BBB and accumulate in the brain. Remarkably, RDP-liposome prolonged the survival of meningoencephalitis-bearing mice [198]. Despite the many advantages of using liposomes for delivering antifungal drugs, their disadvantages cannot be overlooked. Liposome nanoparticles are soluble, they have short half-lives, and they oxidate and hydrolyze phospholipids [121].
Nanoemulsions (NEs) are dispersed systems of two immiscible liquids (either oil-inwater or water-in-oil) that are stabilized with an appropriate surfactant [228,229]. Particles of these NEs are typically smaller than 500 nm, and they have good stability, rapid digestibility, controlled release, and good bioavailability. Moreover, their physicochemical characteristics allow the delivery of various drugs. The oily component of NEs varies (e.g., essential oils or triglycerides) based on their distinct physicochemical and biological properties, and the water layer can be one of various aqueous components. For instance, because essential oils have been shown to have antifungal effects, they can be used in the oily component [230,231].
One NE containing essential oil from Pelargonium graveolens displayed anti-candida activity. Candida albicans and C. krusey showed less biofilm formation using this NE compared to the free essential oil [199]. Ocimum basilicum essential oil loaded in an NE showed augmented activity against C. albicans and C. tropicalis [231]. Two additional natural essential oils from Pogostemon heyneanus and Eucalyptus globulus were isolated and loaded into NEs, resulting in excellent inhibitory effects on both yeast growth and biofilm formation [200,201].
Even when plant-isolated essential oils are used, NEs can provide ideal delivery agents for known antifungal drugs. A nystatin-loaded NE has been tested against C. albicans for the treatment of skin candidiasis. Results have shown improved pharmacokinetic release of nystatin with good sustainability in the skin and NE acts as an optimal agent for treating skin candidiasis [202,203]. Additionally, ketoconazole and clove oil loaded into an NE showed strong disturbance of Candida fungal cell membranes and a better ketoconazole release profile compared to ketoconazole alone [204].
Besides conventional NEs in antifungal applications, gelling agent and thioglycolic acid have been used as permeation enhancers to improve ketoconazole's effect on onychomycosis. The optimized NE is referred to as nanoemulgel [206]. Nanoemulgel displayed 77.54 ± 2.88% transungual permeation values for ketoconazole through goat hooves in 24 h, in contrast to conventional NEs without a permeation enhancer (transungual permeation values = 62.49 ± 2.98%). Thus, there is no doubt that ketoconazole loaded nanoemulgel shows a significant zone of inhibitionfor T. rubrum and C. albicans. Nanoemulgel also has good biosafety according to skin irritation and histopathology studies on rat skin.
To improve the antifungal activities against C. albicans and A. niger for topical delivery, AmB-loaded NE was fabricated using Capmul PG8 and lipid surfactants. AmB-loaded NE had zones of inhibition of 19.1 ± 1.4 and 22.6 ± 2.0 mm against A. niger and C. albicans, respectively. Ex vitro experiments demonstrated that this nanosystem enhances AmB release and has the highest rate in penetrating the stratum corneum barrier compared to AmB drug solution and Fungisome ® (Liposomal AmB, commercial drug) [205]. In another system, NE provided an optimized nanosystem for improving AmB's local antifungal effect without theoretical systemic absorption [232]. Drug-loaded NEs have been demonstrated to have great potential therapeutic roles for topical fungal infection, but their antifungal roles in the treatment of invasive fungal infections need further evaluation.
Solid lipid nanoparticles (SLNs) are similar to NEs, but the essential oil component is replaced by solid lipids to improve the control of drug release, increase system stability, and reduce toxicity [233]. They are colloidal systems of submicron size, ranging from 50 to 100 nm [234,235]. The concentration of the solid lipid component ranges from 1% to 30% w/w, whereas that of the surfactants ranges from 0.5 to 5% [236]. Because the solid lipid core is lipophilic, several hydrophobic drugs can be packed into this nanosystem. Antifungal drugs such as miconazole, AmB, fluconazole, ketoconazole, and voriconazole have been carried by SLNs.
Nokhodchi et al. developed fluconazole-loaded SLNs and demonstrated its significant inhibition of fluconazole-resistant strains of Candida [207]. After measuring the antifungal activities, fluconazole was released dramatically from SLNs in the first 30 min followed by a sustained release over 24 h. The MIC 50 of fluconazole-loaded SLNs for fluconazole-resistant strains were evaluated (2 µg/mL, 1 µg/mL and 2 µg/mL for fluconazole-resistant strains of Candida albicans, Candida parapsilosis and Candida glabrata, respectively) This nanosystem based drug delivery system for inhibiting Candida strains exhibited low susceptibility against the conventional formulation of fluconazole.
Miconazole is another common broad-spectrum drug used against various fungal species, but it has poor water solubility. The use of SLNs alleviates this problem. Thus, various approaches have been used to enhance the antifungal efficiency of miconazone, such as adding surfactants or incorporating lipid nanoparticles, including SLNs [208,237,238], the latter of which also improves biodegradability, biocompatibility, and protection of the incorporated drug from chemical degradation [239]. In cutaneous and vulvovaginal candidiasis, ketoconazole dispersed into SLNs showed anti-candida activity [209,210]. Voriconazole, another common anti-fungal drug, was loaded into SLNs, and two studies showed antimycotic activity against C. glabrata when administered ocularly [211,240].
Besides azole drugs, as the antifungal gold standard, AmB has also been utilized as an effective agent for incorporation into SLNs to ameliorate its poor oral bioavailability. Both in vivo and in vitro pharmacodynamic and pharmacokinetic studies show that AmB loaded into SLNs provides better bioavailability and lower nephrotoxicity, and the anticandida efficiency of AmB appeared to be potentiated [212,213,241]. To optimize cutaneous treatments, terbinafine hydrochloride was incorporated into SLNs, resulting in prolonged drug activity and a promising alternative for the treatment of cutaneous candidiasis [213].
Not only can SLNs incorporate single antifungal drugs, they can take on and deliver a combination of antifungal agents. A drug delivery system that simultaneously transports two or more drugs to a target site represents a promising strategy that can improve the therapeutic effectiveness of those drugs and overcome drug resistance. Clotrimazole and alphalipolic acid were loaded into SLN, and various effects were found, including reductions in reactive oxygen species and allergic responses. In vitro, the combination showed prolonged drug release without a burst effect. In total, the growth of 25 strains of C. albicans was inhibited by this dual-drug delivery system. This study demonstrated that the antifungal effect of clotrimazole and the protective effect of alphalipolic acid could be combined using this optimized strategy for the treatment of candidiasis [214].
Finally, SLCs have been studied as a means of delivering natamycin with improved therapeutic effects against fungal keratitis [215]. This lipid-based drug delivery system showed sustained natamycin release and better corneal penetration compared to the drug alone. The SLCs significantly improved the antifungal effectiveness of natamycin against clinical isolates of A. fumigatus and C. albicans without cytotoxic effects on corneal tissues. With this evidence, the properties of SLNs render them the ability to significantly increase the bioavailability of antifungal agents while alleviating their side effects. Such SLN nanosystems are favorable for the treatment of cutaneous, oral, ocular, and vaginal fungal infections.
A nanostructured lipid nanocarrier (NLC) is a nanoscale colloidal system that has a hydrophobic nucleus dispersed in an aqueous membrane and is stabilized by a surfactant. It is considered an improvement over SLNs, although the manufacturing methods are similar [242,243]. The attractive properties of a synergic NLC system include nanoparticle size (<100 nm), broad size distribution, long-term stability, and low cytotoxicity to human cell lines (HaCaT and A431). The hydrophobic nucleus gives an NLC better biocompatibility and a larger drug payload compared to the solid lipids in SLNs. Moreover, substitution of the nucleus can improve drug release properties and the incidence of drug expulsion during development and storage [236,244,245]. Thus, broad-spectrum drugs carried by NLCs have applications in the treatment of fungal infections.
Several azole drugs with poor water solubility (voriconazole, clotrimazole, miconazole, fluconazole, and sertaconazole) have been incorporated into NLCs and used to treat fungal infections. One study indicated that voriconazole loaded onto NLCs significantly alleviated biofilm formation and fungal growth of C. albicans. This shows the critical role of NLCs in facilitating the inhibition effects of voriconazole against yeast-hyphae switching, a known critical virulence characteristic of the Candida species [216].
In another study, fluconazole-loaded NLCs were prepared using probe ultrasonication. The particles were approximately 126 nm in diameter and provided sustained drug release over 24 h, resulting in a significant decrease in minimum inhibitory concentration for all Candida species. In particular, isolates of C. albicans were more susceptible to fluconazoleloaded NLCs than C. glabrata or C. parapsilosis [217].
To optimize the treatment of vaginal fungal infections, clotrimazole was dispersed into NLCs, improving its anti-fungal effects by about four times compared to commercial Fungizone TM [218]. The NLCs produced by ultrasonication were viscosized by the addition of poloxamer P407 gel to obtain a clotrimazole-NLC-gel nanosystem. The most significant advantage of this system is its thermogelling property, by which the temperature of vaginal fluids is increased by the clotrimazole-NLC-gel. This facilitates the ability of clotrimazole to pass through the vaginal mucosa, resulting in improved antifungal effectiveness in the treatment of vaginal candidiasis.
To optimize treatments of oral fungal infections, miconazole has been used as the loaded drug, and this NLC-based hydrogel system controlled the release of miconazole [219]. The encapsulation of miconazole into NLCs improved its antifungal activity against C. albicans, giving this nanosystem the ability to obtain the same therapeutic effect with a 17-fold lower dose than a commercial oral gel formulation.
The therapeutic effects of itraconazole against dermal candidiasis have also improved with the use of NLCs [220]. Itraconazole-loaded NLCs decreased trans-epidermal water loss, an index of cutaneous barrier function, in intact skin and tissues damaged by a linear incision. This indicates that the NLCs improved the ability of itraconazole to localize within the skin. This NLC system was evaluated only on Galleria mellonella larvae, and in that setting, it showed significant antifungal effects against Sporothrix brasiliensis and C. albicans [220].
In another study, an NLC-based nose-to-brain drug delivery system was developed to overcome the poor ability of ketoconazole to penetrate the BBB. A large amount of fluorescent-labelled NLCs accumulated in the brain, and ketoconazole-loaded NLCs significantly inhibited the number of colony-forming units of C. neoformans in mouse brains [221]. The use of NLCs is not limited to delivering single antifungal drugs. A Mediterranean essential oil (Rosmarinus officinalis, Lavandula x intermedia, or Origanum vulgare) was loaded along with clorimazole in an NLC system, which increased the drug's effectiveness in an optimized treatment for Candida infections [222]. The essential oil enhanced the dermal permeability of the clotrimazole, possibly as a result of its excellent interaction with the skin, and the nanoparticles provided prolonged in vitro release of clotrimazole. The abilities of C. albicans, C. krusei, and C. parapsilosis to grow were significantly hampered, thus confirming that NLCs containing Mediterranean essential oils and clotrimazole represent a promising strategy for enhancing drug effectiveness against topical candidiasis.
The antifungal gold standard drug, AmB, has been used for many years. To optimize its effectiveness in the treatment of ocular fungal infections, AmB was loaded into a polyethylene glycol PEGylated NLC system using a high-pressure homogenization technique. The molecular weight of the PEG was varied to find the one that provided the best stability. When the NLC was PEGylated with 2K PEG, the system remained stable for at least 4 weeks at both 4 • C and 25 • C. In vitro, this system provided excellent antifungal activity against C. albicans and A. fumigatus. Compared to commercially available AmB formulations such as Fungizone TM and AmBisome ® , it showed significantly better antifungal activity, and compared to AmB alone, it showed lower cytotoxicity.
This structure facilitated the ocular bio-distribution of AmB when applied topically in animal experiments. The fabricated NLC system has an excellent ability to sustain antifungal agents, control drug release, and reduce Candida activity in ocular regions in animals. This optimized method can provide an innovative strategy for treating various fungal infections.
Compared to SLNs, most NLC-based drug delivery systems can be used to combat invasive and systemic fungal infections as well as those of a topical nature. They can also restrict fungal biofilm formation. In summary, this nanosystem provides a promising method for optimizing current antifungal therapies, given their unique properties of high biocompatibility, optimized bioavailability, and varied routes of administration.
Lipid nanocapsules (LNCs) are lipid-based nanocarriers that have a relatively novel structure, given that they have recently been introduced (in the 2000s). They consist of a core containing medium-chain triglycerides that is stealth-coated with a relatively rigid phospholipid/ethoxylated surfactant capsule [246]. Unlike SLNs and NLCs, LNCs are prepared using a low-energy phase-inversion method. The nanocapsule is less than 100 nm in size and has a narrow size distribution. It has high interfacial stability and excellent stability with the payload [247,248]. Such nanosystems have been developed as an effective cancer drug delivery tool to be used via parenteral [249], topical [250], or oral routes of administration [251,252].
Promising outcomes have been achieved when using LNCs for the delivery of antifungal drugs by various routes of administration. To optimize the treatment of dermal candidiasis, itraconazole-loaded LNCs were compared to itraconazole-loaded NLCs. The lipid nanocapsules showed significantly better in vitro characteristics such as the polydispersity index, itraconazole release profile, and antifungal activity against C. albicans. When applied topically, the nanocapsules showed better dermal retention of itraconazole in excised human skin. They provided a critical therapy for the treatment of induced cutaneous candidiasis in rats while avoiding histopathological changes in the epidermal and dermal layers of the skin during a 14-day treatment.
The study showed that LNCs have superior physicochemical characteristics for permeating the skin and treating dermal candidiasis. Resistance is another major problem with azole treatments. To evaluate fluconazole resistance reversion in C. albicans, C. glabrata, C. krusei, and C. tropicalis isolates, LNCs with modified lipid cores were loaded with fluconazole. The LNCs provided advantages such as a reduced fluconazole dose and resistance reversion in all Candida isolates. This study provided a potential application for an azole-LNC nanosystem in the treatment of fungal infections caused by resistant isolates of Candida [223].
To treat cryptococcosis, LNCs have been evaluated for their anti-cryptococcal effects in murine models [224]. This novel study focused not only on the use of LNCs in the delivery of conventional azole drugs, but also on developing synergic drugs for treating cryptococcosis in mice. The target was a fungal calcium channel and a selected blocker, amiodarone, was used as an alternative therapy. Amiodarone is an antiarrhythmic drug that shows broad-spectrum antifungal effects and is effective against C. neoformans [253]. It can interrupt the calcium-calcineurin signaling pathway, significantly compromising cryptococcal virulence [254,255]. It has also been used to treat fluconazole-resistant fungi [256].
In a recent study, amiodarone and fluconazole were loaded singularly and in combination into LNCs. Each one showed antifungal effects when administered intranasally, but the LNCs improved the anti-cryptococcal effects for amiodarone only. Therefore, the LNC system is a critical strategy for improving drug loading that improves efficiency and optimizes conventional cryptococcosis treatments.
Hydrophobic agents such as AmB and azoles can be dispersed into any of the lipidbased nanosystems: SLNs, NLCs, and LNCs. All of these are used in attempts to circumvent adverse effects and improve bioavailability in the treatment of oral, ocular, vaginal, or dermal fungal infections. Moreover, synergic incorporation of antifungal agents with natural biomolecules, such as plant-isolated essential oils, allows lipid-based nanosystems to boost drug sustainability, prolong drug release, and improve antifungal effects [224]. Thus, these three lipid-based nanosystems provide promising methods for optimizing antifungal therapies. However, in vivo experiments must be evaluated further.

Conclusions and Perspectives
Fungal infections have become a global burden and cause significant mortality. Conventional antifungal drugs have unsatisfactory therapeutic benefits, severe side effects, or unfavorable physicochemical properties. Nanoparticle-based drug delivery systems provide alternative strategies for overcoming these limitations, providing favorable properties such as small size, biocompatibility, and low toxicity.
This review has summarized the applications of four different categories of nanoparticles in antifungal therapy, which possesses with distinct properties. The metallic nanoparticles display chemical stability, adjustable pore size, and potential antifungal activity by inhibiting fungal enzymes and inducing the production of reactive oxygen species. Currently, the antifungal effects of gold, silver, zinc oxide, and iron oxide are mainly studied among metallic nanoparticles. However, the cost of materials is a limitation for the large-scale applications of such nanosystems.
MSNs are highly biocompatible and display satisfactory chemical and thermal stability. Due to their excellent plasticity and large drug-loading capacity, MSNs are suitable for loading large amounts of antifungal agents for treating severe fungal infection. PNs are also commonly used nanomaterials in drug-delivery systems and have been developed over half a century. Synthetic or natural polymers endow PNs with great capability to improve target guiding via surface ligand assembly, and fabricated PNs have been used as effective delivery systems for antifungal agents for decades.
Lastly, lipid-based nanoparticles involve several variations: liposomes, NEs, SLNs, NLCs, and LNCs. Although lipids are the fundamental structural components of these nanoparticles, they have distinct properties for incorporating different antifungal agents with either hydrophobic or hydrophilic properties. Moreover, distinct lipid nanoparticles display different tissue-penetration efficiency for circumventing tissue barriers.
The purpose of developing novel delivery systems for antifungal therapies or antifungal agents is to find an effective, economical, and low-toxicity therapeutic strategy. Although these nanomaterials have distinct properties, they significantly enhance drug bioavailability and stability and display non-toxic biological characteristics that can improve the potency of antifungal drugs. Nevertheless, optimized and novel nanomaterials need to be elucidated with more animal experiments and clinical trials to evaluate their antifungal efficiency and undesirable side effects in complex environments.

Conflicts of Interest:
The authors declare no conflict of interest.