Nanomaterial-based therapeutics for enhanced antifungal therapy

Fang Liu ^a, Yongcheng Chen ^a, Yue Huang ^a, Qiao Jin *^a and Jian Ji *^ab
aMOE Key Laboratory of Macromolecule Synthesis and Functionalization of Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China. E-mail: jinqiao@zju.edu.cn; jijian@zju.edu.cn
^bState Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Rd, Hangzhou, 310009, China

First published on 22nd August 2024

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Fang Liu

Fang Liu received her bachelor's degree from Dalian University of Technology in 2018. She is currently pursuing a master's degree in the Department of Polymer Science and Engineering, Zhejiang University. Her research interests include nanotechnology in the treatment of fungal infection.

Yongcheng Chen

Yongcheng Chen received his bachelor's degree from Shandong University in 2017. He is currently pursuing a PhD in the Department of Polymer Science and Engineering, Zhejiang University. His research interests include antibacterial nanoparticles and their biological applications.

Yue Huang

Yue Huang received his bachelor's degree from Zhejiang University in 2015. He received his PhD from the Department of Polymer Science and Engineering, Zhejiang University in 2024. His research interests include antibacterial nanoparticles and development of metal polyphenol networks.

Qiao Jin

Qiao Jin received his PhD from the Department of Polymer Science and Engineering at Zhejiang University in 2010. He worked as a postdoctoral fellow at the University of Marburg from 2011 to 2012. He joined Zhejiang University at the end of 2012 and was promoted to professor at the end of 2021. His main research direction is the design of intelligent nano-drug carriers and their applications in anti-infection areas.

Jian Ji

Jian Ji received his PhD from the Department of Polymer Science and Engineering at Zhejiang University in 1997. Then he worked at Zhejiang University and became a professor in 2004. He is mainly engaged in the basic research on the application of biomedical materials, and has carried out systematic and in-depth research on the biocompatibility and biological function of the interface between living systems and materials.

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1. Introduction

As of the end of 2022, the World Health Organization (WHO) published the inaugural Fungal Pathogen Priority List (FPPL), identifying 19 fungi, including C. albicans, Aspergillus fumigatus, Candida auris, and Cryptococcus neoformans, as significant threats to global public health. This publication not only marked an unprecedented global attention towards fungal infections but also highlighted the inadequacies in existing antifungal strategies.¹ Fungal infections are broadly categorized into superficial and invasive infections.^2,3 Superficial fungal infections (SFIs), such as dermatophytosis, athlete's foot, and oral thrush, affect approximately 25% of the global population.⁴ Invasive fungal infections (IFIs), such as aspergillosis and fungal pneumonia are associated with high mortality rates, posing severe risks to vital organs or tissues, especially threatening individuals with weakened immune systems, such as cancer patients, HIV/AIDS sufferers, and organ transplant recipients.⁵

Despite increasing concerns about fungal infections, the range of available antifungal drugs remains limited, primarily relying on four types of antifungal antibiotics: polyenes, azoles, echinocandins, and pyrimidine analogs.⁶ These antifungal drugs generally suffer from significant side effects, inadequate oral bioavailability, and limited therapeutic targets, and rapid development of resistance, rendering some IFIs virtually untreatable.⁷ Consequently, the development of novel antifungal strategies has become particularly urgent.⁸ In response to this challenge, the evolution of nanotechnology offers new hope for antifungal therapies. Nanomedicines are gaining increasing attention due to their unique biological properties. These nanomedicines are characterized by small size, large specific surface area, strong drug loading capacity, and facile surface modification, allowing them to navigate freely within the body, selectively target infection sites, and reduce side effects, thereby significantly enhancing the specificity and efficiency of antifungal treatments.⁹ Nanomaterials have been extensively applied in drug delivery and disease diagnostics.¹⁰

In the field of antifungal therapy, nanotechnology is applied mainly in two ways. On one hand, some nanoparticles possess inherent antifungal properties, such as silver nanoparticles,¹¹ titanium dioxide nanoparticles,¹² and chitosan nanoparticles,^13,14 all of which have extensive antifungal applications. On the other hand, nanoparticles can be used as nanocarriers to encapsulate and deliver antifungal drugs to improve drug bioavailability and reduce side effects.^15–17 The encapsulation of antifungal drugs in nanomaterials can be achieved by different driving forces, such as hydrophobic interaction, covalent conjugation, and electrostatic interaction.

Nanotechnology has demonstrated great potential in addressing several critical challenges encountered in traditional antifungal treatments. In recent years, some relevant review articles about nanotechnology against fungal infections were reported.^18–21 In these review articles, they mainly focused on different kinds of nanoparticles for antifungal therapy. However, this review article emphasizes three key strategies to enhance antifungal efficacy by nanotechnology, which provides a new perspective to combat fungal infections by nanotechnology. Firstly, nanomaterials, due to their unique surface properties, can be engineered to adhere to fungal cells. For example, nanoparticles modified by specific ligands or antibodies can specifically recognize and bind to molecules on the fungal cell wall or membrane, enhancing interaction with fungi.^22–24 Secondly, nanomaterials can be designed to respond to specific biochemical signals, such as changes in pH or the presence of enzymes, enabling precise drug release at the infected sites.^25,26 The smart drug release in pathological tissues not only increases therapeutic efficacy but also reduces systemic side effects. For example, pH-responsive delivery systems can trigger drug release in the acidic environment caused by fungal infections. Additionally, nanomedicines can non-invasively penetrate natural barriers within the body, such as the blood–brain barrier (BBB) or skin, which is particularly crucial for treating infections that are difficult to solve with traditional methods.^15,17,27 This review will comprehensively explore the application of nanotechnology in the field of antifungal therapy, particularly focusing on enhancing interaction with fungi, stimuli-responsive drug release in fungal infected tissues, and crossing biological barriers (Fig. 1). It will also delve into innovative antifungal nanotechnologies, providing future directions of antifungal therapies.

2. Fungal infections

2.1 Classification of fungal infections

Fungi, a widely distributed group of organisms, vary from unicellular microsporidia and yeasts to multicellular molds, filamentous fungi, mushrooms, lichens, rusts, and smuts. These organisms contribute significantly to human life such as medicine, food and beverage industries. Most of fungi are harmless to humans. However, a minority of fungi can cause severe diseases that impact the health of plants and animals, significantly affecting ecology and agriculture.²⁸ For humans, fungal infections are categorized into superficial fungal infections (SFIs) and invasive fungal infections (IFIs).^2,3 SFIs such as dermatophytosis, athlete's foot, and oral thrush, are generally not life-threatening but can spread to other skin areas and are highly contagious. Occasionally, these infections may lead to secondary bacterial skin infections and permanent hair loss. The primary pathogens include Dermatophytes, Malassezia spp., Candida spp., and molds spp.²⁹ In contrast, IFIs are more prevalent among individuals with compromised immune systems, such as patients with HIV/AIDS, severe combined immunodeficiency (SCID), endocrine metabolic disorders, or those who have undergone organ transplantation.³⁰ It is estimated that over 6.5 million people are affected by IFIs annually, with 3.8 million deaths.^31,32 The main pathogens include Candida spp., Aspergillus spp., Cryptococcus spp. and Fusarium spp.³³

Particularly, Candida spp. is one of the most common fungal pathogens, causing diseases ranging from vulvovaginal candidiasis and oral thrush to deep infections and bloodstream infections such as intra-abdominal candidiasis, Candida endocarditis, osteomyelitis, and candidemia.^34,35 Candida albicans (C. albicans) has become a predominant pathogen in hospital-acquired infections, accounting for 15% of all sepsis cases and 40% of clinical bloodstream infections.³⁶ Among other Candida spp., Candida glabrata has emerged as a primary culprit in invasive candidiasis, with an increasing number of cases reported in recent years.³⁷ Additionally, Candida auris, due to its high transmissibility in clinical settings and multidrug resistance, has been labeled a “superbug”.³⁸

The Aspergillus spp., particularly Aspergillus fumigatus (A. fumigatus), is commonly found in environments such as decaying plant material and soil.^39,40 Typically, this fungus does not cause diseases in individuals with normal immune function. However, Aspergillus can cause severe invasive diseases in immunocompromised patients, such as those undergoing cancer chemotherapy, receiving organ transplants, or those treated with corticosteroids for chronic obstructive pulmonary disease (COPD). The most common form is invasive pulmonary aspergillosis (IPA), where the fungus invades the lung alveoli and may further spread to blood vessels and other organs, such as the brain and heart. Additionally, Aspergillus can also cause allergic bronchopulmonary aspergillosis, characterized by asthma-like symptoms and impaired lung function.³⁹

Cryptococcus spp. infections, primarily caused by Cryptococcus neoformans and Cryptococcus gattii, are predominantly transmitted via the respiratory route and can cause latent infections in humans that reactivate when immunity is compromised.⁴¹ In immunosuppressed patients, Cryptococcus infections can evolve into IFIs, such as cryptococcal meningitis, which accounts for approximately 181000 deaths globally each year, particularly in resource-poor regions.⁴²

Fusarium spp. cause a wide range of infections in humans, including superficial, locally invasive, and disseminated infections. Onychomycosis is a common condition in the healthy population, with up to 10% of cases caused by Fusarium.⁴³ Fusarium spp. is a primary source of fungal keratitis, initially observed mainly among farmers and outdoor workers, but the widespread use of contact lenses has significantly increased the incidence of keratitis among urban populations.⁴⁴ Furthermore, the incidence of Fusarium infections is rising among patients with compromised immune systems.⁴⁵

2.2 Antifungal antibiotics

Unlike bacteria, fungal cells share structural similarities with mammalian cells, such as phospholipid bilayer plasma membranes, and common organelles including the nucleus, ribosomes, Golgi apparatus, mitochondria, and endoplasmic reticulum (Fig. 2(A)). While both mammalian and fungal cells possess cell membranes, their lipid compositions differ; mammalian cell membranes are rich in cholesterol, whereas fungal cells primarily contain ergosterol. Additionally, fungal cells are encased in a robust cell wall (Fig. 2(B)).⁴⁶ Due to the shared eukaryotic nature of fungi and mammalian cells, similarities in cell structure and metabolic pathways often lead to excessive cytotoxicity of antifungal drugs. Consequently, the repertoire of antifungal drugs is not as extensive as that of antibacterial agents, and there has been little significant progress in discovering new antifungal agents in recent years. Currently, clinical treatment of fungal infections mainly relies on four major classes of antifungal drugs: polyenes, azoles, echinocandins, and pyrimidine analogs.^6,47,48 These drugs target fungi through distinct mechanisms (Fig. 2(C)), detailed as follows:

1. Polyene drugs. Polyene antifungals, such as Amphotericin B (AmB), function by binding to ergosterol in the fungal cell membrane, forming channels or pores that lead to leakage of cellular contents and ultimately fungal cell death. AmB is widely used to treat severe fungal infections like cryptococcosis, aspergillosis, and invasive candidiasis due to its broad antifungal spectrum and potent efficacy. However, its significant side effects, including severe nephrotoxicity, fever, chills, myalgias, hypokalemia, and potential cardiac toxicity, limit its widespread usage. Liposomal formulations or polymers containing AmB have been developed to reduce these side effects. There are three available formulations of AmB, which are AmB-lipid complex (ABLC), AmB colloidal dispersion (ABCD), and liposomal AmB (AmBisome).⁵⁰

2. Azole drugs. Azoles, including fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole, primarily inhibit the activity of lanosterol 14α-demethylase (CYP51 or Erg11p), thereby blocking the biosynthesis of ergosterol, a key component of the fungal cell membrane. Therefore, azoles can impair the structure of the fungal cell membrane, affecting its growth and reproduction. Azoles are extensively used to treat Candida infections (such as candidemia), aspergillosis, skin mycoses, and nail fungus. However, azoles can cause liver toxicity, gastrointestinal discomfort and headaches.⁵¹

3. Echinocandin drugs. Echinocandins, including micafungin, caspofungin, and anidulafungin, are a newer class of antifungals that inhibit the activity of β-1,3-D-glucan synthase in the fungal cell wall, thereby blocking cell wall synthesis. Therefore, echinocandins can result in an incomplete fungal cell wall structure, leading to fungal cell death. Echinocandins are primarily used to treat invasive candidiasis and aspergillosis, and also serve as alternative treatments when other antifungal therapies fail or are not tolerated.⁵²

4. Pyrimidine analogs. Pyrimidine analogs, such as 5-flucytosine, mimicking the structure of pyrimidine, interfere with the synthesis of RNA and DNA, which blocks the replication of genetic material and protein synthesis in fungal cells. Flucytosine is primarily used to treat candidiasis and cryptococcosis, often in combination with other antifungals to enhance efficacy. However, flucytosine can lead to side effects such as bone marrow suppression and hepatotoxicity, and resistance can develop easily.⁵³

In recent years, ongoing efforts have been made to develop new antifungal drugs. However, clinical use is still predominantly reliant on triazole antifungals (fluconazole, itraconazole), AmB, and 5-fluorocytosine.^31,54 The adaptive response of fungi to chemical attacks from these antibiotics often leads to treatment failures, highlighting the urgency of developing new antifungal drugs amid increasing fungal resistance.

2.3 Challenges in treating fungal infections

The treatment of fungal infections faces multiple challenges, primarily due to the limited variety of available antifungal drugs, antibiotic unitary therapeutic target, potential side effects, and resistance issues. The continual emergence of drug-resistant fungal strains exacerbates these problems, further restricting the options for effective treatment strategies. Resistance to existing antifungal drugs primarily develops through various mechanisms, including genetic mutations, alterations in drug targets, overexpression of efflux pumps, and biofilm formation. The formation of fungal biofilms significantly reduce the penetration of drugs, thus complicating treatment efforts.⁶

Moreover, current antifungal drugs, particularly potent ones like AmB, are often associated with severe side effects, including nephrotoxicity and hematologic toxicity. These adverse effects not only severely limit the dosage and duration of drug use but also negatively impact the life quality of patients and may necessitate the discontinuation of treatment.^48,55 Given these limitations with existing antifungal medications, the development of new antifungal drugs and antifungal strategies, especially those with novel modes of action, has become critically urgent. Researchers are actively exploring various new therapeutic targets and drug pathways, including the use of nanotechnology to enhance drug targeting and reduce toxicity. The application of nanotechnology not only improves drug delivery efficiency but may also provide new strategies for treating stubborn fungal infections.

2.4 Feasibility of nanomedicine for fungal infections

The application of nanotechnology in the medical field has significantly enhanced the efficacy and safety of various drugs.⁵⁶ The introduction of nano-delivery systems opens a new avenue for the use of traditional antibiotics. Commonly used nano-delivery systems in antifungal treatment include liposomes,⁵⁷ dendrimers,⁵⁸ metal nanoparticles,⁵⁹ carbon quantum dots,⁶⁰ and polymer nanoparticles.⁶¹ For instance, antibiotics encapsulated in liposomes have become a widely used clinical strategy.⁵⁵ Nano-delivery systems offer several advantages in the treatment of fungal infections:^18–21

1. Enhanced bioavailability and solubility. Many antifungal drugs have poor water solubility, limiting their effectiveness. Nano-delivery systems significantly improve the solubility and bioavailability of hydrophobic drugs.

2. Increased drug stability. Nano-delivery systems protect drugs from environmental degradation, thereby prolonging the half-life of drugs in vivo.

3. Improved oral bioavailability of hard-to-swallow drugs. Through nanotechnology, drugs that were previously administered via intravenous injection can be reformulated for oral administration.

4. Reduced side effects. Nano-delivery systems can deliver drugs directly to the site of infection, reducing their impact on healthy tissues and lowering systemic side effects.

5. Controlled release and targeted delivery. Nano-delivery systems can be designed to respond to specific pathological microenvironment, enabling targeted drug release to enhance therapeutic effects.

The subsequent review will delve into three strategies for combating recalcitrant fungi using nanomaterials: enhancing interaction with fungi, stimuli-responsive drug release in fungal infected tissues, and crossing biological barriers. It will demonstrate how these innovative technologies can improve the efficacy and safety of antifungal therapy.

3 Enhancing interaction with fungi

The introduction of nanotechnology significantly enhanced the interaction between drugs and fungal cells, thereby improving treatment targeting and efficiency. Utilizing nanodelivery systems, researchers have developed various strategies to optimize these interactions, including electrostatic binding to fungi, targeting fungal cell walls, targeting fungal cell membranes, and cell-mimicking strategies.

3.1 Electrostatic binding to fungi

Given that fungal cell membranes and cell walls are generally negatively charged,^62,63 different strategies were developed to enhance the interaction between nanoparticles and fungal cells through electrostatic interactions. Positively charged nanoparticles, such as metal–organic frameworks (MOFs), chitosan nanoparticles, and dendrimer drug delivery systems, can effectively bind to fungal cells, thereby enhancing drug penetration.

3.2 Targeting fungal cell walls

Unlike mammalian cells, fungal cells are encased in a dense cell wall, a crucial protective structure for the survival and pathogenicity of fungi.⁷¹ The complex structure of fungal cell walls primarily consists of glucans (50–60%), glycoproteins (20–30%), and smaller amounts of chitin, which impart mechanical strength and flexibility while forming a natural barrier to drugs (Fig. 2(B)).^49,72,73 The fungal cell wall, as the outermost structure, plays a crucial role in interactions mediated with host cells, such as adhesion or phagocytosis processes.⁷⁴ Due to its unique composition, which is absent in human cells, the fungal cell wall represents an ideal target for antifungal therapy. Various strategies were developed to disrupt or dismantle fungal cell walls or to enhance drug delivery efficiency by targeting specific components of the cell wall.

Liposomes, as drug delivery systems, typically rely on passive targeting to reach primary organs, but this approach does not distinguish between normal and target cells. In contrast, targeted liposomes are modified with specific ligands, including antibodies, peptides, or other ligands that bind specifically to specific cell membrane antigens, enabling “active delivery” of drugs to infection sites, thus improving delivery efficiency and reducing side effects.^75,76 Anticancer drugs like iridium(III),⁷⁷ paclitaxel,⁷⁸ as well as pain relief medications such as indomethacin, have already been delivered using targeted liposomes. Dectin-1 is a mammalian protein that binds to β-glucan polysaccharides in nearly all fungal cell walls. Liposomes loaded with AmB and modified with Dectin-1, called as DEC-AmB-LL, significantly enhanced binding to fungal cells, reduced the required dose of AmB, and effectively inhibited A. fumigatus. The binding efficiency of DEC-AmB-LLs was 10 times higher than unmodified liposomes (Fig. 4(A)).²² However, Dectin-1-coated liposomes bind poorly to C. albicans, presumably due to the presence of a thick mannan polysaccharide and mannoprotein outer layers masking their β-glucans. Dectin-2, a mammalian innate immune membrane receptor that binds to mannans in a dimeric form and signals fungal infection, was used to modify liposomes loaded with AmB, creating targeted liposomes DEC2-AmB-LL, which could effectively bind to C. albicans, C. neoformans, and A. fumigatus. The binding efficiency of DEC2-AmB-LLs was 50–150 times higher than that of unmodified liposomes (Fig. 4(B)).²³

Chitosan, a deacetylated form of chitin, naturally exists in many fungal cell walls, providing a crucial target for antifungal strategies.⁷⁹ The chitosan-binding peptide (CP) can be used as a targeting ligand for targeted adhesion of nanoparticles to fungi. For example, Tang et al. identified a CP by phage display. Subsequently, CP was conjugated to the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which served as the itraconazole carrier (CP-NPs). CP-NPs were incubated with free chitosan (C-CP-NPs), allowing the noncovalent binding of chitosan to adhere to mucosal layers. This adherence significantly enhanced the penetration of nanoparticles through the oral absorption barrier into the bloodstream. Once in circulation, the targeting ligand was re-exposed to recognize the fungal pathogen at the site of infection. C-CP-NPs significantly cleared pulmonary infections caused by C. neoformans in a mouse model (Fig. 4(C)).²⁴

3.3 Targeting fungal cell membranes

The fungal cell membrane is one of the most important targets for treating fungal infections. Different antifungal nanoparticles were developed by targeting fungal cell membranes, including improving drug delivery efficacy and direct intervening in the structure and function of the cell membrane.

3.4 Cell-mimicking strategies

Rapid developments in biomimetics have introduced cell membrane camouflage as a promising strategy. Nanoparticles coated with cell membranes have garnered significant attention in targeted delivery of antimicrobials. For example, nanoparticles disguised with platelet membranes were designed to enhance the targeted delivery of antimicrobials to invasive microorganisms.^86,87 Additionally, therapeutic nanoparticles coated with macrophage membranes can specifically adhere to bacteria to exert their bactericidal effects.⁸⁸

Drawing inspiration from the natural binding capabilities between C. albicans and vaginal epithelial cells, Chen et al. developed a “three-in-one” biomimetic nanoplatform called VM(IR780)-PFC(O₂). The photosensitizer IR780 and oxygen-filled perfluorocarbon (PFC) were encapsulated into vaginal epithelial cell membrane coated nanoparticles. The cell membrane coating allowed the nanoplatform to specifically bind with C. albicans in both superficial and deep vaginal epithelia, thereby protecting the host cells from toxin-mediated cytotoxicity. Upon candidalysin sequestration, pore-forming on the surface of the nanoplatform accelerated release of photosensitizer and O₂, resulting in enhanced antifungal power under NIR irradiation (Fig. 6(A)).⁸⁹

Considering the asymmetric biological properties of the membranes, the nanocarriers modified with cell membranes need to ascertain that the membranes are successfully coated on the nanoparticulate cores and they are in the correct orientation. To address this, Li et al. developed a concise and effective “molecular affinity” strategy using the intracellular domain of transmembrane receptors as “grippers” during membrane coating. They modified the surfaces of cationic liposomes with peptide ligands derived from the cytoplasmic protein P4.2. P4.2 could recognize the cytoplasmic domain of band 3, a key transmembrane receptor of erythrocytes. Therefore, P4.2-derived peptide would interact with the isolated RBC membrane, forming a “hidden peptide button”, which ensured the right-side-out orientation. Liposomes coated with cell membranes specifically targeted C. albicans through the interaction between the pathogenic fungus and host erythrocytes, serving to neutralize the hemotoxins secreted by the fungi (Fig. 6(B)).⁹⁰

Besides host cells, immune cells also exhibit strong binding capabilities with fungi.^86,87 Therapeutic nanoparticles coated with macrophage membranes can effectively exhibit fungicidal activity through specific adhesion to fungi.⁸⁸ In a typical example, lanthanide-doped upconversion nanoparticles (UCNPs) loaded with a photosensitizer methylene blue and DNA sensing elements were wrapped by macrophage cell membranes, which exhibited fungal diagnostics, antifungal adhesion, precise fungal eradication, and cytokine isolation. Cell membrane camouflage not only improved the stability and biocompatibility of UCNPs, but also avoided the rapid clearance of immune cells, thus enhancing its fungicidal efficacy. The resulting nanocomposite could perform multiple functions including fungi detection, PDT, and promotion of wound healing (Fig. 6(C)).⁹¹

4 Stimuli-responsive nanoparticles for antifungal applications

A critical feature of fungal infections is the formation of biofilms, comprising various morphological forms of fungal cells such as hyphal cells (chains of cylindrical cells), pseudohyphal cells (ellipsoidal cells joined end to end), and round yeast cells (Fig. 7(A)).⁹² Owing to the formation of fungal biofilm, fungal infections create unique microenvironments at infection sites, characterized by altered physiological and biochemical properties that can be strategically targeted for treatment. These environments typically display reduced pH levels, elevated reactive oxygen species (ROS), and increased enzyme activity, which are crucial for fungal survival and pathogenicity. Lower pH levels, often a byproduct of fungal metabolism and the host inflammatory response, help fungi like C. albicans thrive, facilitating biofilm formation.⁹³ Elevated ROS levels, generated excessively during infections as part of the immune response to destroy pathogens, can damage both fungal and host tissues, exacerbating tissue damage and chronic inflammation.^94,95 Increased activity of fungal enzymes, such as proteases and lipases, helps break down host tissues, aiding fungal invasion and immune evasion.^25,96 Given the distinctive features of fungal biofilms and the inflammation at infection sites, the development of nanoparticles that respond to specific physiological signals, such as acidic pH and ROS changes, and release drugs within the infected microenvironment has emerged as an innovative strategy. This targeted approach ensures that the drug is released directly at the site of infection, maximizing efficacy while minimizing systemic exposure and potential side effects.

4.1 pH-responsive antifungal nanoparticles

The microenvironment of fungal infections and the resulting biofilms typically exhibit lower pH values compared to normal tissues. The pH-responsive antifungal nanomaterials should be stale in normal physiological environment with minimal drug leakage. However, antifungal drugs can be effectively released from the nanoparticles in the fungal infected microenvironment to exert the fungicidal activity. Based on the chemical structures of pH-responsive molecules, pH-responsive mechanisms can be categorized into three types: (1) protonation/deprotonation of amine groups and carboxyl groups; (2) cleavage of chemical bonds; (3) supramolecular assembly/disassembly.⁹⁹

Protonation of amine groups is a common strategy to fabricate pH-responsive nanoplatforms. Specifically, pH-responsive micelles based on poly(ethylene glycol) ethyl ether methacrylate (PEGMA) and poly 2-(diethylamino) ethyl methacrylate (DEAEMA) block-copolymers of P(PEGMA-b-DEAEMA) were developed and loaded with the antifungal drug itraconazole (ICZ) to combat C. albicans biofilms. In the acidic microenvironment of C. albicans biofilms, the pH-sensitive tertiary amine modules of DEAEMA were protonated, leading to the release of ICZ (Fig. 7(B)).⁹⁷

pH-responsive polymers as antifungal carriers have enormous potential. For example, various pH-cleavable chemical bonds such as hydrazone bonds, boronic esters, acetal linkages, and Schiff bases can be introduced into polymers.¹⁰⁰ In our previous study, 2,3-dimethylmaleic anhydride (DA)-grafted low molecular weight chitosan (CS) nanoparticles could convert from negatively charged to positively charged in the acidic infection microenvironment, releasing encapsulated antibiotics (Fig. 7(C)).⁹⁸

Another pH-responsive mechanism involves the assembly and disassembly of supramolecular structures, where metal–polyphenol networks are well-known for their acid-triggered antimicrobial properties.^101,102 Additionally, research directly assembled nanoparticles (TP NPs) using the antifungal agent tebuconazole (TEB) and the plant immune inducer poly(salicylic acid) (PSA). The binding force between nanoparticles is non-covalent interactions. In specific acidic microenvironments caused by plant pathogen invasion, TP NPs exhibited acid-responsive behavior, synergistically exerting antimicrobial effects of the antifungal agent and plant immune inducer. Stimulus-responsive co-delivery of fungicidal nanosystems not only had a longer effective duration but also significantly reduced the genetic toxicity of TEB to plants (Fig. 7(D)).²⁶

4.2 ROS-responsive antifungal nanoparticles

The accumulation of reactive oxygen species (ROS) is a common phenomenon of fungal infections. Therefore, it is promising to design nanomaterials that can respond to ROS levels and release drugs accordingly to effectively treat fungal infections. These nanomaterials can change their structure or solubility in high level ROS, thus releasing their payloads to act directly in the infectsourced sites to eliminate fungi. Futhermore, materials that can consume ROS have been proven to effectively alleviate oxidative stress.¹⁰³ Oxidative stress and inflammation are primary causes of tissue necrosis in fungal keratitis, and reducing ROS and inflammatory responses is a key target in developing treatments for fungal keratitis.

For example, the researchers designed and prepared a GC-EB-VOR nanodrug-based eye drops to treat fungal keratitis. This nanocarrier contained glycol chitosan (GC) as the base, modified with 4-carboxyphenylboronic acid pinacol ester (EB) as the ROS-responsive group, loaded with the antifungal drug voriconazole (VOR). During inflammation or infection, ROS could react with borate esters, resulting in cleavage or change of borate structure, thus triggering the release of VOR. Experimental results demonstrated that GC-EB-VOR exhibited high permeability through the corneal barrier and was able to achieve controlled drug release. To sum up, GC-EB-VOR could respond to ROS and eliminate ROS through drug release and the antioxidant properties of carriers, thus providing a new strategy for the treatment of fungal keratitis.¹⁰⁴

4.3 Enzyme-responsive antifungal nanoparticles

In the microenvironment of fungal infecton sites, increased activity of specific enzymes presents unique opportunities for enzyme-responsive nanomedicine delivery systems. For instance, matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, a key member of the MMP family, is highly expressed in the infectious microenvironment and provides a foundation for targeted drug delivery against fungal infections.¹⁰⁵ Li et al. developed a MMP-3 responsive micro-to-nano (MTN) system for targeted drug delivery against complex fungal infections. This system utilized bovine serum albumin (BSA), a natural ligand for SPARC, as the base for the nanoparticles and constructed microspheres through a specific MMP-3 responsive peptide linkage, achieving precision drug delivery. This design allowed the MTN system to be mechanically captured by the smallest capillaries in the lungs after intravenous injection, then hydrolyzed by MMP-3 in the infectious microenvironment (IME) into BSA NPs, further targeting lung tissue, brain, and infected macrophages (Fig. 8(A)).¹⁰⁵

Lipase is another significant upregulated enzyme in the fungi-infected microenvironment, secreted by various fungi.¹⁰⁷ Poly(ethylene glycol)-poly-(ε-caprolactone) (PGL) polymers, known for their amphiphilicity, excellent biocompatibility, and sensitivity to lipase degradation, were explored in drug delivery. Dong et al. utilized PGL to loaded diketopyrrolopyrrole (DPP) and fluconazole (FLU), preparing nanoparticles named PGL-DPP-FLU NPs. DPP could generate ROS and heat upon light activation. C. albicans secreted lipase at the infection site could degrade PGL polymer and trigger the release of FLU. The PGL-DPP-FLU NPs enabled simultaneous photothermal therapy (PTT), photodynamic therapy (PDT), and antibiotic treatment (ABT), exhibiting excellent antifungal properties (Fig. 8(B)).²⁵

Similarly, a study has designed a novel fungal pathogen-responsive assembly of Cu₂O nanoparticles (Cu₂O–PE–BSA).¹⁰⁸ Cu₂O–PE–BSA, initially formed from Cu₂O nanoparticles and coated with phosphatidylethanolamine (PE) and bovine serum albumin (BSA), were driven by hydrophobic/electrostatic interactions. When the nanoparticles encountered C. albicans, lipase and protease secreted by C. albicans recognized PE and BSA, and then catalyze their decomposition. The decomposition of PE and BSA resulted in the reduction of the stability of the assembly and triggered the assembly process. After disassembly, Cu₂O nanoparticles could contact the cell wall of C. albicans more directly and exert their antifungal effect.

Fungi can also secrete hyaluronidase, which specifically hydrolyzes hyaluronic acid (HA). Candidal vaginitis, a common fungal vaginal inflammation disease typically caused by C. albicans. Vaginal microflora plays a key role in maintaining vaginal microenvironment and health, especially Lactobacillus. For example, Hyaluronidase (HAase) secreted by C. albicans and bacteria can degrade HA. Wei et al. developed a HA hydrogel named rGO@FeS₂/Lactobacillus@HA to treat candidal vaginitis and reduce recurrence. In the vaginal microenvironment, HA was enzymatically decomposed, locally releasing Lactobacilli and rGO@FeS₂ nanozymes. On one hand, Lactobacilli fermented and produced lactic acid, normalizing the vaginal microenvironment and reducing vaginal pH to 4 to 4.5. On the other hand, the rGO@FeS₂ nanozymes could catalyze Lactobacillus-produced H₂O₂ to generate a large amount of ˙OH to kill C. albicans (Fig. 8(C)).¹⁰⁶

5 Crossing biological barriers

Crossing tissue-specific barriers, such as the skin, corneal, and blood–brain barrier (BBB), is crucial for enhancing the effectiveness of antifungal therapies. Nanotechnology offers an effective way capable of crossing these barriers and reaching the infection sites directly.

5.1 Crossing fungal cell walls

Firstly, the fungal cell walls and extracellular polysaccharides of biofilms act as dense barriers that hinder drug entry. The polysaccharides in fungal cell walls, such as β-1,3-glucan, play a crucial role in fungal drug resistance.⁶² The formation of fungal biofilms further enhances their resistance to drugs as the extracellular matrix (ECM) within the biofilm promotes fungal adhesion and protects the internal fungi. β-1,3-glucan, as the primary polysaccharide in the ECM, acts as a permeability barrier, preventing antifungal agents from reaching their intracellular targets.¹⁰⁹ To break through the fungal cell wall and extracellular polysaccharide barriers, Zhou et al. proposed a strategy that involved degrading extracellular polysaccharides, particularly β-1,3-glucan, to increase the sensitivity of antifungal strategies. They developed an integrated nanosystem (MLPGa) incorporating lyticase and Ga ions that can degrade the extracellular polysaccharides in cell walls and biofilms. MLPGa eradicated C. albicans and mature biofilms through the release of gallium ions. Finally, the MLPGa-based antifungal strategy achieved satisfactory therapeutic effects in a fungal keratitis mouse model, showcasing the potential for overcoming critical fungal resistance mechanisms through nanotechnology-enabled interventions (Fig. 9(A)).¹¹⁰

5.2 Crossing skin barrier

Fungal skin infections, as the most common fungal infections, fall under SFIs.²⁹ When fungal pathogens colonize and settle on the epidermal surface of the skin and hair follicles, both superficial and skin tissues are subject to fungal infection. Dermatophytes are among the most prevalent pathogens in skin fungal diseases.¹¹³ For effective combat against superficial fungal infections, antifungals must reach the stratum corneum with sufficient concentration to target the pathogen and retain there long enough to inhibit the fungal pathogens. The development of various antifungals has significantly improved the ability to combat skin fungal diseases. However, penetrating the skin to reach the infection sites remains a significant barrier to antifungal treatment.¹¹⁴ To address this issue, nano-delivery systems such as micelles, liposomes, submicron emulsions, and polymer nanoparticles were developed to enhance the transdermal absorption capabilities of antifungal carriers.^57,115,116 Furthermore, many chemical, biochemical, and physical methods were developed to enhance the effectiveness of transdermal drug penetration.¹¹⁷ Transdermal delivery of nanoparticles has sparked considerable interest in local antifungal treatment due to its high drug permeability, controllability, and ability to sequentially or trigger the release of encapsulated payloads.

5.3 Crossing corneal barrier

Fungal keratitis is an important eye infection disease primarily caused by pathogens such as Fusarium spp., Aspergillus spp., and Candida spp. It occurs when fungi enter the corneal stroma through defects in the corneal epithelium. Once fungi infiltrate the tissue, they replicate within the stroma, gradually spreading to surrounding areas and eventually reaching the anterior chamber.¹²⁵ Without timely treatment, fungal keratitis can lead to corneal perforation, endophthalmitis, and even blindness. It is estimated that over a million cases of fungal keratitis are diagnosed each year, with over half of the affected patients experiencing vision loss or unilateral blindness.¹²⁶ Fungi secrete various toxins and enzymes, such as serine proteases and matrix metalloproteinases, which damage corneal epithelial cells.¹²⁷ Additionally, the corneal epithelium has potential fungal binding sites such as laminin, fibronectin, and collagen, making fungi easily to invade and colonize the cornea, thus fungal keratitis is difficult to cure.^128,129 Topical antifungals are the mainstay of treatment for fungal keratitis, such as 5% natamycin, voriconazole, and AmB. However, these antibiotics struggle to penetrate the ocular surface barriers (tear film barrier and corneal barrier), and their effect against resistant fungi is still limited.^130,131

To overcome the ocular surface barriers, Li and colleagues utilized positively charged nonantibiotic nanoplatform, which not only prolonged eye retention time through electrostatic attraction but also easily traversed the corneal epithelium via extracellular pathways due to their small size. These positively charged small carbon dots (SP-CDs) were effectively internalized by fungi, then released ROS and disrupted fungal cell membranes, exhibiting excellent antifungal activity at low concentrations. In vivo experiments showed that SP-CDs could eradicate fungi and cure keratitis. Additionally, SP-CDs exhibited a favorable ability to penetrate the corneal barrier by temporarily opening the corneal epithelial tight junctions (ZO-1, β-catenin), resulting in enhanced therapeutic efficacy in the deep stroma (Fig. 10(A)).⁶⁰

Given the constant washing by tears, antifungal drugs have a very short retention time on the ocular surface, Only less than 5% of eye medications are utilized effectively.¹³³ To extend the drug retention time on the ocular surface and enhance drug delivery, Shi and colleagues developed a multi-enzyme-mimicking nanozyme-thixotropic anionic hydrogel coating (NHC) for the treatment of fungal keratitis, which was achieved through a Schiff base reaction between a self-synthesized polyaldehyde oligomer (PAO) and amino-functionalized hyaluronic acid (AHA). The thixotropic hydrogel could alter viscosity or flow properties in response to variations in stress or strain. NHC notably increased the retention time and permeability of voriconazole, enabling a minimal dosage to reach effective therapeutic levels. Furthermore, NHC promoted corneal wound healing by stimulating cell proliferation with a derivative of hyaluronic acid (HA). In addition, it combated ROS through the catalase-like and superoxide dismutase-like activities of CuPC, which enhanced the therapeutic efficacy in the treatment of fungal keratitis (Fig. 10(B)).¹³²

5.4 Crossing blood–brain barrier

The blood–brain barrier (BBB) is formed by endothelial cells of brain capillaries creating extremely tight junctions, making it one of the most challenging biological barriers to penetrate.¹³⁴ The BBB effectively blocks most drugs from entering the brain. In recent years, fungal infections of the central nervous system (CNS) have become more prevalent.¹³⁵ Fungal infections of the CNS can manifest in various clinical forms, including meningitis, encephalitis, hydrocephalus, brain abscess, and stroke syndromes. However, the presence of the BBB limits drug concentrations in the brain, hindering antifungal efficacy. It is estimated that over 98% of small molecule drugs and nearly 100% of large molecule drugs do not cross the BBB.¹³⁶ To address this challenge, several nanoparticle-based drug delivery systems capable of targeting the brain were designed.

Angiopep-2 is a ligand that binds to the low-density lipoprotein receptor-related protein and has shown higher endocytosis capacity and parenchymal accumulation compared to transferrin, lactoferrin, and avidin. Angiopep-2 was demonstrated to effectively facilitate the transport of nanoparticles across the BBB and accumulate within the brain.¹³⁷ Taking advantage of Angiopep-2, Jiang et al. prepared PEG micelles loaded with the AmB and surface-modified with Angiopep-2. Both in vitro and in vivo experiments indicated that Angiopep-2 modified micelles exhibited higher efficiency in penetrating the BBB compared to unmodified micelles.¹⁵

The adsorption of apolipoprotein E (Apo E) onto the surface of nanoparticles could increase brain uptake since Apo E could be recognized by low density lipoprotein receptors (LDLR) expressed on brain capillary endothelial cell membranes.¹³⁸ Thus, it is an effective approach for BBB penetration to modify nanoparticle-based drug delivery systems with Apo E. For example, Fricker and colleagues prepared itraconazole loaded poly(butyl cyanoacrylate) polymeric nanoparticles (PBCA-NCAs) to modify PBCA-NCAs was modified with DSPE-PEG(2000)-maleimide and then conjugated with Apo E. The Apo E conjugated nanoparticles enabled the delivery of high concentrations of poorly soluble itraconazole across the BBB to the therapeutic target.¹³⁹

Additionally, rabies virus glycoprotein (RVG29) could specifically bind to nicotinic acetylcholine receptor proteins (nAchR) on neuronal cells. A novel brain delivery system using RVG29 as a targeting ligand was able to deliver the antifungal drug itraconazole to the brain.¹⁴⁰ In addition to peptide modifications, some small molecules can also be used to enhance BBB permeability. Borneol (BO) is a highly lipophilic bicyclic terpene extracted from the resin of Dryoblanops armarica Gaertn. f. BO can facilitate the distribution of CNS drugs in the brain by opening the BBB.¹⁴¹

Importantly, BO-induced BBB opening is a reversible physiological process, unlike the pathological damage caused by stroke. Therefore, BSA nanoparticles (PEG/BO-ITZ-NPs) encapsulating itraconazole and modified with BO and PEG were designed to deliver antifungals to the brain. Compared to unmodified nanoparticles (ITZ-NP), BO modified nanoparticles were expected to have higher drug loading, longer blood circulation time, and better BBB penetration (Fig. 10(C)).¹⁷

6. Novel strategies in nanotechnology against fungal infections

In the field of nanotechnology for treating fungal infections, in addition to the various nanotechnologies already mentioned, many innovative strategies are being developed to enhance antifungal efficacy.

6.1 Fungal vaccines

Vaccines are one of the most effective measures to prevent and eradicate fungal infections.¹⁴² Since Dromer et al. demonstrated the protective effect of a monoclonal antibody (mAb) against C. neoformans in a lethal mouse infection model, the importance of fungal antibodies in preventing fungal diseases has been increasingly recognized.¹⁴³ Given the rapid development of resistance to antifungal drugs and the high mortality associated with invasive fungal infections, the development of effective fungal vaccines for high-risk populations is particularly urgent. Most research currently focuses on developing subunit vaccines. The antigens in subunit vaccines typically have little inherent antigenicity and thus require administration with adjuvants. The most commonly used adjuvant in clinical settings is aluminum salts (alum), which usually induce a strong antibody response to co-administered antigens. Considering that CD4 T helper cells (Th) mediated immunity is crucial in defending against various fungal diseases, studies suggest including adjuvants that trigger strong Th1 or Th17 cell-mediated responses in fungal vaccines.^144–146 Research has also shown that using components of the fungal cell wall, such as β-1,3-glucan and mannan, as adjuvants can enhance the immune response.¹⁴⁷ Successful studies used glucan particles and glucan–chitin particles extracted from Saccharomyces cerevisiae and red algae to develop different types of vaccines. These vaccines induced persistent specific antibody responses and CD4 T cell responses biased towards Th1 and Th17 in mice, providing protective effects against fungal infections. This strategy offered a novel approach to combating fungal infections and laid the groundwork for future vaccine development against fungal diseases.^147,148

6.2 Modulating macrophages

The host innate immune system is the first line of defense against fungal infections. Host innate immune cells recognize invading fungi through pattern recognition receptors (PRRs) and subsequently initiate a series of effector mechanisms and adaptive immune responses to mediate fungal clearance. Among these cells, macrophages play a critical role in recognizing pathogenic fungi.¹⁴² Macrophages engulf pathogens through various PRRs, including Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), and C-type lectin receptors (CLRs),¹⁴⁹ which recognize pathogen-associated molecular patterns (PAMPs) on fungal cell walls, such as lipopolysaccharides, peptidoglycans, and β-glucans.¹⁵⁰ This recognition triggers immune responses to destroy the ingested pathogenic fungi. Therefore, the fabrication of nanoparticles that can specifically target macrophages is a promising strategy for treating fungal infections.

The mouse EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1), also known as F4/80, is a cell surface glycoprotein highly expressed on mature macrophages, including Kupffer cells, Langerhans cells, and microglia. An anti-F4/80 antibody functionalized itraconazole encapsulated core–shell polymer nanoparticle was prepared to achieve targeted drug delivery to macrophages, which significantly enhanced antifungal activity against Haemophilus influenzae-infected mouse macrophages compared to bare nanoparticles (Fig. 11(A)).¹⁵¹

The mannose receptor (MR) is an endocytic protein found on macrophages and dendritic cells. Its extracellular region includes three domains: (i) carbohydrate recognition domains (CRDs), (ii) fibronectin type II domains, and (iii) cysteine-rich domains (CysD). CRDs bind mannose and N-acetyl-glucosamine, while CysD binds sulfated polysaccharides such as carrageenan.¹⁵⁵ Thus, nano-delivery systems functionalized with ligands like glucan, mannose, and fucoidan can effectively target macrophages and activate their phagocytic clearance function. For example, Vyas et al. developed AmB-encapsulated multilamellar vesicles (MLVs) coated with O-palmitoylated mannan (OPM). Compared to unmodified liposomes, OPM-coated formulations showed higher accumulation levels in macrophages and demonstrated better antifungal activity.¹⁵⁶ Furthermore, another study conjugated AmB loaded gelatin A NPs to carboxymethylated i-carrageenan (CMC-AmB-GNPs) to target macrophages through the interaction between sulfated polysaccharides and CysD on macrophages. CMC-AmB-GNPs exhibited significantly higher antifungal effects than bare AmB and AmB-GNPs (Fig. 11(B)).¹⁵²

However, macrophages face challenges when combating pathogenic fungi, as certain fungi develop strategies to evade host defenses. For instance, some fungi mask PAMPs that macrophages recognize with their dense cell walls. Therefore, breaking the immune inertia of pathogenic fungi instead of using immune stimulants can restore macrophage–fungus interactions. Xing et al. reported a micafungin–encapsulated conjugated polymer nanoparticle (CPNs-M) to activate pathogenic fungi-macrophages immune response for intracellular infections eliminations. Under NIR irradiation, CPNs-M exposed β-glucans on the surface of fungal conidia through photothermal damage and drug release. The exposed β-glucans triggered macrophage recognition, activating the Ca²⁺–calmodulin protein (Ca²⁺–CaM) signaling pathway followed by the LC3-associated phagocytosis (LAP) pathway to kill fungal conidia (Fig. 11(C)).¹⁵³

Macrophages exist in two polarization states: M1 and M2. M1 macrophages play a major role in host defense against various microbial pathogens through phagocytosis and antigen presentation, while M2 macrophages, expressing high levels of CD206 and Arg-1, contribute to immunosuppression. TLRs on macrophages, especially TLR-4 activation through conventional recognition of LPS or chitin, play a key role in guiding macrophage polarization towards the M1 phenotype.¹⁵⁷ Jiang et al. designed a mannosylated nanotrinity carrying imatinib with mannosylated chitosan oligosaccharides. Imatinib blocked the STAT6 phosphorylation pathway to reduce the M2 macrophage population. Chitosan oligosaccharides mediated TLR-4 pathway activation, promoting macrophage repolarization to the M1 phenotype. Mannose motifs enhanced macrophage targeting. The nanotrinity could induce in situ remodeling of macrophages, “turning on” M1 phenotype polarization while “turning off” M2 phenotype polarization, thereby eradicating C. albicans infections (Fig. 11(D)).¹⁵⁴

6.3 Phage therapy

Phages are obligate predators of their bacterial hosts, capable of recognizing and hijacking host bacteria to produce progeny, ultimately leading to bacterial lysis.¹⁵⁸ It should be emphasized that phages can not only exhibit direct fungicidal activity, but also identify and engineer peptides that can specifically target fungal pathogens via phage display technology. In 1985, George Smith pioneered phage display technology to present short peptides on the surface of phages.¹⁵⁹ Simply put, this strategy involves selecting specific phages from a large phage library that can target desired biomolecules or cells.¹⁶⁰ Phage display technology has been widely used to screen specific peptide sequences that can target fungal pathogens.

On one hand, phages, due to their nanoscale size, can enter cells and can be engineered to deliver target antigens to stimulate an immune response, thus exhibiting some antifungal effects. For example, secreted aspartyl proteases (Saps) produced by C. albicans are considered key virulence factors. Saps include three different groups: Sap2, Sap1, and Sap3. Studies have utilized phage display technology to screen for two nanobodies (scFvs phages) that can target Sap2. Compared to the control group, scFvs phages treatment significantly improved overall survival rates while reducing colony counts and infection foci.¹⁶¹

On the other hand, phages can serve as targeting peptides after conjugated to the surface of nanoparticles, enhancing drug targeting and achieving precise and rapid antifungal activity. Additionally, phages as vaccine carriers can enhance immune responses against C. albicans infection, as extensively studied. Specific epitopes LKVIRK, DEPAGE, YGKDVKDLFYAQE, SLAQVKYTSASSI, and VKYTS were genetically inserted into the phage pVIII or PIII respectively.¹⁶² These phage-displayed epitopes on pVIII induced the specific antibody response, enhance delayed-type hypersensitivity (DTH) response, stimulated splenocyte proliferation, reduced fungus load, and improved the mice survival rate.^163–165 Phages represent an ideal strategy for drug and vaccine delivery to treat deep-seated fungal infections, with significant research potential in the future.

6.4 Combination therapy

In the face of the growing issue of fungal resistance, combination therapy represents a highly promising strategy in clinic. Combining treatments can reduce the required dosage, thereby minimizing side effects on the host and has become a potential alternative for treating invasive fungal infections.^166,167 Moreover, nanotechnology allows for the integration of multiple antifungal therapies into a single nanoparticle. This combination harnesses multiple antifungal mechanisms to enhance therapeutic efficacy and reduce the development of drug resistance.

For example, a lipase-triggered drug release nanoplatform (PGL-DPP-FLU NPs) was developed for multimodal antifungal therapy. This nanoplatform utilizes the synergistic effects of PDT, PTT, and antibiotic therapy (ABT) to treat azole-resistant C. albicans infections.²⁵ Zhou et al. also proposed AgCu₂O–EDTA nanoparticles, which effectively kill fungi and treat fungal keratitis. AgCuE NPs disrupted the cell structure through an internal cascade of synergistic effects, including metal ion, CDT, PDT, and mild PTT, thereby eradicating C. albicans (Fig. 12(A)).⁶³ Sun et al. developed CuO nanoparticle composites (CuONP@ALGNP@PL) with ε-polylysine–alginate nanogels, which exhibited synergistic fungicidal effects of ε-PL and copper ions in treating A. alternata infections in plants (Fig. 12(B)).¹⁶⁸ Currently, strategies for combination therapy against fungal infections often involve integrating bactericidal agents such as metal ions, antibiotics, and cationic polymers, along with multiple therapeutic methods including CDT, PDT, PTT, and SDT.

6.5 High-throughput screening technologies

High-throughput screening technologies offer a pivotal advantage in the rapid development of antifungal therapies by efficiently identifying potent antifungal agents from vast libraries of small molecules and polymers.¹⁷¹ For example, computational docking studies, like those conducted by V. Aparna, utilize virtual screening to select optimal excipients for drugs such as Amphotericin B (AmB), pinpointing Gelatin A from a set of amino-functionalized polymers due to its superior binding energy.¹⁵² Additionally, a study used high-throughput screening to evaluate 281 (meth)acrylate polymers, identifying several that reduce the adhesion of the human pathogen C. albicans, the crop pathogen Botrytis cinerea, and other fungi (Fig. 12(C)).¹⁶⁹ Expanding the screening library to thousands of compounds, another study used a high-content screening assay based on 96-well microtiter plates to identify 38 pharmacologically active agents that inhibit C. albicans biofilm formation from a library of 1200 FDA-approved non-proprietary drugs. They further tested the potency and efficacy of these drugs, identifying three with novel antifungal activities (Fig. 12(D)).¹⁷⁰

Building upon these foundations, the integration of nanotechnology can further enhance the effectiveness of these newly identified antifungal agents. By encapsulating these agents in nanocarriers, their bioavailability and target specificity can be substantially improved, while reducing systemic toxicity. Nanotechnology not only enables the precise delivery of antifungals to the infection site but also optimizes the release profiles, ensuring sustained therapeutic action. Thus, the synergy between high-throughput screening and nanotechnology not only speeds up the discovery and development of new antifungal drugs but also enhances their efficacy and safety profiles. This combined approach holds great promise of delivering more effective antifungal therapies to the market more quickly, potentially transforming the landscape of antifungal treatment.

7. Conclusion and future prospects

Fungal infections, as one of the most challenging clinical diseases to treat, have become a significant public health threat. Although available antifungal antibiotics (polyenes, azoles, echinocandins, and pyrimidine analogs) are effective in treating fungal infections, the rapid development of resistance renders these antifungal antibiotics less effective. With the increase of fungal infection cases, high mortality rate, limited drug choice, and the shortage of existing drugs, there is an urgent need to solve the disadvantages of the use of existing antifungal drugs. Nanotechnology provides an effective solution to this requirement. Nanotechnology significantly enhances antifungal therapy through multiple mechanisms. Firstly, nanocarriers such as liposomes, chitosan nanoparticles, and dendrimers enhance drug–fungus interaction via electrostatic interactions and specific targeting mechanisms. Secondly, pH, ROS, and enzyme-responsive nanomaterials enable precise drug release in the fungal infected sites, thereby improving therapeutic outcomes. Furthermore, nanotechnology shows great potential in crossing biological barriers by enhancing drug penetration, effectively addressing challenges posed by the skin, cornea, and blood–brain barriers.

The development of fungal vaccines offers a new avenue for preventing and treating fungal infections, with significant progress made in subunit and nano-vaccine research. Nanotechnology strategies that modulate phagocyte function have shown advantages in enhancing host immune responses and clearing fungal infections. Phage therapy, as an emerging antifungal strategy, is gaining attention for its high specificity and targeting capabilities. Combination therapy, which integrates multiple antifungal mechanisms, has effectively improved overall therapeutic outcomes. High-throughput screening technologies accelerate antifungal drug screening and development, facilitating the discovery of new antifungal treatments.

Despite the great potential of nanotechnology in antifungal therapy, there are still many challenges. Currently, most of the studies on antifungal nanoparticles are limited to in vitro experiments. Establishing animal models of fungal infections that closely resemble human diseases is challenging, restricting the evaluation and optimization of nanoparticles in preclinical studies. Data on the biodistribution, metabolic pathways, and long-term biosafety of nanomedicines are still insufficient. The biocompatibility, long-term biological effects, and potential toxicity of nanomedicines also require further evaluation. Additionally, the interaction mechanisms between nanoparticles and fungal cells are not yet fully understood, limiting the precise design of nanoparticles with high antifungal efficiency.

With the development of advanced nanotechnology, safer and more effective nanomedicines will be developed. Researchers may optimize the targeting and therapeutic efficiency of nanoparticles through advanced surface modification techniques and smart responsive systems. Moreover, integrating multiple therapeutic strategies could achieve more effective fungal infection prevention and treatment. In conclusion, nanotechnology offers a promising pathway for antifungal therapy. With continuous advancements and optimizations in nanotechnology and a deeper understanding of fungal infection mechanisms, nanotechnology is expected to play an increasingly important role in antifungal treatments in future.

Author contributions

Fang Liu: conceptualization, data curation, methodology, writing – original draft; Yongcheng Chen: validation; Yue Huang: formal analysis, visualization; Qiao Jin: funding acquisition, writing – review & editing; Jian Ji: funding acquisition, writing – review & editing.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the National Natural Science Foundation of China (52293381, 52022090, 52273154) and the Key Project of Natural Science Foundation of Zhejiang Province (LZ23B040002) is gratefully acknowledged.

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