A Review On Beyond Conventional: Itraconazole Niosomal Gel As A Novel Approach For Candida Albicans Management

The delivery of medication molecules to their intended locations in biological systems has developed into a highly specialized and advanced field of pharmaceutical research. The role of a revolutionary drug delivery system extends beyond the convenience and ease of administration of drug packages; by delivering drug molecules to the target site in the most convenient way, it is also necessary to improve therapeutic efficacy and safety. These innovative carriers offer long-term, sustained medication release in the targeted tissue, improving therapeutic efficacy and reducing adverse effects [1]. The most prevalent places for superficial and moist Candida infections on the skin are these locations [2]. Any part of the body, including mucosal membranes, cuticles, vaginal regions, and moist skin folds, might experience its development [3]. Global concerns surround the health of women's reproductive systems, and one common type of acute fungal infection that causes inflammation of the vulva and vagina is called candidal vulvovaginitis (CV) [4]. Accompanied by several species of Candida (C. albicans, C. glabrata, C. tropicalis, and C. krusei) and, more recently, by the appearance of disease spectra that form biofilms attached to epithelium, it has gained the undeserved reputation of being a difficult drug target for scientists and clinicians [5]. Itraconazole belongs to the imidazole class of synthetic antifungal agents, which function by inhibiting the growth of infection-causing fungus. To treat fungal infections, it is utilized. Triazole medication targets the particular fungal production of lipids in membranes. Itraconazole selectively binds to fungal membranes, impairing their ability to function. 5-fluorocytosine targets DNA replication unique to fungi [6].

DISCUSSION

1. Niosomes –

When cholesterol and non-ionic surfactant of the alkyl or dialkyl polyglycerol ether class are mixed together and then hydrated in aqueous conditions, microscopic lamellar structures known as niosomes or non-ionic surfactant vesicles are created. The same basic procedure is used for preparation: the lipid phase's aqueous phase hydrates the surfactant (which can be either pure or a combination of surfactant and cholesterol) [7]. When applied to topical drug delivery systems, niosomes improve penetration, function as a local drug storage facility for prolonged release, make poorly soluble pharmaceuticals more soluble, and operate as a rate-limiting membrane barrier for controlled administration systems. Numerous niosome–skin interaction mechanisms, including diffusion through skin, contact with skin lipids, and modification of stratum corneum structure to increase skin penetration, have been proposed in the literature for niosomes [8]. The surfactants act as an essential component of niosomes structure which act as permeation enhancers and direct fusion of vesicles with stratum corneum [9]. Niosomes are a better method than liposomes since they are comparatively more stable and cost-effective. Consequently, it is anticipated that using niosomes will enhance fusidic acid's skin penetration. Many nanosystems, such as nanoemulsions, solid lipid nanoparticles, liposomes, ethosomes, niosomes, polymeric nanoparticles, dendrimers, micellar systems, and carbon-based nanostructures, have previously been investigated for the treatment of wounds and related illnesses [10]. Because of their sturdy bilayer structure, niosomes increase the stability of the product by shielding the therapeutic ingredient inside from proteolytic enzymes, external pH changes, and osmotic agents. In contrast to liposomes, noisome has comparatively leaky vasculature. Despite their similar characteristics, niosomes have a few key advantages over liposomes. These include greater skin penetration, which makes them appropriate for the treatment of cutaneous and dermal mycosis, increased chemical stability, longer product shelf life, and reduced costs. Furthermore, a wide range of medicines can be entrapped by niosomes because of their special amphiphilic features. Niosomal preparation's additional shape, size, fluidity, and surface functionalization can be readily adjusted by modifying the formulation's composition and preparation technique. It was discovered that encasing ketoconazole in niosomes enhanced its antifungal effectiveness [11].

       
           
       
    Fig 1. Structure of a Niosome

Formulation Components of Niosomes -

a. Non-ionic Surfactant

A non-ionic surface-active agent serves as the fundamental ingredient in the niosome preparation process. Amphiphilic in nature, non-ionic surface-active molecules have a non-polar tail and a polar head [12]. Compared to anionic, cationic, and amphoteric surfactants, these surfactants are less harmful, more stable, and compatible because they are charge-free. Less haemolysis and surface irritation are brought on by these surface-active compounds on cellular surfaces. These can be employed as emulsifiers, wetting agents, and to increase permeability and solubility. The ability of non-ionic surfactants to inhibit p-glycoprotein can improve absorption and targeting, which is a crucial characteristic [13]. Another significant component influencing the entrapment efficiency may be the phase transition temperature. As an illustration, span 60 shows a high entrapment efficiency, which could be related to a high transition temperature [12].

b. Cholesterol

An essential ingredient in the creation of niosomes is cholesterol. Although cholesterol per se is not necessary for the formation of niosomes, its presence influences numerous noisome characteristics. It may have an impact on the membrane's permeability and stiffness, entrapment effectiveness, stability, rehydration ease for freeze-dried niosomes, duration of storage, and toxicity. When cholesterol is combined with low HLB surfactants, the vesicle's stability can be increased. If the HLB value is more than 6, bilayer vesicles can be formed. When cholesterol is added, the preparation becomes more viscous and therefore harder [12].

c. Charge Inducer Molecule

The formulation of niosomes involves the use of certain chemicals that induce charges. By adding a charge to the surface and stabilizing the niosomes by electrostatic repulsion, these chemicals aid in delaying coalescence. Dicetylphosphate (negatively charged), phosphotidic acid (negatively charged), stearyamine (positively charged), and other compounds are a few instances of molecules that induce charge [12].

d. Hydration Medium

In addition to the components listed above, the hydration medium is another crucial element needed for the niosome preparation process. Phosphate buffer is typically employed as a hydration medium. However, the solubility of the medicine contained determines the pH of the buffer [14]. Zidovudine niosomes (stable vesicles) were generated using the film hydration process in phosphate buffer saline at pH 7.4. Drug leakage increased along with the hydration medium volume, but entrapment efficiency increased when the hydration period was extended from 20 to 45 minutes [15].

Fig 2. A Schematic Diagram of Niosomes showing a Non-ionic Surfactant-based Bilayer Vesicle. Cholesterol and Ionic Amphiphiles could be incorporated into the bilayers for additional Stability.

METHODS OF PREPARATION –

Niosome preparation starts with the hydration of a lipid mixture and surfactant at high temperatures. Optional niosome size reduction is then performed to produce a colloidal suspension [16]. Niosome preparation can be done using a number of well-researched conventional techniques. Among the techniques are sonication, microfluidization, ether injection, and thin-film hydration [17]. Then, using centrifugation, gel filtration, or dialysis, the unentrapped drug is removed from the entrapped drug [18].

a. Ether Injection Method

The dissolution of surfactant in diethyl ether is the initial stage in the niosome manufacturing process by ether injection. After that, the solution is injected using a 14-gauge needle into a drug aqueous solution that is kept at 60°C. Ether vaporization causes singlelayer vesicles to develop, which have sizes ranging from 50 to 1000 nm [19].

b. Thin-film Hydration Technique

The hand-shaking approach, sometimes referred to as the thin-film hydration technique, involves dissolving cholesterol and surfactant in a volatile organic solvent before moving the mixture to a rotary evaporator. A thin layer of the solid mixture is left on the flask wall following evaporation. The target medication is then hydrated into the dry layer using an aqueous phase. This procedure can be completed with mild stirring at room temperature [19].

c. Sonication

Another method for making niosomes is to sonicate a solution containing the medication, cholesterol, and surfactant for three minutes at 60°C. The vesicles created with this technique are often uniformly tiny in size [19].

d. microfluidization

Another repeatable method for achieving this size homogeneity is microfluidization. In operation, two fluidized streams pass through a precisely defined microchannel at a very high speed while interacting with one another [19].

e. Multiple Membrane Extrusion Method

Since then, many techniques for niosome preparation have been established. Using the multiple membrane extrusion approach, a thin film is created by evaporating a mixture of surfactant, cholesterol, and dicetyl phosphate in chloroform. After hydrating the film with an aqueous drug solution, the suspension is extruded through a succession of polycarbonate membranes that can accommodate up to eight passageways [19].

f. Reverse-phase Evaporation Technique

In addition to ether and chloroform, the reverse-phase evaporation method uses a combination that includes cholesterol and surfactant in a 1:1 ratio. The combination is mixed with an aqueous phase containing the target medication, and then it is sonicated at a temperature of 4-5°C. After a little amount of phosphate-buffered saline is added to the mixture, sonication is carried out once again. The residual suspension is diluted with phosphate-buffered saline after the organic solvent is extracted at 40°C under light pressure. The mixture is heated to 60°C for 10 minutes to produce the final product, niosomes [19].

g. bubble Method

The "bubble" approach can be used to generate niosomes without the use of organic solvents. A round-bottomed flask with three necks submerged in water makes up a "bubbling unit." The first and second necks of the flask are filled with a thermometer and a water-cooled reflux condenser, respectively, while the third neck is used to supply nitrogen. Using a "bubbling unit," cholesterol and surfactant combined at 70°C in a buffer are homogenized and "bubbled" at that temperature [20].

h. Emulsion Method

Another way for making niosomes is the emulsion approach, which makes use of an oil-in-water emulsion made from an organic solution of cholesterol, surfactant, and medication in an aqueous solution. The finished product is obtained by evaporating the organic solvent. In contrast, the lipid injection method involves melting and injecting a mixture of lipids and surfactant into a heated aqueous phase that contains the medication [19].

Problems faced during Formulation

• Aggregation, fusion, and leakage are common issues throughout the niosome manufacturing process. These issues are influenced by the physiochemical characteristics of vesicles, such as their size, charge, lamellarity, elasticity, and thermodynamic phase. The afore mentioned restrictions may be circumvented by preparing proniosomes, a dry form of niosomes that hydrate instantly before usage to produce an aqueous niosome dispersion [20].
• The charge and rigidity of the niosome's bilayer structure are indirectly influenced by the interactions that encapsulated medicines often have with the surfactant's head group. In general, hydrophobic medications increase the stability of niosomes, whereas hydrophobic medications decrease it. It's interesting to note that amphiphilic medications don't visibly alter the bilayer structure of niosomes [21].
• The creation of niosomes' size and shape as well as the assembly of surfactants into vesicles are both influenced by the temperature of hydration. The temperature selected should ideally be higher than the gel-to-liquid phase transition temperature [22].
• Niosome size grows in direct proportion to the rise in surfactant hydrophilic–lipophilic balance (HLB). It is deemed that the niosome vesicle formation is relatively steady and optimal if the HLB value is between 4 and 8 [23].
• The surfactant employed in the niosome production process must have a hydrophilic head and a hydrophobic tail. For the preparation of niosomes, surfactants having hydrophobic tails and alkyl (chain length from C12 to C18), perfluoroalkyl, or steroidal groups are often appropriate [24].

2. Niosomal Gel –

It is possible to load drugs into niosomal vesicles, which can then be combined with the right gel base to create niosomal gel. Novel gel compositions may offer the following benefits: delivery of drugs at a certain location, staying away from first-pass metabolism removal of gastrointestinal discomfort brought on by specific medications, reduced frequency of dosage and maintenance of a controlled and prolonged drug level Self-administration and direct distribution to the intended site of action avoid drug-related side effects and hasten the end of the drug's action. Comparing niosomal gel to normal carbopol gel, another study showed that the former shows approximately 6.5 times higher drug localization in the skin, suggesting improved niosomal gel target accumulation. To improve the stability of the vesicles, charge inducers like cationic (cetyl pyridinium chloride and sterylamine) or anionic (diacetyl phosphate and lipoamine acid) components are frequently added to the formulation. It functions by preventing vesicles from aggregating because of net repulsive forces [26]. Niosomes' special properties enable application via a variety of topical routes, including ophthalmic, mucosal, and vaginal. Ning and colleagues looked at the clotrimazole-loaded niosomal gel's antifungal properties. The findings showed a regulated and prolonged release pattern in rats that was well tolerated at the tissue level for appropriate local vaginal treatment [27]. In comparison to standard niosomal formulations, negatively charged niosomes integrated into hydroxyethyl cellulose gel exhibit superior physical and chemical stability [28].

The following is the mechanism of niosomal gel absorption:

Lipophilic drugs penetrate the stratum corneum by way of niosomes, which diffuse from the stratum corneum layer of skin overall. Niosomes interact with the stratum corneum through aggregation, fusion, and adhesion to the cell surface, resulting in a high thermodynamic activity gradient of the drug at the vesicle-stratum corneum surface. The structure of the stratum corneum may be altered by niosomes, which would result in a looser and more permeable intercellular lipid barrier [29].

3. Itraconazole –

Itraconazole is an orally active triazole antifungal agent, that demonstrates broad spectrum activity against a number of fungal species which includes dermatophytes, Malassezia furfur, Candida species, and Histoplasmacapsulatum [30]. It is also recommended for systemic infections in cases where other antifungal medications are inappropriate or ineffective, such as aspergillosis, candidiasis, and cryptococcosis. Itraconazole acts by preventing the synthesis of ergosterol, which is a crucial part of the fungal cell membrane [31]. Because of its somewhat acidic pKa of 3.7, this salt can only be ionized at extremely low pH levels. Its aqueous solubility is also low, being slightly greater than 4 ng/ml at pH 1 and nearly equal to 1 ng/ml at pH 7. Accordingly, it falls into class II of the Biopharmaceutical Classification System, which includes compounds with poor solubility and high permeability for which the dissolution rate is the factor limiting absorption. It is therefore categorized as a highly lipophilic molecule. Itraconazole is not well absorbed following oral administration, similar to other medications with low water solubility. This results in a wide range of absorption extent and rates, which in turn causes variations in blood levels and Area Under the Curve (AUC) values. Since P-glycoprotein controls the pre-systemic first pass effect, its role in the oral absorption of itraconazole may also contribute to the substantial variability in the drug's bioavailability. Owing to Itraconazole's limited bioavailability (maximum of 55% when taken with a full meal), transdermal administration is the favoured alternate mode of administration. The stratum corneum is the primary barrier to drug delivery via the skin, and several strategies have been proposed to overcome it [32]. Itraconazole uses a variety of methods to carry out its mission. It prevents the formation of fungal-mediated ergosterol by inhibiting lanosterol 14?-demethylase [33]. It is distinct in that it suppresses angiogenesis and the hedgehog signaling pathway, which connects it to the suppression of trafficking, glycosylation, phosphorylation of VEGFR2, and cholesterol production [34]. Owing to its efficacy, Itraconazole is used in a number of systemic oral marketed treatments, including Fungitraxx® and Sporanox® [35].

4. Candida albicans

Globally, systemic infections are primarily caused by Candida albicans and other newly discovered NAC species, including as Candida glabrata, Candida krusei, Candida tropicalis, and Candida parapsilosis. These microbes are the most frequent cause of mucosal or superficial vaginal infections. In certain cases, they can also penetrate the bloodstream and cause deep-tissue infections [36]. As a diploid polymorphic yeast of mucosal surfaces, Candida albicans is a frequent microflora member found in the human gastrointestinal (GI), respiratory, and genitourinary systems. Normally benign, this commensal fungus has the ability to transform into an opportunistic pathogen in people with weakened immune systems or immunodeficiency. This microbe can interact with a range of host cells, including Th17 cells, over the course of illness symptoms [37]. Within the damage response framework, the results of C. albicans infections can be categorized into six groups based on the host immune response, the anatomical site of infection, the virulence of Candida, and the morphology of hyphae and hyphae-specific gene expression as important virulence factors [38].

       
           
       
    Fig. 3 The morphological switches and transitions of Candida Albicans during the infection process

There are various morphological forms of Candida albicans, including hyphae, pseudohyphae, and blastospores (Fig 3). By budding, bacterospores divide asexually [39]. On the surface of the blastospore, new cell material forms during that process. The new bud, which starts the growth phase, develops from a tiny, carefully chosen blastospore and is typically found away from the location of a birth scar. Following the completion of the growth phase, the cells divide, causing the daughter to create a partition that divides it from the parent cell [39]. Pseudohyphae are characterized by elongated yeast cell chains, while hyphae are characterized by branched tubular cell chains that do not narrow at the sites of septation [40]. Higher than 37 °C, an alkaline pH, serum, and high CO2 concentrations all promote filamentation [41]. Similar to this, it is also improved when N-acetylglucosamine (GlcNAc) is present but nitrogen and carbon are absent [40]. When a blastospore turns into a hypha, a complex regulatory network of signal pathways, including numerous transcription factors, is activated [41]. The hypha wall contains somewhat more chitin than the yeast wall, which is the primary compositional difference between the two [42]. The vital component of the cell membrane that gives it stiffness, stability, and resistance to external stresses is sterol [42]. The most prevalent type of sterol, ergosterol is found in fungal cell membranes. It is produced on lipid bodies and the endoplasmic reticulum [43]. There is a phospholipid bilayer in the cell membrane that contains proteins that function as receptors as well as those that are involved in signal transduction and transport [44]. Candida albicans employs amino acids as a supply of nitrogen and glucose as a source of carbon in its metabolism [45]. Chitin, protein, and glucan comprise the cell wall. Its function is to shield the cell from environmental stressors such osmotic shifts, dehydration, and temperature swings as well as from the host's immune system [46].

1. Size, morphology and size distribution of niosomes

Numerous methods, including electron microscopic examination, photon correlation spectroscopy, light microscopy, coulter counters, and Scanning Electron Microscopes (SEM), Transmission Electron Microscope (TEM) [47], The size and morphology of niosomes can be ascertained using the freeze-fracture replicator, light scattering, zeta sizer and metasizer [12]. Because the two methods employ different measuring methodologies, the particle size determined by the transmission electron microscope is smaller than that determined by the dynamic light scattering (DLS) approach [48].

2. Entrapment Efficiency

It can be calculated by subtracting the amount of unloaded drug from the total amount of drug added [1]. Techniques such thorough dialysis, filtration, gel chromatography, or centrifugation can be used to determine the unloaded medication [49]. By dissolving niosome in 50% n-propanolol or 0.1% Triton X-100, the concentration of loaded medicines can be determined. The resulting solution can then be tested using any particular technique [50]. Following equation can be used to calculate the % entrapment efficiency [12] -

%Entrapment Efficiency = Quantity of drug-loaded in the niosome/ Total quantity of drug in the suspension X 100

3. Charge on niosome and zeta potential

The charge on niosomes makes them repel one another. Additionally, they remain stable because this electrostatic repulsion stops them from aggregating and fusing [51]. Zeta potential is used to determine the charge on a niosome. The DLS apparatus, high-performance capillary electrophoresis, microelectrophoresis, mastersizer, zeta potential analyzer, and pH-sensitive fluorophores are utilized to measure the zeta potential [52]. The equation used to calculate zeta potential is Henry's equation [53] –

£ = ?E?? /?

Where £ = Zeta potential.

?E = Electrophoretic mobility

? = Viscosity of medium

? = Dielectric constant

4. Membrane rigidity

A fluorescent probe's mobility can be utilized to assess the stiffness of the membrane as a function of temperature [54]. Fluorescence polarization can be used to determine the membrane's microviscosity and gain an understanding of the niosomal membrane packing structure [55]. The fluorescence anisotropy (r) of the fluorescence observations (? = 350–425 nm) obtained using a luminescence spectrometer can be calculated using the equation that follows [56]:

Florescence Anisotropy(r) = (IVV - GIVH) / (IVV + 2GIVH)

5. In-vitro release

Dialysis membrane technique is used to study in vitro release. This procedure involves placing niosomes in a dialysis bag, which is then placed in a container containing a buffer or other dissolving liquid. The entire assembly is maintained at a regulated temperature of 37 °C using a magnetic stirrer. A sample is obtained from the receptor compartment at predetermined intervals, and the drug concentration is measured using any technique described in the literature [57][58]. Agarwal et al. employed an alternative technique to investigate the dispersion of morusin from niosomes. They mixed 15 cc of phosphate buffer with a pH of 4.5 and 7.4 to distribute 15 mg of the product. We collected this sample using fifteen eppendorf tubes. For nine days, these tubes were turned nonstop at a temperature of 37 °C at a speed of 130 rpm. The tube is removed at a prearranged interval and centrifuged at 15,000 rpm for 30 minutes. The amount of drug in the supernatant was determined using spectrophotometry [59].

6. Tissue distribution / In-vivo study

The method of distribution, drug concentration, action, and duration of drug presence in tissues such the liver, lung, spleen, and bone marrow all affect in-vivo niosome investigations [12]. Using animal models, the tissue distribution of a medication can be investigated. Animals must be sacrificed in order to remove various tissues, including the liver, kidney, heart, lungs, and spleen, which should then be homogenized, centrifuged, and cleaned with buffer in order to investigate the distribution pattern. The drug content of the supernatant is examined [59].

7. Stability studies

On storage, the drug may leak from the niosomes, because of aggregation and fusion [12]. Stability studies involve frequent evaluations of criteria such as entrapment efficiency, size, and shape [60]. Studying the impact of gastrointestinal enzymes on niosome stability was done by Bayindir and Yuskel [61]. In this investigation, the drug and drug-loaded niosomes were exposed to various gastrointestinal enzymes, including chymotrypsin, trypsin, and pepsin. It was discovered that the medication was shielded from gastrointestinal enzyme destruction by the niosomes.