Development And Evaluation of Drug Loaded Metal Oxide Nanoparticles for Antifungal Topical Application

Dermatophytosis, also known as fungal infections, is one of the most common skin diseases affecting millions of people globally, especially in hot and humid climates where fungi thrive. Dermatophytosis, candidiasis, and tinea infections are common superficial fungal infections that affect the skin, hair, and nails, causing itchiness, irritation, and discomfort. Dermatophytosis is often caused by dermatophytes like Trichophyton, Microsporum, and Epidermophyton, which infect the keratinized structures of the body [1]. The rising prevalence of fungal infections and inefficiencies of conventional topical formulations due to poor penetration, low bioavailability, and the need for frequent applications have led to the development of novel drug delivery systems [2].Tolnaftate is a commonly used antifungal drug formulated into topical products for the treatment of dermatophytic infections, including athlete's foot, ringworm, and jock itch. It works by blocking squalene epoxidase, which in turn blocks ergosterol synthesis and fungal cell membrane production. Yet, Tolnaftate has low water solubility that may affect its efficacy and distribution in traditional formulations.

So, designing new drug delivery systems with improved solubility, stability, and sustained release is crucial to enhance its antifungal activity. Nanotechnology offers a potential solution in drug delivery to improve the therapeutic efficacy of drugs. Zinc oxide nanoparticles (ZnO NPs) have attracted particular interest as nanomaterials because of their physicochemical characteristics, such as high surface area, antimicrobial properties, biocompatibility, and stability [3]. ZnO nanoparticles have inherent antifungal and antibacterial activity and can be used as carriers in topical drug delivery systems. Their small size facilitates drug penetration and enhances the efficacy of antifungal drugs.

The use of plant extracts for green synthesis of nanoparticles is an eco-friendly and economical alternative to chemical synthesis. Bioactive components in plant extracts, including flavonoids, phenolics, and polysaccharides, serve as reducing and stabilizing agents in nanoparticle synthesis. Aloe vera extract is widely used in green synthesis due to its antioxidant, anti-inflammatory, and stabilizing properties [4]. Furthermore, Aloe vera possesses wound-healing and antimicrobial activity, contributing to the therapeutic effectiveness of nanoparticle-based topical gels.

The inclusion of drug-loaded nanoparticles in gel-based formulations provides ease of administration, patient acceptability, extended retention, and sustained drug delivery [2]. Carbopol gels are widely employed in topical drug delivery because of their superior gelling behavior, stability, and compatibility with various drugs and excipients. Adsorption of Tolnaftate onto ZnO nanoparticles can enhance drug loading efficiency and provide sustained drug release, improving therapeutic efficacy. Thus, the current study focuses on the development and characterization of Tolnaftate-loaded ZnO nanoparticle gel synthesized via a green method using Aloe vera extract. The proposed system is anticipated to improve antifungal efficacy, stability, and drug release properties when compared to traditional topical drug products.

Materials-

Tolnaftate was purchased from Yarrow Chem Pvt. Ltd., zinc acetate from CDH, methanol from loba chemie ,naoh from fisher scientific,  Carbopol 934 from pallav, propylene glycol, methyl paraben , propyl paraben, triethanolamine

Methods-

• Preparation of Aloe vera Extract

Fresh Aloe vera leaves were washed thoroughly with distilled water to remove impurities. The outer peel was removed, and the inner gel was collected. Approximately 10 g of gel was homogenized with 100 mL of distilled water and heated at 60–70°C for 30 minutes. The mixture was filtered using Whatman filter paper, and the filtrate was stored at 4°C for further use.[5][6]

• Green Synthesis of ZnO Nanoparticles-

 Zinc acetate (0.1 M) solution was prepared in distilled water. Aloe vera extract was introduced dropwise while stirring the solution. Then NaOH solution was added to adjust the pH to 10-12. The mixture was heated at 60-80°C for 2-3 hours until a white precipitate was formed. The precipitate was washed with distilled water and ethanol, and dried at 60°C. The sample was calcined 2 hours at 400-500°C to yield ZnO nanoparticles.[7][8]

• Drug Loading by Adsorption Method –

A clear solution of Tolnaftate was prepared in methanol. An appropriate amount of ZnO nanoparticles (e.g., 1:5 drug:ZnO) was added to the drug solution. This was stirred for 24 hours at room temperature to allow adsorption. Centrifugation of the suspension was done, and the supernatant was examined using UV spectroscopy to check for unentrapped drug content. The pellet with the drug-loaded nanoparticles was dried.[9][10]

Entrapment efficacy-EE(%)=(Total Drug−Free Drug)/ Total Drug ?×100

• Preparation of Carbopol Gel-

Carbopol 934 (1% w/v) was added to distilled water and was allowed to swell for 2-3 hours. Drug-loaded ZnO nanoparticles were dispersed in propylene glycol and mixed with Carbopol dispersion while stirring. The pH was adjusted (6-7) with triethanolamine to get a uniform gel.[11]

Table 1- Formulation table of drug loaded metal oxide nanogel.

UV Spectroscopy of Tolnaftate in methanol

A stock solution of 1mg/mL of standard drug was prepared, later dilutions were made with methanol. From this stock solution 10, 20, 30, 40, 50 µg/mL dilutions were prepared using methanol. The λmax of the drug was determined by scanning the dilutions between 200 to 400 nm using a UV-Visible spectrophotometer. [12]

Characterization of drug loaded nano particles-

• Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) is widely used to study the surface morphology, shape, and particle size of drug-loaded Zinc Oxide Nanoparticles, especially when loaded with Tolnaftate.

• FTIR-

FTIR analysis is performed to identify functional groups and interactions between tolnaftate and ZnO nanoparticles.

Evaluation of Gel Formulation –

1. Physical Appearance

The prepared gel is visually inspected for color, clarity, homogeneity, and presence of any particulate matter or phase separation. A good gel should be smooth, uniform, and free from lumps or grittiness.

2. pH Determination

The pH of the gel is measured using a calibrated digital pH meter. Approximately 1 g of gel is dispersed in 10 mL of distilled water and pH is recorded at room temperature.
Ideal pH: 5.5–7.0 (compatible with skin)

3. Viscosity Study

Viscosity is determined using a Brookfield viscometer at different spindle speeds. The gel should exhibit pseudoplastic (shear-thinning) behavior, which is important for easy application and spreadability.

4. Spreadability

Spreadability indicates how easily the gel spreads on skin. It is calculated using:

S=M.L/T

Where:

• M = weight tied to upper slide
• L = length of glass slide
• T = time taken to separate slides

Higher spreadability indicates better patient compliance.

5. In Vitro Diffusion Study

A Franz diffusion cell was used for the in-vitro diffusion investigation, and the cellophane membrane was immersed in distilled water for the entire night. The membrane was fixed to the donor compartment and was placed on the reservoir compartment of the Franz diffusion cell which held 150 milliliters of phosphate buffer with a pH of 7.4. The donor compartment's cellophane membrane was covered with one gram of fluconazole gel. The magnetic stirrer was used to hold the entire set. The experiment was carried out with 12 hours at 100 rpm and at 37°C. Samples were removed periodically, at an interval of 10 minutes, out of the sampling port of the reservoir compartment and the absorbance at 257 nm measured using a Shimadzu 1800 UV visible spectrophotometer.[13]

RESULT-

Standard calibration curve of tolnaftate in UV spectrophotometer- The UV absorbance of tolnaftate standard solutions was in the range of 10-50µg/mL of drug in methanol showed linearity at 257 nm. The linearity was plotted for absorbance (Abs) against concentration (µg/mL) with R2 value of 0.999 and with the slope equation y =0.065x-0.0056.

Fig no.1- Uv of Tolnaftate

Scanning Electron Microscopy-

Fig no.-2 SEM image of drug loaded ZnO

The SEM-based results indicated that the drug coated ZnO nanoparticles were appeared as a crystal with similar size and shape and it is also arranged in a periodic pattern

FTIR-

Fig no.-3 FTIR of drug

Entrapment efficiency-

Table 2- Entrapment efficiency of formulation

pH-

Table 3- pH of gel formulations

Viscosity and spreadability-

Table 4- Viscosity and spreadability

In vitro study-

Table 5- In -Vitro study of formulations

Fig 4- Graph of invitro study