Formulation and Evaluation of Niosomal Gel Containing Itraconazole And Tea Tree Oil for Enhanced Topical Antifungal Therapy

Fungal infections represent one of the most common dermatological disorders globally, affecting millions of individuals across different climates and age groups. Dermatophytes, yeasts, and molds are the primary etiological agents responsible for superficial and cutaneous mycoses. Common infections such as tinea corporis, tinea pedis, and candidiasis are not only recurrent but also difficult to eradicate due to the limited penetration of antifungal agents through the stratum corneum barrier [1]. According to recent epidemiological data, superficial fungal infections account for nearly 40% of all skin diseases worldwide, with an estimated prevalence exceeding one billion cases annually [2]. Warm and humid environments, immunosuppression, diabetes, and prolonged antibiotic use contribute to the rising incidence of fungal infections. Conventional antifungal treatments are primarily administered through topical or systemic routes. While systemic therapy ensures deeper tissue penetration, it often results in undesirable side effects such as hepatotoxicity, gastrointestinal disturbances, and drug–drug interactions. On the other hand, topical formulations—such as creams, lotions, or ointments—offer localized treatment with fewer systemic effects. However, their clinical efficacy is compromised by poor drug solubility, inadequate skin retention, and frequent reapplication requirements [3,4]. Many azole antifungals, including itraconazole, clotrimazole, and ketoconazole, possess limited water solubility, which hinders effective delivery to the target site. Itraconazole (ITZ), a potent triazole derivative, acts by inhibiting cytochrome P450-dependent 14α-demethylase, an enzyme critical for ergosterol biosynthesis, thereby disrupting fungal cell membrane integrity [5]. Despite its superior antifungal spectrum compared with fluconazole or ketoconazole, the topical delivery of itraconazole is limited due to its high lipophilicity and poor aqueous solubility (1 ng/mL at 25 °C) [6]. Consequently, systemic administration is often preferred, but this route exposes patients to hepatic metabolism and systemic side effects. Therefore, developing an optimized topical carrier that enhances skin permeation while maintaining localized drug concentration has become a significant focus of pharmaceutical research. To address these challenges, researchers have explored vesicular systems such as liposomes, ethosomes, transfersomes, and niosomes. Among them, niosomes—non-ionic surfactant-based vesicles—have gained attention for their structural stability, low cost, and ability to encapsulate both hydrophilic and lipophilic drugs [7]. The amphiphilic nature of niosomes allows them to integrate into the lipid domains of the stratum corneum, promoting better penetration of encapsulated drugs. Additionally, the presence of surfactants like Span 60, Tween 80, and cholesterol contributes to enhanced vesicle rigidity, controlled release, and improved drug retention within skin layers [8]. Recent studies have shown that niosomal formulations of itraconazole significantly enhance antifungal efficacy compared with conventional gels or creams. For instance, Kaoud et al. [9] demonstrated that itraconazole-loaded niosomal gel exhibited superior in vitro release and ex vivo skin permeation, achieving sustained drug deposition over 12 hours. The entrapment efficiency exceeded 80%, with particle sizes in the range of 150–250 nm, which favored uniform skin distribution. Furthermore, antifungal assays against Candida albicans and Aspergillus niger revealed an increased zone of inhibition, confirming enhanced therapeutic performance. Incorporating tea tree oil (TTO) into the niosomal formulation further augments antifungal potency due to its intrinsic antimicrobial and anti-inflammatory activities. TTO, derived from Melaleuca alternifolia, primarily contains terpinen-4-ol, α-terpineol, and cineole—bioactive monoterpenes known to disrupt microbial cell membranes and enhance drug permeability [10]. Several studies have reported synergistic effects between azole antifungals and essential oils, leading to increased sensitivity of resistant fungal strains [11]. Therefore, a combination of itraconazole and tea tree oil encapsulated within niosomes presents a promising strategy for achieving dual antifungal action and improved skin retention. The formulation of a niosomal gel provides additional advantages over simple niosomal dispersions. Gelling agents such as Carbopol 940 or hydroxypropyl methylcellulose (HPMC) improve the viscosity and spreadability of the formulation, ensuring better patient compliance and prolonged contact time at the infection site [12]. The niosomal gel not only provides controlled release of itraconazole and tea tree oil but also reduces irritation and enhances drug stability. The present review aims to comprehensively analyze the development, characterization, and evaluation of itraconazole and tea tree oil-loaded niosomal gels for topical antifungal therapy. It explores formulation parameters, optimization methods, and physicochemical evaluation techniques, alongside discussions on in vitro and in vivo antifungal performance. Furthermore, it assesses the potential mechanisms contributing to improved efficacy, including synergism between the drug and essential oil, enhanced penetration through vesicular delivery, and sustained release kinetics. The paper also compares these advanced systems with conventional antifungal formulations to highlight their superior therapeutic performance and discusses the regulatory and commercial aspects of integrating herbal–synthetic combinations into modern drug delivery platforms. Ultimately, this work emphasizes the transformative potential of niosomal gels in dermatological therapy, particularly for chronic and resistant fungal infections, paving the way toward next-generation antifungal nanomedicines.

2. ITRACONAZOLE: A POTENT ANTIFUNGAL AGENT

Itraconazole (ITZ) is a second-generation triazole antifungal agent with a broad therapeutic spectrum, effectively targeting both superficial and systemic fungal infections. Chemically, it is a synthetic triazole derivative characterized by a molecular formula of C??H??Cl?N?O? and a molecular weight of 705.64 g/mol. Its chemical structure consists of a triazole ring system linked to a dichlorophenyl moiety and a dioxolane ring, which together confer lipophilic properties essential for its activity against fungal cell membranes [13]. Itraconazole is practically insoluble in water but soluble in organic solvents such as methanol and ethanol, which presents challenges in topical drug formulation. Its pKa is approximately 3.7, and it exhibits high protein binding (>99%), further complicating systemic bioavailability when administered orally [14]. Pharmacologically, itraconazole exerts its antifungal effect by inhibiting the cytochrome P450-dependent enzyme lanosterol 14α-demethylase, an essential catalyst in the ergosterol biosynthetic pathway of fungal cells. Ergosterol, analogous to cholesterol in mammalian cells, maintains the integrity, fluidity, and function of fungal cell membranes. Inhibition of ergosterol synthesis leads to the accumulation of toxic sterol intermediates, increased membrane permeability, and ultimately cell death [15]. This mechanism distinguishes itraconazole from polyene antifungals such as amphotericin B, which directly bind to ergosterol, and from allylamines like terbinafine, which inhibit squalene epoxidase. Itraconazole demonstrates remarkable potency against dermatophytes (Trichophyton, Microsporum, Epidermophyton species), yeasts (Candida albicans, Candida glabrata), and molds (Aspergillus fumigatus, Aspergillus niger) [16]. Its broad-spectrum activity and favorable safety profile have established it as a first-line agent for chronic fungal infections such as onychomycosis, systemic aspergillosis, and histoplasmosis. However, systemic administration is constrained by first-pass metabolism in the liver and variability in gastrointestinal absorption. The bioavailability of oral itraconazole is highly pH-dependent, with reduced absorption in patients using proton pump inhibitors or those with gastric achlorhydria [17]. Despite its efficacy, topical application of itraconazole remains limited due to several physicochemical challenges. The drug’s extreme lipophilicity restricts its diffusion through the hydrophilic layers of the stratum corneum, while its crystalline nature limits dissolution in aqueous-based topical systems. Additionally, itraconazole is susceptible to oxidative degradation under light and humidity, which reduces formulation stability [18]. These limitations necessitate the development of novel delivery systems capable of solubilizing itraconazole, maintaining its stability, and ensuring sustained drug release at the target site. One promising strategy involves encapsulating itraconazole into niosomal vesicles, which protect the drug from degradation while enhancing its solubility and permeability through the skin. Niosomes, formed by non-ionic surfactants such as Span 60 and cholesterol, create bilayer vesicles that entrap hydrophobic drugs within the lipid domain. The presence of surfactants lowers surface tension, facilitating enhanced drug diffusion through the stratum corneum’s lipid matrix [19]. In a comparative study by Kaoud et al. [9], itraconazole-loaded niosomes demonstrated improved entrapment efficiency (83.6%) and prolonged drug release (up to 12 hours) compared with conventional gels. Similarly, Nikam and Maniyar [8] reported that incorporating itraconazole into a niosomal matrix enhanced its antifungal activity by 1.5–2-fold against Candida albicans. Another significant advantage of using vesicular systems is the controlled release behavior of itraconazole. Traditional topical formulations often result in rapid drug diffusion, causing transient therapeutic effects and necessitating frequent reapplication. In contrast, niosomal encapsulation provides a sustained release mechanism where the drug diffuses gradually through the lipid bilayer, maintaining a steady-state concentration at the infection site [20]. Controlled release not only enhances antifungal efficacy but also reduces the risk of systemic absorption and potential adverse effects such as hepatotoxicity. Itraconazole’s pharmacokinetic profile further supports its suitability for vesicular encapsulation. The drug exhibits strong tissue affinity and accumulates preferentially in keratinized tissues such as skin, hair, and nails, which are common sites of fungal colonization [21]. Studies have demonstrated that itraconazole concentration in stratum corneum remains detectable for up to 28 days post-treatment, reflecting its high lipophilicity and tissue retention [22]. Encapsulation in niosomes can further enhance this retention by ensuring localized deposition, thereby improving therapeutic outcomes with reduced dosing frequency. Recent innovations have also explored the co-delivery of itraconazole with natural antifungal agents such as tea tree oil (Melaleuca alternifolia) to exploit potential synergistic effects. Tea tree oil not only contributes direct antifungal activity but also acts as a natural permeation enhancer, facilitating deeper drug penetration through lipid membranes [10,11]. The combination of itraconazole with tea tree oil in a niosomal system can therefore address two critical issues simultaneously: low drug solubility and inadequate skin permeation. The physicochemical properties of itraconazole, such as its partition coefficient (log P ≈ 6) and low melting point, make it an ideal candidate for lipid-based and surfactant-based carriers [23]. Its strong lipophilic character allows for efficient entrapment within the hydrophobic bilayers of niosomes, while the addition of cholesterol stabilizes the vesicle structure. Optimization studies typically focus on adjusting surfactant type (Span 40, Span 60, Tween 80), surfactant-to-cholesterol ratio, and hydration conditions to achieve the desired particle size and encapsulation efficiency [24]. A representative study by Belete and Nagasa [5] compared conventional itraconazole gels, liposomal gels, and niosomal gels, revealing that niosomal formulations exhibited superior antifungal activity, lower irritation potential, and enhanced patient acceptability. Furthermore, in vitro diffusion studies using Franz diffusion cells indicated a biphasic release pattern—an initial burst followed by sustained release—which is desirable for maintaining prolonged local activity against fungal pathogens. In summary, itraconazole remains one of the most effective antifungal agents available, but its therapeutic potential in topical formulations is hindered by solubility and permeability limitations. The encapsulation of itraconazole within niosomal vesicles represents a strategic advancement that not only overcomes these barriers but also enhances stability, bioavailability, and antifungal efficacy. When combined with natural permeation enhancers such as tea tree oil, this approach opens new avenues for developing effective, safe, and patient-friendly topical antifungal therapies.

3. TEA TREE OIL (MELALEUCA ALTERNIFOLIA)

Tea tree oil (TTO) is an essential oil obtained by steam distillation of the leaves of Melaleuca alternifolia, a plant native to Australia. The oil has been recognized for more than a century for its broad-spectrum antimicrobial, antifungal, antiviral, and anti-inflammatory properties. Chemically, TTO is a complex mixture of over 100 components, primarily monoterpenes and sesquiterpenes, with terpinen-4-ol, γ-terpinene, α-terpinene, and 1,8-cineole being the major constituents. Among these, terpinen-4-ol is considered the principal bioactive compound responsible for antifungal activity [25]. According to the International Organization for Standardization (ISO 4730:2017), high-quality tea tree oil should contain at least 30% terpinen-4-ol and no more than 15% 1,8-cineole to ensure optimal therapeutic efficacy and minimal irritation potential [26]. The mechanism of action of TTO is multifaceted and involves both physicochemical disruption of microbial membranes and modulation of cellular signaling pathways. The lipophilic terpenes penetrate the fungal cell wall and interact with membrane lipids, leading to increased permeability and leakage of vital intracellular contents such as potassium ions and nucleotides. Transmission electron microscopy studies have revealed extensive membrane damage, cytoplasmic coagulation, and organelle disintegration in Candida albicans exposed to TTO [27]. Furthermore, TTO interferes with the mitochondrial respiratory chain, causing energy depletion and oxidative stress in fungal cells [28]. The antifungal efficacy of TTO has been demonstrated against a wide range of pathogenic species including Candida albicans, Candida glabrata, Trichophyton rubrum, Trichophyton mentagrophytes, and Aspergillus niger [29]. Hammer et al. reported minimum inhibitory concentrations (MICs) of 0.12–0.50% v/v for dermatophytes and 0.25–1.0% v/v for yeasts, values comparable to conventional azole antifungals [30]. Beyond its direct fungicidal effect, TTO exhibits potent anti-inflammatory properties by down-regulating pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-8, which are elevated in chronic fungal dermatitis [31]. This dual antifungal–anti-inflammatory mechanism makes TTO particularly valuable for topical formulations aimed at reducing infection-associated erythema and irritation. From a formulation perspective, TTO’s physicochemical characteristics pose challenges in terms of volatility, oxidation, and solubility. The oil is highly lipophilic (log P ≈ 4.7) and prone to oxidative degradation in the presence of heat, light, or oxygen, leading to the formation of peroxides and allergenic by-products [32]. Encapsulation within vesicular systems such as niosomes or liposomes can overcome these limitations by stabilizing volatile constituents, reducing evaporation, and controlling the release profile. Vesicular encapsulation also masks the strong odor of TTO, improving patient acceptability [33]. Recent advances in nanocarrier systems have highlighted the benefits of incorporating TTO into niosomal and nanoemulsion formulations. Joshi et al. [10] developed a microemulsion gel combining clove oil and tea tree oil for the topical delivery of luliconazole; the formulation achieved enhanced drug permeation and a 2.3-fold increase in antifungal activity compared with conventional gels. Similarly, Nikam and Maniyar [8] reported that niosomal formulations containing TTO and eucalyptus oil exhibited higher entrapment efficiency and prolonged release profiles, attributing this to the partitioning of volatile terpenes within the lipid bilayer. Tea tree oil acts not only as an active antifungal agent but also as a natural penetration enhancer. Terpenes disrupt the highly ordered structure of stratum-corneum lipids, increasing skin permeability without causing irritation when used in controlled concentrations [34]. The presence of α-terpineol and terpinen-4-ol has been shown to fluidize lipid bilayers, thereby facilitating the transdermal delivery of co-encapsulated drugs such as itraconazole [35]. In the context of a niosomal gel, TTO’s penetration-enhancing ability complements the surfactant-mediated permeation of niosomes, creating a synergistic enhancement in dermal absorption. Several studies have explored synergism between tea tree oil and azole antifungals. Osanloo et al. [11] observed that combining TTO with itraconazole or fluconazole reduced the MICs of both agents by up to fourfold against Candida albicans and Trichophyton rubrum. This synergistic interaction was attributed to membrane disruption by TTO, which increased intracellular access of azoles to their target enzyme, lanosterol 14-α-demethylase. Such synergy is particularly advantageous in combating azole-resistant fungal strains, where membrane permeability is often diminished. In topical drug delivery, one of the major drawbacks of using essential oils is their instability during storage. Incorporating TTO into a niosomal gel matrix addresses this issue effectively. The surfactant-cholesterol bilayers protect the oil from oxidation, while the gel network (typically based on Carbopol 940 or hydroxypropyl methylcellulose) provides a controlled-release environment. Kaoud et al. [9] demonstrated that an itraconazole–TTO niosomal gel maintained more than 90% of its initial drug and oil content after three months of accelerated stability testing at 40 °C ± 2 °C and 75% ± 5% RH, indicating excellent stability. The gel also showed superior spreadability and patient compliance compared with commercial creams. TTO’s anti-inflammatory and wound-healing effects further contribute to its therapeutic value in antifungal formulations. Terpinen-4-ol modulates macrophage activity and suppresses nitric oxide production, thereby reducing inflammation at the infection site [36]. Studies in murine wound models have shown that TTO accelerates re-epithelialization and collagen deposition, which may aid in faster recovery from fungal lesions [37]. This makes the combination of itraconazole and tea tree oil particularly effective for conditions such as tinea pedis and candidal intertrigo, where both infection and inflammation coexist. Safety assessments indicate that TTO is generally well tolerated in topical applications at concentrations below 5%. Patch-testing studies involving over 2 000 subjects have reported mild irritation in fewer than 2% of participants, primarily due to oxidized components in improperly stored oils [38]. The incorporation of antioxidants such as α-tocopherol or ascorbyl palmitate, along with niosomal encapsulation, can further minimize oxidative degradation and irritation potential [39].

4. NIOSOMAL DRUG DELIVERY SYSTEM

The concept of vesicular drug delivery systems has revolutionized topical and transdermal pharmacotherapy, offering improved bioavailability, targeted delivery, and controlled drug release. Among these systems, niosomes have emerged as a versatile and stable alternative to liposomes. Niosomes are microscopic, lamellar structures composed primarily of non-ionic surfactants combined with cholesterol and, in some cases, charge-inducing agents such as dicetyl phosphate or stearylamine. These amphiphilic vesicles possess a bilayer configuration capable of entrapping both hydrophilic and lipophilic drugs, thereby accommodating a wide variety of pharmaceutical compounds [40]. Niosomes were first introduced in the 1970s as a more chemically stable and cost-effective substitute for phospholipid-based liposomes. The structural stability of niosomes arises from the use of non-ionic surfactants such as Span (sorbitan esters), Tween (polyoxyethylene sorbitan esters), or Brij series surfactants, which are less prone to oxidation and hydrolysis than phospholipids [41]. These surfactants self-assemble into closed bilayers upon hydration, entrapping aqueous drug solutions within their hydrophilic core while incorporating lipophilic molecules within the hydrophobic bilayer.

Mechanism of formation and structure.
The formation of niosomes involves hydrophobic and van der Waals interactions among surfactant molecules, leading to the generation of bilayer vesicles (Figure 1). Cholesterol is typically added to the formulation to enhance membrane rigidity and prevent leakage of entrapped drug molecules. The size of niosomes can vary from 100 nm to several micrometers, depending on formulation parameters and preparation methods [42].

Figure 1: Schematic representation of a niosome structure showing hydrophilic core, surfactant bilayers, and entrapped drug molecules (adapted from Goyal et al., 2020 [43]).

Several methods are employed to prepare niosomes, including thin-film hydration, ether injection, reverse-phase evaporation, and microfluidization. Among these, the thin-film hydration method remains the most widely used due to its simplicity and reproducibility. In this technique, surfactant and cholesterol are dissolved in an organic solvent (such as chloroform or methanol), followed by solvent evaporation under reduced pressure to form a thin film on the flask wall. Hydration with an aqueous drug solution at an appropriate temperature leads to the spontaneous formation of niosomal vesicles [44].

Advantages over liposomes. While both liposomes and niosomes share structural similarities, niosomes exhibit several distinct advantages that make them more suitable for large-scale pharmaceutical applications. Niosomes are chemically more stable because non-ionic surfactants are resistant to oxidation, whereas phospholipids used in liposomes are prone to hydrolytic degradation. Moreover, surfactants are inexpensive and widely available, allowing for cost-effective manufacturing [45]. Niosomes also display improved shelf-life, lower toxicity, and a greater ability to encapsulate both hydrophobic and hydrophilic drugs in a single carrier.

Composition and physicochemical behavior.
The key components of niosomes include:

• Non-ionic surfactant: Determines vesicle size, stability, and entrapment efficiency. Surfactants such as Span 60 (sorbitan monostearate) and Span 40 (sorbitan monopalmitate) are frequently used because of their high phase transition temperatures and long alkyl chains, which contribute to more stable bilayers [46].
• Cholesterol: Regulates membrane fluidity and permeability. Higher cholesterol content generally increases membrane rigidity and reduces drug leakage.
• Charge inducers (optional): Agents such as stearylamine or dicetyl phosphate can be incorporated to create surface charge, preventing aggregation through electrostatic repulsion [47].

The ratio of surfactant to cholesterol is a critical formulation parameter influencing vesicle size, encapsulation efficiency, and release characteristics. A typical ratio ranges from 1:1 to 2:1, although optimization is required depending on the drug and surfactant type [48].

Mechanism of skin penetration. The enhanced skin permeation achieved through niosomal delivery arises from several mechanisms. First, the surfactants in niosomes act as penetration enhancers, temporarily disturbing the lipid arrangement in the stratum corneum, thereby facilitating drug diffusion. Second, the small vesicular size allows niosomes to accumulate in hair follicles and skin appendages, forming drug reservoirs that enable sustained release [49]. Third, niosomes can fuse with the lipid domains of the stratum corneum, allowing direct drug transfer into deeper epidermal layers. These mechanisms collectively result in higher drug deposition within the skin and reduced systemic exposure. In a comparative study of liposomal and niosomal gels containing fluconazole, Shirsand et al. observed that niosomal formulations demonstrated 2.4-fold greater skin deposition and prolonged antifungal activity [50]. Similarly, Kaoud et al. [9] reported that itraconazole-loaded niosomal gels exhibited higher permeation flux and antifungal efficacy against Candida albicans than conventional formulations.

Characterization parameters. Comprehensive physicochemical characterization is essential for evaluating the performance of niosomal formulations. Key parameters include:

• Vesicle size and polydispersity index (PDI): Determined using dynamic light scattering (DLS); an optimal size range (100–300 nm) ensures efficient skin penetration and stability.
• Zeta potential: Reflects surface charge; values greater than ±30 mV typically indicate good colloidal stability.
• Entrapment efficiency (EE%): Measures the amount of drug encapsulated within the vesicles relative to total drug content.
• Morphology: Examined using scanning electron microscopy (SEM) or transmission electron microscopy (TEM), which confirm spherical and uniform vesicular structures.
• In vitro release studies: Conducted using dialysis membranes to assess sustained drug release behavior [51].

Table 1: Commonly used surfactants and additives in niosomal formulations for topical delivery.

Applications in antifungal therapy. Niosomes have demonstrated remarkable potential for delivering antifungal agents such as itraconazole, fluconazole, ketoconazole, and terbinafine. Their ability to localize the drug in skin layers while minimizing systemic absorption makes them ideal for the treatment of superficial mycoses. Niosomal delivery also mitigates drug resistance by maintaining a steady therapeutic concentration at the infection site [52]. In antifungal therapy, vesicular encapsulation prolongs drug retention, ensures controlled release, and enhances patient compliance by reducing application frequency.

Recent advances and trends. Recent research has focused on developing hybrid vesicular systems that combine the advantages of niosomes with other nanocarriers. Examples include niosomal–nanoemulsion hybrids, ethoniosomes (ethanol-containing niosomes), and niosomal hydrogels. These systems further enhance drug permeation and stability. In a 2024 study by Ahuja and Bajpai [23], hybrid niosomal gels incorporating itraconazole and essential oils demonstrated significantly improved drug retention and antifungal activity compared to standard gels. Additionally, computational modeling has been used to predict drug–surfactant interactions and optimize formulation parameters [53]. In conclusion, niosomes represent a highly adaptable and efficient nanocarrier system for topical antifungal drug delivery. Their superior stability, biocompatibility, and ability to encapsulate diverse drugs provide a solid foundation for designing advanced therapeutic formulations. The incorporation of itraconazole and tea tree oil into niosomal gels exemplifies how combining synthetic and natural agents within this platform can overcome solubility, permeability, and stability limitations, ultimately leading to safer and more effective treatments for fungal infections.

5. Formulation of Itraconazole And Tea Tree Oil-Loaded Niosomes

The formulation of a niosomal delivery system containing itraconazole (ITZ) and tea tree oil (TTO) requires careful optimization of surfactant type, cholesterol ratio, hydration medium, and preparation conditions to achieve the desired vesicle size, entrapment efficiency, and stability. The goal is to encapsulate the hydrophobic itraconazole efficiently within the lipid bilayers while simultaneously incorporating TTO as both an antifungal co-agent and a natural permeation enhancer.

5.1. Selection of Components

a) Non-ionic surfactants

The choice of surfactant plays a pivotal role in determining vesicle characteristics such as lamellarity, rigidity, and permeability. Surfactants commonly used include sorbitan esters (Span 20, Span 40, Span 60) and polyoxyethylene derivatives (Tween 20, Tween 80). Among these, Span 60 (sorbitan monostearate) is preferred for antifungal formulations because of its high phase transition temperature (Tc ≈ 53 °C) and low hydrophilic–lipophilic balance (HLB ≈ 4.7), which result in more ordered bilayers and higher entrapment efficiency for hydrophobic drugs like itraconazole [54]. The inclusion of Tween 80 in certain ratios with Span 60 produces a mixed-surfactant system that improves vesicle deformability and enhances drug permeation through the stratum corneum. Such combinations yield vesicles with smaller particle sizes (100–200 nm) and moderate polydispersity indices (PDI < 0.3), indicating uniform vesicular distribution [55].

b) Cholesterol

Cholesterol is essential for modulating membrane fluidity and preventing vesicular aggregation. It intercalates between surfactant molecules, reducing bilayer permeability and leakage of encapsulated drug. The typical Span 60: cholesterol ratio ranges from 1:0.5 to 1:1 (molar basis). Increasing cholesterol concentration beyond 1:1 result in rigid vesicles with reduced elasticity but improved stability [56].

c) Tea Tree Oil (TTO)

TTO serves a dual function—acting as an active antifungal component and as a penetration enhancer. The incorporation of TTO into the surfactant–cholesterol matrix modifies the hydrophobic region of the bilayer, increasing flexibility and facilitating diffusion of the drug through the skin. Optimal incorporation levels are typically 0.5–1.5% w/w relative to total lipid content [57]. Excess TTO may destabilize the vesicles by disrupting bilayer packing, resulting in increased PDI and leakage [58].

d) Hydration Medium and Additives

The hydration medium often comprises phosphate-buffered saline (PBS, pH 7.4) or distilled water containing 0.1–0.5% w/v glycerol or propylene glycol to maintain osmotic balance and enhance hydration efficiency. The use of hydrating media containing ethanol (5–10%) has been shown to reduce vesicle size and improve entrapment by promoting surfactant solubilization during film hydration [59].

Figure 2: Schematic representation of the formulation process for itraconazole and tea tree oil-loaded niosomes using thin-film hydration method (adapted from Moghassemi & Hadjizadeh, 2014 [60]).

5.2. Methods of Preparation

Several methodologies have been developed for preparing niosomes, but for itraconazole–TTO systems, the thin-film hydration method remains most widely adopted because it offers reproducible vesicle size and high entrapment efficiency.

Step 1: Preparation of lipid phase.

Span 60, cholesterol, and TTO (in specified molar ratios) are dissolved in a chloroform–methanol mixture (2:1 v/v) in a round-bottom flask. The flask is attached to a rotary evaporator and rotated under reduced pressure at 55–60 °C until a thin, uniform lipid film is formed on the flask wall.

Step 2: Film hydration.

The dry lipid film is hydrated with 10–15 mL of PBS (pH 7.4) containing a pre-dissolved itraconazole solution in a small amount of ethanol or DMSO to ensure complete drug dispersion. Hydration is performed for 30 minutes at 60 °C with intermittent agitation to yield a milky suspension of niosomal vesicles.

Step 3: Size reduction and homogenization.

The obtained suspension is subjected to probe sonication (50 W, 5 minutes, in an ice bath) or high-pressure homogenization to reduce vesicle size and achieve uniformity. Subsequent filtration through a 0.45 µm membrane removes unentrapped drug crystals. Alternative preparation techniques include reverse-phase evaporation and microfluidization, which produce smaller unilamellar vesicles with higher entrapment efficiencies but require specialized equipment. Reverse-phase evaporation involves forming a water-in-oil emulsion followed by solvent removal under vacuum, resulting in highly deformable vesicles suitable for transdermal delivery [61].

5.3. Optimization Parameters

The physicochemical performance of niosomal formulations depends on multiple interdependent variables that require optimization through factorial or Box–Behnken experimental designs.

Entrapment Efficiency (EE%)

Entrapment efficiency is a critical indicator of formulation success. For itraconazole, EE values above 80% have been achieved using Span 60: cholesterol ratios of 1:1 and hydration at 60 °C [9,62]. Increasing surfactant concentration enhances EE up to a point, beyond which drug leakage occurs due to increased bilayer fluidity.

Vesicle Size and Polydispersity Index (PDI)

The mean vesicle size of optimized itraconazole–TTO niosomes ranges between 120 and 250 nm, with PDI < 0.3, ensuring narrow size distribution [63]. Vesicle size can be reduced through probe sonication or extrusion through polycarbonate membranes. Smaller vesicles exhibit better skin penetration, while larger multilamellar vesicles provide sustained drug release.

Zeta Potential

Surface charge influences colloidal stability by preventing aggregation. A zeta potential magnitude greater than ±30 mV typically indicates electrostatic stabilization. Incorporating charge inducers such as stearylamine (positive charge) or dicetyl phosphate (negative charge) enhances stability during storage [64].

Effect of Hydration Time and Temperature
Hydration at temperatures above the surfactant’s phase transition temperature (Tc) ensures proper bilayer organization. For Span 60, hydration at 60 °C for 30–45 minutes yields optimal results. Shorter hydration times may result in incomplete vesicle formation, while excessive hydration promotes aggregation [65].

Table 2: Optimization parameters affecting niosomal characteristics.