Nanotechnology-Based Diagnosis and Treatment of Fungal Infections

Fungal infections are a global health problem, especially in immunocompromised individuals such as physicians, organ transplant recipients, or people with HIV/AIDS (1). These infections, primarily caused by pathogenic species like Candida, Aspergillus, Cryptococcus, and Histoplasma, can lead to a wide range of clinical manifestations, from superficial skin infections to life-threatening systemic conditions (2). The incidence of fungal infections has increased due to the emergence of various types of antifungal drugs that reduce the effectiveness of antifungal medications. (3).

Figure: 1. Nanotechnology-based drug delivery systems. (20)

1.1 Limitations of Conventional Antifungal Treatments and Diagnostics

Conventional antibiotics, which generally include polyenes (e.g. amphotericin B), azoles (e.g. fluconazole), and echinocandins (e.g. caspofungin), often have many limitations, including high toxicity, poor solubility, limited bioavailability, and the occurrence of drug reactions (4). Amphotericin B is still one of the most effective antibiotics, but its use is limited due to its nephrotoxicity (5). Furthermore, fungal pathogens have resisted azoles, the most commonly used antifungals, which has driven an urgent need for alternative treatment options (6). In addition to medical problems, fungal diseases also face significant limitations. Current methods such as culture methods, polymerase chain reaction (PCR), and serological tests are often time-consuming and not sensitive enough to detect early. They cannot distinguish between colonization and infection (7). Considering these limitations, there is a need for new methods in the diagnosis and treatment of fungal diseases. Nanotechnology offers a revolutionary tool to solve these complex problems in this context.

1.2 Role of Nanotechnology in Addressing Fungal Infections

Nanotechnology is manipulating matter at the atomic, molecular, or supramolecular level, usually at 1–100 nanometres. Artifacts of this size have chemical, physical, and biological properties that differ from larger materials (8). These properties make nanomaterials ideal candidates for many biomedical applications, including drug delivery, diagnosis, and treatment. Nanotechnology offers new solutions for fungal diseases that overcome the limitations of antibiotics and diagnostic methods. Nanoparticles can be designed to increase drug solubility, select fungal pathogens, penetrate biofilms, and increase drug bioavailability (9). Clinical applications of nanotechnology are equally promising. Nanosensors provide rapid, sensitive, and specific detection of fungal diseases, enabling early diagnosis and treatment. (10).

1.3 Classification of Nanomaterials in Fungal Infection Management

Nanomaterials used in fungal infection management can be broadly classified based on their composition and application. Each class of nanomaterial offers distinct advantages and challenges depending on the context of their use.

1.3.1 Organic Nanomaterials

Organic nanomaterials mainly include carbon-based structures such as liposomes, dendrimers, micelles, and polymeric nanoparticles. These materials are biodegradable and biocompatible, making them ideal for drug delivery.

• Liposomes are spherical vesicles composed of lipid bilayers, capable of encapsulating both hydrophilic and hydrophobic drugs. Liposomal formulations such as liposomal amphotericin B (AmBisome) have lower toxicity compared to drug formulations when administered clinically. (11). Liposomes can also be functionalized with specific targeting ligands to deliver antibodies to the site of infection and reduce side effects. (12).
• Polymeric nanoparticles are versatile carriers that can be engineered to release drugs in a controlled manner. These nanoparticles have been used to deliver antibiotics such as itraconazole, increasing the solubility and bioavailability of the drug. (13). Due to their biocompatibility and tunable release properties, polymeric nanoparticles are promising tools for antifungal therapy.
• Dendrimers are highly branched synthetic polymers that can be functionalized with multiple ligands or drugs. Dendrimers exhibit several effects that enable targeted drug delivery and increase solubility and cellular uptake (14). Research into dendrimer-based formulations continues with a focus on increasing their specificity and reducing potential toxicity.

1.3.2 Inorganic Nanomaterials

Inorganic nanomaterials are made of metal or metal oxide nanoparticles such as silver, gold, zinc oxide, etc. These materials can prevent many fungal diseases due to their ability to produce reactive oxygen species (ROS) and disrupt fungal cell walls and membranes (15).

• Silver nanoparticles (AgNPs) are one of the most studied inorganic nanomaterials in the field of antibiotic use. AgNPs exhibit broad-spectrum antifungal activity by inducing oxidative stress and disrupting the integrity of fungal cell membranes (16).  AgNPs also prevent biofilm formation, which is a major challenge in the treatment of persistent fungal infections (20). AgNPs are effective against bacteria such as Candida auris (16).
• Gold nanoparticles (AuNPs) can be used in combination with phototherapy to selectively target and kill fungal pathogens due to their photothermal properties (17). When working with antibodies or target molecules, AuNPs show greater selectivity and efficacy, reducing the potential for side effects.
• Zinc oxide nanoparticles (ZnO NPs) have also emerged as effective antibacterial agents. ZnO NPs induce oxidative stress and disrupt fungal biofilms, which is important for the control of persistent infections caused by Candida and Cryptococcus (18). These nanoparticles have the potential for therapeutic and preventive applications.

1.3.3 Hybrid Nanomaterials

 Hybrid nanomaterials combine organic and inorganic components to create multifunctional systems with enhanced antifungal properties. Nanomaterials can be developed to have diagnostic and therapeutic functions, allowing for the detection and treatment of fungal diseases. (13).

• Metal-organic frameworks (MOFs) are hybrid nanostructures containing metal ions coordinated with organic ligands. MOFs have been used to encapsulate antibodies and deliver drugs in a controlled manner, with the added benefit of ROS release to promote drug resistance (18).
• Nanocomposites are another example of hybrid materials that combine an organic matrix with inorganic nanoparticles. For example, polymer composites loaded with silver nanoparticles have been used to increase the stability and antifungal activity of conventional drugs (19). These hybrid systems provide a versatile platform for the development of next-generation vaccines.

1.4 Applications of Nanotechnology in Fungal Infections

The application of nanotechnology in fungal infection management can be broadly divided into two major categories: diagnostics and therapeutics.

1.4.1 Nanotechnology in Diagnostics

Early diagnosis of fungal infections is important to improve patient outcomes, especially in immunocompromised individuals, as early diagnosis can prevent infection. Nanotechnology is leading to the development of unique and specific diagnostic tools, such as nanosensors that can detect fungal pathogens at low concentrations. Proteins and other biomarkers can provide rapid and accurate diagnosis, even in the early stages of disease.

1.4.2 Nanotechnology in Therapeutics

Nanoparticle-based drug delivery systems are revolutionizing the way antifungal drugs are administered. By encapsulating antifungal agents in nanocarriers, such as liposomes, dendrimers, and polymeric nanoparticles, it is possible to enhance the solubility, stability, and bioavailability of these drugs. Nanocarriers can also be engineered to target fungal cells selectively, reducing off-target toxicity and improving therapeutic efficacy (20). The ability of nanoparticles to overcome drug resistance mechanisms further highlights their potential as next-generation antifungal therapies. Mechanisms of action, and the potential challenges of integrating nanotechnology into clinical practice.

2. Current Limitations of Traditional Diagnostics and Antifungal Treatments

2.1 Limitations in Diagnostics

Fungal infections are notoriously difficult to diagnose due to the non-specific symptoms they present and the limitations of current diagnostic methods (20). Traditional culture techniques, while still considered the gold standard, are slow, taking up to several days or even weeks to yield results. Serological tests, though faster, often lack the sensitivity needed for early detection, particularly in immunocompromised patients where fungal loads may be low (20). PCR-based molecular diagnostics have improved the speed and accuracy of fungal detection; however, these methods are still expensive and require specialized equipment, limiting their accessibility in resource-constrained settings (21). Moreover, traditional diagnostic methods often fail to differentiate between colonization and infection, leading to unnecessary or delayed treatments.

Figure: 2.  Nanosensors for Fungal Detection. (21)

2.2 Challenges in Antifungal Therapy

Antifungal therapies are currently based on three main drug classes: polyenes, azoles, and echinocandins. All groups face significant challenges. Polyenes, such as amphotericin B, are effective against many fungi but are severely nephrotoxic. Azole drugs, such as fluconazole, are widely used but have become ineffective due to the increasing number of antifungal agents. Echinocandins target fungal cell wall synthesis and have limited oral bioavailability, limiting them to intravenous injection (22). In addition, biofilms formed by fungal pathogens are difficult to treat. Biofilms protect fungal cells from antibiotics, allowing them to survive high doses of antibiotics (23). The emergence of antifungal agents such as C. auris adds to these challenges, highlighting the need for new therapeutic strategies (25).

3. Nanotechnology for the Diagnosis of Fungal Infections

Nanotechnology holds promise for addressing the limitations of traditional diagnostic methods. Nanomaterials exhibit unique properties due to their size and large surface area, making them ideal candidates for the development of sensitive devices and specialized diagnostics. Nanosensors are of particular interest due to their ability to detect fungal infections at an early stage.

3.1 Biosensors Based on Quantum Dots

Quantum dots (QDs) are semiconductor nanoparticles with unique optical properties, such as size-tunable light emission and resistance to photobleaching, making them ideal for diagnostic applications (28). Quantum dot-based biosensors have been developed to detect fungal diseases and enable accurate and specific information with optimal, sensitive, and specific clinical samples.

The sensor achieved specificity for Candida species with bacterial DNA detection as low as 10 pg/mL, far exceeding the sensitivity of traditional PCR-based diagnostics (29). This technology has the potential to improve early detection of fungal infections, especially in individuals with early immunity.

3.2 Nanosensors for Fungal Detection

For example, graphene oxide nanosheets have been developed to detect Candida bacteria in clinical samples. These nanosheets show excellent sensitivity and can detect fungal infections at levels as low as 10^2 CFU/mL, allowing for early and accurate diagnosis. Another example is the use of quantum dots conjugated to antibodies to prevent Aspergillus infection in immunocompromised patients (29). The use of high fluorescence quantum dots provides important results in the diagnostic process by allowing immediate monitoring of fungal diseases. Nanosensors are sensitive devices that can detect low concentrations of biomarkers, making them useful for early detection of fungal infections (36). These sensors typically use materials such as graphene oxide, carbon nanotubes, and quantum dots, which have been shown to detect fungal pathogens with high sensitivity and specificity (36).

Table 1: Comparison of Traditional and Nanotechnology-Based Diagnostic Methods for Fungal Infections

4. Nanotechnology in the Treatment of Fungal Infections

Nanoparticles hold great promise in overcoming the limitations of antibiotic therapy. Due to their small size and tunable surface properties, nanoparticles can be designed to improve the solubility, stability, and bioavailability of antibiotics, while also reducing toxicity and improving target specificity.

4.1 Nanoparticle-Based Drug Delivery Systems

Nanoparticles such as liposomes, dendrimers, and polymeric nanoparticles are widely investigated as pharmaceutical agents for antimicrobial activity. These nanocarriers improve the pharmacokinetics of anti-inflammatory drugs, allowing for greater delivery and reducing side effects. For example, liposomes have been used to encapsulate amphotericin B, one of the most potent antibiotics available. Liposomal amphotericin B (AmBisome) reduces formulation-associated nephrotoxicity while maintaining efficacy. Similarly, polymeric nanoparticles have been used to improve the solubility and bioavailability of poorly water-soluble antifungal agents such as itraconazole, thereby increasing their therapeutic efficacy (38). Chemically synthesized polymers provide another promising platform for drug resistance. Dendrimers can be functionalized with antibiotics and targeting ligands due to their distinct orientations, allowing for specific targeting of fungal pathogens. In addition, dendrimers can increase the solubility of hydrophobic drugs, improving their bioavailability and therapeutic effects.

Table 2: Types of Nanocarriers Used in Antifungal Therapy

4.2 Metal Nanoparticles for Antifungal Therapy

Metal nanoparticles, including silver (AgNP), gold (AuNP), and zinc oxide (ZnO), have been shown to have potent antibacterial properties against various fungal pathogens (26). These nanoparticles can inhibit fungal cells, induce oxidative stress, and prevent biofilm formation, making them particularly useful in anti-inflammatory treatments.

4.2.1 Silver Nanoparticles (AgNPs)

Silver nanoparticles have attracted widespread attention due to their broad-spectrum antimicrobial properties. AgNPs act as antibiotics by generating reactive oxygen species (ROS) that damage fungal cell walls and membranes, ultimately leading to cell death. Studies have shown that AgNPs are effective against Candida species, including resistant strains such as Candida auris.

4.2.2 Gold Nanoparticles (AuNPs)

Gold nanoparticles are effective and efficient options for treating fungal infections when used in conjunction with photothermal therapy. AuNPs can be functionalized with antibodies or targeting ligands, allowing them to bind specifically to fungal cells. When exposed to near-infrared (NIR) light, AuNPs generate local heat that kills fungal cells without damaging surrounding tissue.

4.2.3 Zinc Oxide Nanoparticles (ZnO NPs)

ZnO nanoparticles exhibit anti-inflammatory properties by increasing oxidative stress and inhibiting fungal cell walls. These nanoparticles are particularly effective against Candida and Cryptococcus. Zinc oxide nanoparticles have also been shown to inhibit fungal biofilm formation, which is important in the treatment of persistent infections.

5. Mechanisms of Action of Nanoparticles in Antifungal Therapy

Nanoparticles exert their anti-inflammatory effects through different mechanisms depending on the type of nanomaterial used, including the production of reactive oxygen species (ROS), disruption of fungal cell membranes, and inhibition of biofilm formation (12).

5.1 Reactive Oxygen Species (ROS) Generation

Many metal nanoparticles, including silver and zinc oxide, produce reactive oxygen species when in contact with fungal pathogens. These ROS cause oxidative stress by damaging important cellular components such as lipids, proteins, and DNA. This causes the fungal cell wall and membrane to deteriorate, ultimately leading to cell death.

5.2 Disruption of Fungal Cell Membranes

Nanoparticles, particularly metal nanoparticles, can directly interact with fungal cell membranes, causing structural damage and loss of membrane integrity. This disrupts the normal function of the cell membrane, leading to leakage of cellular contents and eventual cell death.

5.3 Inhibition of Biofilm Formation

One of the biggest problems in treating fungal infections is the formation of biofilms, which protect fungal cells from attack by antibiotics and the immune system. Nanoparticles have been shown to inhibit biofilm formation by preventing fungal pathogens from adhering to surfaces and by disrupting the extracellular matrix that supports biofilm structure.

Table 3: Mechanisms of Action of Nanoparticles in Antifungal Therapy