Gallic acid loaded self-nano emulsifying hydrogel-based drug delivery system against onychomycosis

Abstract

Aim: To developed and investigate gallic acid (GA) loaded self-nanoemulsifying drug delivery systems (SNEDDS) for treating onychomycosis via transungual route.

Materials & methods: The SNEDDS were prepared by direct dispersion technique and were evaluated for characteristics parameters using Fourier transform infrared, differential scanning calorimetry, confocal microscopy, transmission electron microscopy and zeta sizer. Furthermore, the safety of prepared formulation was evaluated via Hen’s egg test-chorioallantoic membrane study and stability was confirmed using different parameters. Also, its effectiveness was evaluated against fungal strain Trichophyton mentagrophytes.

Results: The SNEDDS displayed a particle size of 199.8 ± 4.21 nm and a zeta potential; of -22.75 ± 2.09 mV. Drug release study illustrated a sustained release pattern with a release of 70.34 ± 0.20% over a period of 24 h. The penetration across the nail plate was found to be 1.59 ± 0.002 µg/mg and 0.97 ± 0.001 µg/mg for GA loaded SNEDDS and GA solution respectively. An irritation score of 0.52 ± 0.005 and 3.84 ± 0.001 was reported for GA loaded SNEDDS hydrogel and GA solution, indicating a decrease in the drug’s irritation potential from slightly irritating to non irritating due to its entrapment within the SNEDDS.

Conclusion: GA loaded SNEDDS has potential to address limitations of conventional treatments, enhancing the drug’s efficacy and reducing the likelihood of resistance in the treatment of Onychomycosis.

Graphical Abstract

Plain language summary

Article highlights.

• The study investigated impact of gallic acid-loaded self-nanoemulsifying drug delivery systems (SNEDDS) hydrogel for treatment of onychomycosis.

• Permeation potential of the prepared hydrogel was analyzed ex vivo using the bovine hoof membrane.

• Nail clipping study demonstrated deeper drug permeation into the nail layers, supporting both in vivo & ex vivo data.

• Irritation potential & biological safety of the SNEDDS were evaluated using the Hen’s egg test-chorioallantoic membrane study.

• An antifungal study was conducted to evaluate the formulation's activity against Trichophyton mentagrophytes.

• Gallic acid-loaded SNEDDS has the potential to enhance the drug's efficacy in treating onychomycosis.

1. Introduction

Among the most prominent fungal infections in humans, infection affecting the nail plate and its matrix is one of the highly prevailing infections affecting the population worldwide. Each year 20% of the general population and 70% of the people above 60 years of age are affected by nail infection, alone in the USA [1]. The nail plate is composed of protein keratin, which is aligned in a cross-linked manner, imparting hardness to the plate. Onychomycosis is a chronic nail infection characterized by nail thickening, discoloration and deformity [2]. It is generally caused by any of these microorganisms, including Trichophyton rubrum, Trichophyton mentagraphytes or Candida albicans. Onychomycosis is generally preceded by asymptomatic, dry hyperkeratotic tenia pedis [3]. Over the period, the moist, dark, warm conditions of shoes and repeated exposure to water lead to microtraumatic compression over the nail which compresses and ultimately disrupts the hyponychium of the nail plate, facilitating the penetration of the dermatophyte into the nail bed. Once the hyponychium of the nail is breached, the nail bed gets infected by the dermatophytes, spreading proximally as hyperkeratosis and onycholysis [4].

Recently, scientists have developed various topical antifungal formulations for the treatment of onychomycosis. However, their effectiveness gets compromised due to lack of permeability, which in turn leads to a lesser amount of drug reaching the nail bed [5]. Moreover, the liquid formulations are wiped out easily upon application to the nail plate, which eventually leads to a decrease in its efficacy. Hence, a drug delivery system to mitigate the aforementioned problems and serve as a potential treatment strategy for onychomycosis needs to be developed [6].

Gallic acid (GA) (3,4,5-tri hydroxyl benzoic acid) is a phytopolyphenolic compound present in various plant sources for example green tea, pomegranates, red wines etc [7].

It is a crystalline solid that is either colorless or slightly yellow. It has a molecular weight of 170.11954 g/mol and a molecular formula of C₇H₆O₅. The melting point of this drug is 210°C, and it decomposes between 235 and 240°C, releasing carbon dioxide and carbon monoxide [8].

GA is mostly recognized as a potent antibacterial, antiviral, antifungal, anticarcinogenic etc. However, when administered topically, its poor bioavailability limits its efficacy. Therefore, formulating nanoparticles of GA and administering via transungual or topical route may resolve the existing issues of low bioavailability [9].

Nanotechnology in the field of medical sciences is a revolution for the treatment of various infections and diseases as it offers low dosage, lower side effects and enhanced therapeutic capability [10,11]. Nanocarrier systems have recently evolved in the diagnosis of several skin and nail infections. Additionally, nano-based delivery systems along with reducing the drug dose, enhance the permeation of the drug across different membranes and are biocompatible compared with the traditional formulations [12]. Along with the advantages of these nanoparticles, certain obstacles are also reported in terms of their encapsulation efficiency and stability that could be alleviated by developing, a self-nano emulsifying drug delivery system (SNEDDS). SNEDDS are thermodynamically stable, isotropic combination of oil, water, surfactants and co-surfactants that form oil in water nanoemulsion. The advantages of these systems include easy preparation, high thermodynamic stability, high drug loading and enhanced bioavailability [13].

SNEDDS can accumulate in skin appendages and provide sustained drug release. Furthermore, SNEDDS can enhance the moisture content of the skin and nails, thereby improving drug permeability. The liquid oil used in SNEDDS reduces crystallization, resulting in a disorganized lipid matrix of nails, which allows for higher drug entrapment efficiency [8].

Along with SNEDDS, other lipid based nanoformulations are also gaining much attention for transungual delivery. In a study by Abobakar et al., terbinafine hydrochloride was incorporated into solid lipid nanoparticles containing thiourea as a penetration enhancer, resulting in the production of extremely small particles with improved penetrability [14]. Additionally, ketoconazole combined with Ucuùba (an Amazonian fat) was encapsulated in lipid carriers, which illustrated high biocompatibility and bioactivity [15].

GA has low binding affinity with keratin which reduces the sterol demethylase C450 and squalene epoxidase, responsible for the growth of the bacteria, hence inducing its antifungal activity. Moreover, there is scarce research available on the utilization of GA for the treatment of onychomycosis [13]. In the presented study, GA loaded SNEDDS have been designed and developed. The drug loaded SNEDDS were further incorporated into hydrogel that increases the drug penetration into the nail keratin and provides a higher residence time of the drug onto the nail plate, altogether leading to sustained release of GA for a prolonged time duration. Also, it has been reported that hydration causes the disulphide bonds of keratin networks between them to loosen which creates spaces for the drug to pass [12]. Carbopol was used as a gelling agent for the formulating hydrogel. Carbopol was selected because of its biocompatibility, aqueous solubility and good permeation. It is primarily employed in cosmeceuticals as a thickening agent for semi-solid preparations [16]. GA loaded SNEDDS hydrogel was compared against the plain drug loaded hydrogel. The antifungal action and effectiveness of the developed formulation were illustrated via in vitro and ex vivo permeation studies and nail clipping experiments. Also, the antifungal activity of GA loaded SNEDDS hydrogel was assessed using fungal strain T. mentagraphytes.

2. Materials & methods

2.1. Materials

GA was received as a gift sample from Central Drug Research Institute, Lucknow, India. Tween 40, labrasol oil, potassium dihydrogen phosphate, disodium hydrogen chloride and sodium chloride were obtained from TCI. PEG 400 was purchased from SRL Chemicals. Rhodamine, mannitol, triethanol amine (TEA), carbopol 940, methanol and ethanol were procured from SD fine, Chennai, India.

2.2. Solubility analysis & analytical method development

The GA solubility was determined in various solvents, and a method using a UV-visible spectrophotometer was developed for analysis. For this, 1 mg of the drug was added to Eppendorf tube containing 1 ml of each solvent (methanol, ethanol and water). The tubes were then mixed using a vortex mixer. After sealing, the Eppendorf tubes were placed in a shaker incubator maintained at a constant temperature of 37°C. Samples were withdrawn after 48 h of continuous shaking. The samples were then centrifuged, and their absorbance was measured using a UV visible spectrophotometer. The UV analyzer. Shimadzu UV-1601 from Kyoto, Japan, was configured within the range of 200–400 nm to evaluate the drug content in the respected solvent at approx. 215 nm [17].

2.3. Construction of pseudo ternary phase diagram

The pseudo ternary phase diagram was created through the water titration method to determine the concentration of each component in the provided spectrum of the SNEDDS [18]. For analysis, surfactants, co-surfactants and oil were grouped in several ways. Every group was assigned a distinct ratio of weights (1:1, 2:1, 3:1 and 4:1) for the surfactant and co-surfactant combination (S_mix). These S_mix ratios were set to elevate the concentration of surfactants in comparison to the co-surfactant.

For every phase diagram, the S_mix ratio and oil were blended in separate centrifuge tubes at different ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1) [19]. Additionally, to establish the boundaries of each phase, various S_mix and oil phase ratios were prepared. Subsequently, the water phase was raised by 5% in order to achieve an aqueous phase concentration of 5–95% of the total. These mixtures were subsequently vortexed for approximately 2 min before being left to reach equilibrium following the addition of the aqueous phase. The three components are based tertiary phase diagram, in which S_mix, oil and water, are each represented along a separate axis. The different phase diagrams were created using the Tri Draw software and it serves as a visual tool for analyzing and identifying the transitions between turbid and transparent states and vice versa [20].

2.4. Development of GA loaded SNEDDS

To prepare the 20 ml of GA loaded SNEDDS, firstly, the solution of 4 ml of Tween 80 and added to the mixture of 1 ml labrasol and 1 ml of PEG400 and kept for constant stirring for 5 min over a magnetic stirrer. Further, the oil phase was bath sonicated for 1 h and a separate solution of 30% of GA was prepared by solubilizing GA into 14 ml of distilled water. To this GA solution, the oil phase was incorporated dropwise. The mixture was then subjected to constant stirring at 1000 rpm for 120 min to prepare GA loaded SNEDDS.

The liquid formulation was then subjected to freeze–drying. This process starts with rapid freezing at temperatures usually ranging from -50°C to -80°C to create small ice crystals. Subsequently, primary drying occurs at reduced pressure and temperatures ranging from -20°C to -10°C, which facilitates the sublimation of ice into vapor. This is followed by secondary drying at slightly higher temperatures, around 20°C to 25°C, to remove the remaining bound water. Throughout the process, a high vacuum of 100–1000 mTorr is maintained to prevent the collapse of the SNEDDS structure. Optionally, an inert gas was used to create an oxygen-free environment. Monitoring temperature, pressure and product appearance is crucial to ensure the quality and stability of the final freeze-dried SNEDDS product [21].

Distinctive physicohemical characterization studies as well as the in vitro drug release study were conducted following the development of the formulation [18].

2.5. Evaluation of physiochemical properties of GA loaded SNEDDS

2.5.1. Particle size, polydispersity index (PDI) & zeta potential

To assess the particle size, surface charge and PDI of the developed SNEDDS, DLS technique powered with a computerized inspection system with Malvern zeta sizer software was used. The sample of 0.887 centipoise viscosity and Refractive Index 1.33 was transferred to the quartz cuvette of the zeta sizer and wavelength was set to 630 nm for particle size (Z-average), surface charge analysis and PDI determination [22].

2.5.2. Transmission electron microscopy

For structural characterization of the SNEDDS, the formulation underwent transmission electron microscopy (TEM) analysis using a high-resolution TEM (HR-TEM) instrument, specifically the TECNAI G2 operating at 200 kV at All India Institute Of Medical Science, New Delhi. After dilution the formulation was placed on a copper grid, the mixture was stained with phosphotungstic acid (2%) and allowed to dry for 30 s. The grid was further examined and analyzed under the microscope [23].

2.5.3. Drug loading & entrapment efficiency determination

The entrapment efficiency and drug loading were assessed to determine the extent of drug entrapment and the quantity of drug loaded into the prepared SNEDDS [24].

Methanol (9 ml) was dispersed in SNEDDS formulation (1 ml) to prepare the ratio of 9:1; the quantity of GA in SNEDDS (C_total) was verified with the developed UV curve. Further, the obtained solution was centrifuged for 30 min at 12,000 rpm. At last, the supernatant nonencapsulated drug was withdrawn and the concentration of the untrapped drug (C_free) was determined using UV spectrophotometer [25]. The following formula was employed to compute the %DL and %EE:

$$\begin{array}{c}\%\text{DL}=\frac{\left({\text{C}}_{\text{total}}-{\text{C}}_{\text{free}}\right)}{\text{Weight of total nanoparticles}}\times 100\\ \%\text{EE}=\frac{\left({\text{C}}_{\text{total}}-{\text{C}}_{\text{free}}\right)}{{\text{C}}_{\text{total}}}\times 100\end{array}$$

2.5.4. Thermal analysis (DSC)

Differential scanning calorimetry (DSC) study was conducted using the instrument TA-1-Ekalasanat (Rodgau, England). Roughly 4–8 mg of lyophilized nanoparticles were placed inside sealed aluminum pans for DSC analysis. The thermograms were recorded over a temperature limit of 30–400°C, with the instrument set to a speed and cooling rate of approximately 10°C/min [26].

2.5.5. Fourier transform infrared spectroscopy (FT-IR)

Similar to Differential Scanning Calorimetry, the Infrared spectra of the GA-loaded SNEDDS were examined. Formulation admixed with potassium bromide was subjected to pellets formation and then analyzed through Perkin Elmer Spectrophotometer (Rodgau, Germany), at a range of 400–4000 cm^-1 [26].

2.5.6. Thermodynamic stability studies

A screening method was employed to confirm the stability of Plain SNEDDS and GA loaded SNEDDS. The different SNEDDS formulation were subjected to various stress-stability studies.

2.5.6.1. Heating cooling cycle

The selected formulations were subjected to three heating and cooling cycles within a temperature range of 4–45°C. The formulations were stored at each temperature for 48 h and were examined for any physical instabilities, such as flocculation, cracking, precipitation and phase separation [27].

2.5.6.2. Centrifugation

Formulations that passed the initial test were then subjected to centrifugation at 3500 rpm for 30 min to eliminate any metastable systems. They were subsequently observed for any changes in homogeneity.

2.5.6.3. Freeze–thaw cycle

Finally, the formulations were stored at temperatures ranging from -21°C to +25°C for 48 h, for up to three cycles. Any changes in the homogeneity of the emulsions were monitored.

2.6. Preparation of GA loaded SNEDDS hydrogel

The Hydrogel was formulated through the direct dispersion technique. The gelling agent carbopol 940 was added into the lukewarm water and kept for constant stirring at 500 rpm, overnight [28]. Pre-determined quantity of GA containing SNEDDS was suspended in the minimum quantity of water and was then dissolved into the aqueous solution of carbopol. Further, TEA was added, while keeping the carbopol solution at constant stirring of 500 rpm. The homogenous lump-free hydrogel was prepared [29].

2.7. Assessment of physiochemical properties of hydrogel

2.7.1. pH determination

The variations in the pH values of formulation indirectly impact the drug ionization and thus directly influence the nail permeability. The pH of the nail is reported to be 4.8–5.4 [24]. The pH of the formulated preparation was measured using a pH meter.

2.7.2. Spreadability assessment

Spreadability was evaluated employing the wooden apparatus recommended by Rifkin et al. [30]. A pulley was affixed to one end of a wooden block, and based on the drag and slip properties of the hydrogel, spreadability was assessed. An excess of hydrogel (2 g) was positioned among two identical slides. One slide was securely attached to the pulley, while the other remained stationary. Subsequently, to achieve a uniform layer of hydrogel between the slides, a weight of 500 mg was applied on top of the slides for the next 5 min. The time taken for the upper slide to move across the lower slide under the influence of weight through the pulley was then recorded [24]. The spreadability was calculated in triplicate using the given formula

$$\text{S}=\text{M}\times \text{L}/\text{T}$$

where, S indicates spreadability, M represents the force applied to the upper slide, L is the length of glass slide and T represents the time taken by the slides to separate from each other [31].

2.7.3. Extrudability assessment

This test determines the amount of force needed to extrude the gel from the tube. The quantity of gel extruded was assessed by applying a 500 mg weight onto the silicone tube. The following equation was used to calculate the extrudability

$$\text{E}=\text{M}/\text{A}$$

where, E: extrudability, A: area, M: weight applied to expel gel from the tube.

At least 0.5 cm ribbon gel should be obtained in 10 seconds for optimum extrudability [32].

2.7.4. Viscosity & texture estimation

The viscous nature of the hydrogel was determined with a Brookfield viscometer. Spindle number 3 of the viscometer was set at a speed of 100 rpm/min and the readings were recorded at the end of the 10 min [33]. In addition, the texture of the SNEDDS hydrogel was examined through a texture analyzer (TA.XT Plus, New York, USA) [34].

2.8. In vitro drug release study

A comparative drug release study was conducted on GA-loaded SNEDDS and the plain drug solution to analyze the quantity of drug released from both formulations over 24 h. A dialysis membrane (membrane no. 61) with a pore size of 0.8 μm, (Himedia®, Mumbai, India) was chosen for the study due to its appropriate porosity and biocompatibility. The authenticated use of this membrane has already been established for evaluating the release of drugs from several formulations developed for nail fungus [35,36]. The membrane was primarily activated involving various steps which included washing the membrane under running water for 5–6 h. Further, it was properly washed with Na₂S solution (0.3%) for 1 min and was further immersed in a beaker containing hot water of temperature 70–80°C for 3–4 min. The membrane was then acidified using 0.2% sulphuric acid for 2.3 min and was washed again for 3–4 min using hot water. At last, the membrane was immersed in phosphate buffer saline pH 7.2, overnight [24,35].

Subsequently, the membrane was carefully positioned on the Franz diffusion cell's diameter, ensuring the absence of any air bubbles. A solvent mixture of Phosphate-buffered saline (PBS): methanol (7:3) was added to the receptor compartment of the cell. Also, the donor compartment was loaded with GA solution and GA loaded SNEDDS, and this setup was then kept for 24 h at 100 rpm, with temperature maintained at 37 ± 2°C [37]. Now, parafilm was used to seal any openings, preventing the loss of water through evaporation. Furthermore, samples of 0.9 ml were collected at regular intervals of 0.1, 2, 4, 8, 12, 16 and 24 h, while maintaining the sink conditions with the addition of the same amount of solvent (0.9 ml) back simultaneously. At last absorbance of withdrawn samples was recorded maintaining the UV spectrophotometer [38].

Furthermore, the release kinetic model was evaluated by placing the obtained data into Higuchi model, Korsmeyer–Peppas, first order, Zero order release kinetic model. The model that exhibited the highest correlation coefficient value was deemed the most optimal model [39,40].

2.9. Transungual permeation study

Bovine Hoof was obtained from the local butcher house. The hoof membrane bears a resemblance to the human nail plate releasing keratin protein on incubation with keratinase. As a result, hoof membrane is frequently employed in nail permeation studies [41]. Before using it for the study, the hoof membrane was immersed in PBS pH7.4 for 24 h [42].

The receptor compartment of the Franz cell was filled with solvent PBS: methanol (7:3) and the hoof membrane was then precisely placed onto the mouth of the Franz cell. Five milligram of GA loaded SNEDDS into the donor compartment [43]. Now, the assembly was placed on a magnetic stirrer for 24 h at 100 rpm, at 37 ± 2°C temperature. Parafilm was used to seal any openings, preventing the loss of water through evaporation. Furthermore, samples of 0.9 ml were collected at regular intervals of 0, 1, 2, 4, 8, 12, 16 and 24 h, maintaining the sink conditions by the addition of 0.9 ml solvent back immediately. At last, absorbance was recorded maintaining the UV spectrophotometer [44,45].

2.10. Ex vivo confocal laser scanning microscopy (CLSM)

Rhodamine-loaded SNEDDS hydrogel and rhodamine hydrogel were formulated for this study. The bovine hoof membrane was installed on Franz diffusion, pre-filled with PBS (pH 7.0) and was then subjected to hydrogel containing rhodamine SNEDDS for 24 h. Hydrogel with plain rhodamine solution was utilized as the control group [46]. Later, the hoof membrane was rinsed with distilled water and was sliced into small segments to prepare slides for microscopy. Further, to analyze the permeation of the developed SNEDDS into the membrane, CLSM was employed to examine the developed slides. In the current study, rhodamine fluorescence was optically stimulated at 488 nm argon laser beam and fluorescent emission above 532 nm [47].

2.11. Nail clipping study

This study utilized human nail clippings obtained from healthy volunteers. Since nails are a waste product and can be donated, no ethical approval is needed for the same [24,48]. However, the nails were collected after providing a thorough description to the patient and with the consent of the individual volunteer [49,50]. Hand nails and toenails were collected from individuals 20–35 years old age, of thickness about 0.31 mm, length 3.26 mm and width approx. 2.77 mm. The study was performed in a group of two consisting of GA solution and GA loaded SNEDDS. One day prior to nail collection, the volunteers were restrained from obtaining any type of medication. The obtained nails were then cleaned with distilled water and wiped out using a clean tissue. In two separate glass vials of 2 ml each, a nail section was added. Plain GA solution was added to the first vial, while to the other vial GA loaded SNEDDS was added. The vials were then sealed and placed for constant stirring, overnight. Drug loading within the nail plate was assessed using overnight nail digestion [41,51]. Later, the nails were removed and wiped with dry tissue paper and damp cotton. Subsequently, these samples were immersed in a 1 M sodium hydroxide solution with constant stirring for 24 h. They were later neutralized by acidification using 1 M hydrochloric acid, resulting in a neutralized solution. Finally, centrifugation was carried out by mixing it with a (7:3) PBS: methanol solution, and the preserved solution was analyzed using a UV spectrophotometer [52,53].

2.12. Hen’s egg test-chorioallantoic membrane (HET-CAM) assay

Fertilized chick eggs were acquired from a poultry farm in Mewat, Haryana, India. Before incubation, a visual examination was conducted to check for any cracks in the eggshell [54]. Then the eggs were placed in an incubator maintained at a temperature of 37 ± 2°C and relative humidity of 60–70%. On the 12th day, a section of the eggshell was removed aseptically. The sample to be tested was then applied onto the surface of CAM. Further, the sample was allowed to interact for the next 5 min. The test groups included NaCl 0.9% solution as a negative control, 0.1 N sodium hydroxide as positive control, GA solution and GA loaded SNEDDS hydrogel for comparison of irritancy.

Moreover, the irritation potential of each sample was assessed by monitoring three end points for 5 min each: lysis, hemorrhage and coagulation [50]. The results were evaluated by comparing the irritation score (IrS), where a score of 5.0–8.9 indicates moderate irritation, 1.0–4.9 suggests slight irritation and 0.0–0.9 signifies non irritation. The changes were continuously observed for more than 10 min, and the point of irritation was recorded. After capturing observation images, the IrS was calculated using the formula outlined by Weimer et al. [55].

$$\begin{array}{c}\text{IrS}=[(301-\text{H}/300)\times 5]+\\ [(301-\text{L}/300)\times 7][(301-\text{C}/300)\times 9]\end{array}$$

where, IrS: the irritation score, C: start second of coagulation effect, H is the start second of hemorrhage effect, L: start second of lysis effect

2.13. In vitro antifungal activity

The cylinder plate method was utilized to determine the antifungal activity in vitro, using Trichophyton mentagrophytes as the fungal strain [56]. Three formulations, i.e., blank SNEDDS, GA solution and GA loaded SNEDDS were examined for their antifungal action. T. mentagrophytes were cultivated in a sabouraud dextrose agar media for a week at 25°C. Further, into 20 ml of media, the harvested spores were added and then passed through the sterile gauze for filtration. Then, inoculated media was added into the sabouraud dextrose agar (100 ml) as growth media and retained at a temperature 37 ± 2°C. Standard solutions of GA solution and GA loaded SNEDDS were prepared, and then required dilution (50 µg) was prepared from stock [24,52,57].

3. Results

3.1. Solubility analysis & analytical method development

The solubility analysis of GA study was conducted and drug was found to be soluble in methnol (0.876 ± 0.28 mg/ml), ethanol (0.743 ± 0.143 mg/ml) and water (0.34 ± 0.073 mg/ml). The results were in compliance with the previous literatures [58].

A UV analytical method was developed for GA. Where R² (Regression coefficient) for GA at concentration 5 mg/ml was found to be 0.961 at a wavelength of 215 nm and with PBS: methanol (7:3) used as a solvent.

3.2. Construction of pseudo ternary phase diagram

The optimal composition range for the excipient was obtained using the Pseudo ternary phase diagram. This technique is employed to engineer the regions of nanoemulsion. In addition, it illustrates the influence of varying phase volumes on the system’s behavior [59].

The diagram reported that out of 4:1, 3:1, 2:1 and 1:1, the 2:1 is an optimized ratio because it covers the larger nano-emulsified area and hence is opted for further research (Figure 1).

Figure 1.

The Pseudo ternary phase diagrams of the Oil S_mix–Water system at the 1:1, 2:1, 3:1 and 4:1 wt ratio of Labrasol -Tween 80/PEG 400 at ambient temperature, shaded area shows nanoemulsions.

3.3. Development of GA loaded SNEDDS

The GA loaded SNEDDS were synthesized and were later freeze-dried to improve their stability as in liquid samples, there are chances of particle aggregation. Later, the freeze dried product was used for performing various evaluation assays [60,61].

3.4. Evaluation of physiochemical properties of GA loaded SNEDDS

3.4.1. Particle size, PDI & zeta potential

The particle size, surface charge and PDI of the synthesized SNEDDS was examined, and particle size was found to be 199.8 ± 4.21 nm which is also a prerequisite for SNEDDS as nanoparticles of this size range exhibit remarkable penetration into the nails [62]. Whereas, PDI was found to be 0.3358 corresponding to the uniform particle dispersion in the system (Figure 2A).

Figure 2.

Evaluation of physiochemical properties of gallic acid loaded self-nanoemulsifying drug delivery systems: (A) Particle size and polydispersity index (B) Zeta potential.

To analyze the stability of the dispersed system, surface potential was observed. It explains the degree of particle coagulation in the system due to electrostatic repulsion. Furthermore, nonionic surfactants are usually considered for formulating SNEDDS as these curb their toxicity and promote stability and compatibility contrary to ionic or amphoteric surfactants [24]. The GA loaded SNEDDS reported a zeta potential of -22.75 ± 2.09 mv (Figure 2B). Since zeta potential serves as a pivotal indicator of nanoparticle stability, nanoparticles exhibit high zeta potential, whether positive or negative are deemed electrically stable. Hence, it can inferred that the nanoformulation is indeed stable.

3.4.2. Transmission electron microscopy

The uniform shape and spherical structure of the SNEDDS were approved through TEM analysis. It also demonstrated that the particle size of SNEDDS was within the defined limit of 100–200 nm (Figure 3A). This offers valuable insights into its potential to improve drug solubility, bioavailability, stability, targeted delivery, therapeutic effectiveness and safety in pharmaceutical applications. These attributes position SNEDDS as a promising platform for enhancing the delivery of diverse drugs [63].

Figure 3.

(A) Transmission electron microscopy analysis of gallic acid loaded self-nanoemulsifying drug delivery systems (B) Illustration of differential scanning calorimetry thermogram (C) FTIR spectra of prepared formulation.

FTIR: Fourier transform infrared.

3.4.3. Drug loading & entrapment efficiency determination

Entrapment efficiency and drug loading were calculated to find the amount of drug that was present in the SNEDDS According to the calculation the drug loading was found to be 9.21 ± 0.003%, respectively. Whereas encapsulation efficiency was found to be 95.13 ± 0.001%. The high drug loading and entrapment efficiency in SNEDDS formulation can be attributed to the solubility of GA in the oil phase and the structure of the formulation.

3.4.4. Thermal analysis

The alteration in the thermal behavior of GA was observed in the drug loaded SNEDDS, which indicates the distinctive nature of GA and drug entrapment. At 266.603°C, a sharp endothermic peak can be observed, which validates the melting point of GA and this correlates with the DSC data cited by Hazari et al. [64]. Here because of the drug encapsulation the peak has been shifted to 347.2°C. Whereas, the peak detected at 167.192°C corresponds to the presence of mannitol in the formulation, used as a cryoprotective agent during lyophilization. In addition, the presence of the untrapped drug caused a sharp peak at 123.495°C (Figure 3B).

3.4.5. Fourier transform infrared spectroscopy (FT-IR)

The FT-IR analysis of GA loaded SNEDDS illustrated distinctive absorption bands; it revealed an absorption band at 164.39 cm^-1 which could be attributed to the C-0 stretching. A peak at 1734.07 cm^-1 indicated the presence of a carboxyl group (C-O); indicating GA has been coated with mannitol. The sharp peak for GA at 1697 cm^-1 was displaced to broad, and peptide peak at 1734 cm^-1 indicated drug entrapment in the SNEDDS (Figure 3C).

3.4.6. Thermodynamic stability studies

Table 1 summarizes the results of stability studies performed on plain SNEDDS and GA loaded SNEDDS. It was confirmed from the results that GA loading in nanoparticles does not hinder the stability of the formulation and that the formulation can be further used for preparation of hydrogel.

Table 1.

Observing thermodynamic stability of plain SNEDDS and gallic acid loaded SNEDDS.

Formulation	Turbidity	After 24 h turbidity	Heating–cooling cycle	Centrifugation	Freeze–thaw cycle
Plain SNEDDS	No	No	Pass	Pass	Pass
GA loaded SNEDDS	No	No	Pass	Pass	Pass

SNEDDS: Self-nanoemulsifying drug delivery systems.

3.5. Preparation of GA SNEDDS loaded hydrogel

The SNEDDS were synthesized using GA, which were further added to hydrogel formulated using carbopol. The formulation was homogenous, lump-free, viscous and transparent.

3.6. Assessment of physiochemical properties of hydrogel

3.6.1. pH determination

The pH of the prepared formulation was found to be 5.26 ± 0.25, indicating its compatibility with the skin and unlikely to cause skin irritation upon application.

3.6.2. Spreadability assessment

This study was used to examine the spreading ability of the prepared hydrogel. The spreadability was noted to be 3.47 cm for normal weight, 3.54 cm for a weight of 50 g, 3.71 cm for 100 g and 4.58 cm for 1 kg, demonstrating the spreadable characteristic of prepared hydrogel.

Appropriate spreadability ensures uniform drug distribution and absorption through topical route. Uneven spreadability could lead to imbalance drug delivery and altered efficacy [65].

3.6.3. Extrudability assessment

This test assesses the extrusion of gel from the silicon tube under the application of a specific weight. The criterion for a successful test is the extrusion of at least 0.5 cm of gel ribbon from the tube within a 10 s timeframe [66]. The extrudability of GA loaded hydrogel was determined to be 2.5 ± 0.49 cm², a measurement considered optimal.

3.6.4. Viscosity & texture estimation

The hydrogel’s viscosity, as determined by a Brookfield viscometer, was approximately 976.264 cP. Additionally, the texture analyzer demonstrated that the hydrogel formulation had the appropriate texture (Figure 4). The analysis of the hydrogel formulation’s mechanical properties included cohesiveness, consistency and firmness. The reported characteristics had a direct effect on the application of the gel onto the affected surface of the patient. Furthermore, the stickiness of the gel was analyzed using viscosity index, whereas the strength of the gel was reported by its firmness value, a high value representing excellent gel strength [67]. The values of cohesiveness, firmness, consistency and cohesion of the formulated hydrogel are reported to be -34.72 g, 60.37 g, 95.94 g/s and -67.98 g/s, respectively.

Figure 4.

Representation of texture analysis of drug loaded nanoparticles.

3.7. In vitro drug release study

The release of the drug from both the GA containing SNEDDS and GA solution was evaluated over 24 h. A graph between percent drug release v/s time was plotted for the same. Quantities of 79.33 ± 0.057% were released from plain drug solution, whereas, 70.34 ± 0.20% was released from the GA loaded SNEDDS (Figure 5A). It was reported that of GA solution showed burst release at the initial stage and no discernible pattern of controlled drug release was observed throughout the study. However in the case of drug containing SNEDDS, a sustained release was noticed. The drug release was consistent even after 12 h, illustrating the better efficacy of the prepared SNEDDS.

Figure 5.

(A) In vitro drug release assessment from gallic acid hydrogel V/S gallic acid loaded SNEDDS hydrogel (B) Transungual permeation study between GA hydrogel and GA loaded SNEDDS hydrogel (ex vivo assessment).

GA: Gallic acid; SNEDDS: Self-nanoemulsifying drug delivery systems.

Further, the data of drug release was fitted into different kinetic models to demonstrate the kinetic and mechanism of drug release and to analyze the best-fitted model. The R² value was computed from the linear curve obtained through regression analysis of the aforementioned models. Higuchi model gave the R² value near 1, and it can explain the release mechanism of the drug from the GA loaded SNEDSS.

3.8. Transungual permeation study

A bovine hoof membrane was used to perform the transungual permeation study which demonstrated results consistent with in vitro drug release. The release study of drug from the GA loaded hydrogel displayed burst release and unregulated release until the completion of the study. This is likely because of the degradation of gel matrix, independent of time. Comparatively, GA loaded SNEDDS hydrogel illustrated sustained drug release throughout the 24 h study. 79.01 ± 0.30% was released from the plain drug containing hydrogel. Whereas, 63.72 ± 0.006% of the drug was released from the GA loaded SNEDDS hydrogel (Figure 5B). The sustained release of the drug from the SNEDDS is the result of versatile structure of the SNEDDS that results in drug release in a time dependent and steady manner. At initial time points, small nanoemulsion particles dispersed in the gel matrix were released. As the study proceed, drug particles that were tightly bound within the hydrogel prevents rapid diffusion. The obtained result is desirable in the treatment of fungal infections as the active agent penetrates deeply beneath the nail or skin. Therefore, sustained release of the drug indicates enhanced antifungal activity resulting in root-seated eradication of the fungus [68].

3.9. Ex vivo confocal laser scanning microscopy (CLSM)

To assess the penetration of rhodamine loaded SNEDDS hydrogel and rhodamine hydrogel confocal microscopic analysis was done. The red color intensity indicates the amount of drug deposited deeper in the membrane of the hooves. The finding of the CLSM investigation demonstrated that rhodamine solution could permeate the hooves membrane only upto 20 um. Whereas, rhodamine loaded SNEDDS hydrogel was highly permeable (upto 30 um) through the membrane of the bovine hooves (Figure 6). Thus, the findings illustrate that the drug may effectively penetrate the hoof membrane, which is essential for the diagnosis of onychomycosis.

Figure 6.

Confocal laser scanning microscopy penetration for (A) plain rhodamine hydrogel (B) SNEDDS loaded hydrogel.

SNEDDS: Self-nanoemulsifying drug delivery systems.

3.10. Nail clipping study

The investigation demonstrated the quantity of drug penetrated across the nail plate in both samples was found to be 1.59 ± 0.002 µg/mg and 0.97 ± 0.001 µg/mg for GA loaded SNEDDS and GA solution respectively. The GA loaded SNEDDS penetrated easily into the rigid matrix of nail keratin due to their nanometric range. Therefore, from the results it can be concluded that the SNEDDS lead to the enhanced permeation of the drug into the deeper layers of the nail in comparison to the free drug. The small-sized globular shaped particles of the formulation result in its increased permeation into the nails leading to the superior affinity of encapsulated GA with the nail keratin. In addition, the nail clipping study aligns with the in vitro and ex vivo examination results. Hence, it was inferred that following the application of GA containing SNEDDS a significant amount of the drug penetrated the nail plate and the bed matrix [69].

3.11. HET CAM assay

Tolerability and irritability of the GA loaded SNEDDS hydrogel was examined using the HET-CAM test and was compared with 0.1 N NaOH (positive control) and 0.9% w/v normal saline (negative control). Compared with positive control, GA loaded SNEDDS hydrogel and negative control both were remarked as non-irritant using IrS. The score of 0.59 ± 0.001 was reported for normal saline, whereas, for 0.1 N NaOH 15.84 ± 0.057 Irs was observed which is considered to be severely irritant. A mean irritant score of 0.52 ± 0.005 and 3.84 ± 0.001 was reported for GA loaded SNEDDS hydrogel and GA solution respectively. Conclusively, the obtained results suggested that the drug’s irritation potential was decreased from slightly irritating to nonirritating due to its entrapment within the SNEDDS (Figure 7).

Figure 7.

(A) Hen’s egg test-chorioallantoic membrane study done on fertilized eggs treated with (A) Negative control (B) Positive control (C) GA solution (D) GA loaded SNEDDS hydrogel.

GA: Gallic acid; SNEDDS: Self-nanoemulsifying drug delivery systems.

3.12. In vitro antifungal activity

Antifungal activity conducted in vitro on the T. mentagrophytes strain demonstrated that throughout incubation, free GA showed colony forming unit lower than the control group (Figure 8). However, when it was encapsulated inside the SNEDDS, colony forming unit reduced to a further extent indicating the enhancement of combined antifungal activity. This enhanced activity could be explained by the small particle size of the formulation, which leads to the gradual and steady release of the drug from the nanoparticle matrix. As previously reported for treating onychomycosis antifungal activity of the drug is essential and the prepared formulation would enhance the therapeutic procedure for managing the disease.

Figure 8.

In vitro antifungal activity of the free GA and GA loaded SNEDDS performed on Trichophyton mentagrophytes, the main causative agent of onychomycosis.

GA: Gallic acid; SNEDDS: Self-nanoemulsifying drug delivery systems.

4. Discussion

Onychomycosis, the most prevalent nail infection globally, is a fungal infection that leads to discoloration and thickening of the affected nail plate. Onychomycosis was initially believed to be mainly caused by dermatophytes. However, recent studies have shown that mixed infections and infections caused by non-dermatophyte molds are more common than previously thought, particularly in warmer climates. Treatment options include topical and oral antifungals, treatments and devices. Oral antifungals offer higher cure rates and shorter treatment durations compared with topical treatments, but they can cause adverse side effects such as hepatotoxicity and drug interactions [70].

In the presented work, an exploration into the use of an antifungal drug was undertaken to assess its effectiveness against onychomycosis. Since treating such infectious nails is quite challenging, it demands the drug to penetrate the deeper layers of the nail ensuring effective treatment. Therefore, GA when encapsulated within the SNEDDS particles, resulted in greater biocompatibility. Taking into consideration the retention time of the drug containing formulation as a crucial factor in topical/transungual route of administration. The optimal composition range for the excipient was determined using a pseudo ternary phase diagram. This diagram helps in selecting the ideal ratio of components needed to prepare formulations like SNEDDS. Accurate identification of the surfactant/co-surfactant system and oil phase impacts drug solubility, dissolution and consistent drug absorption. In addition, the pseudo-ternary diagram aids in the stability analysis of the drug. It prevents aggregation, chemical interactions or degradation that could compromise the drug’s efficacy [21].

Furthermore, the prepared formulation was characterized using TEM, DSC, FT-IR and zeta analyzer. The SENDDS nanoparticles showed high drug loading and entrapment efficiency which can be attributed to the solubility of GA in the oil phase and the formulation’s structure [71].

Thermal stability is a critical factor in nanoformulation development as it ensures the formulation’s integrity during storage and administration. Any chemical or physical changes in the formulation can ultimately impact the drug’s release kinetics and bioavailability [72]. Similarly, FT-IR spectra reveal the composition and chemical structure of the nano-preparation. Changes in the spectra may indicate chemical interactions, potentially leading to impaired cellular uptake, delayed drug release and altered pharmacological activity [27].

The SNEDDS were made viscous by developing hydrogel, ultimately enhancing its retention over the nail plate. The GA loaded SNEDDS were determined as stable with no reported indications of degradation. While it can be applied and removed with ease, it exhibits a gel-like consistency with adequate spreadability ensures even drug distribution and absorption through the topical route [65].

Also, the release of drug from the GA loaded SNEDDS was reported to be more sustained than the plain GA solution hydrogel, both ex vivo and in vitro. In vitro studies revealed that the GA loaded SNEDDS follows Higuchi model of drug release. The Higuchi model elucidates the mechanism by which drugs are released from a matrix system over time, emphasizing release controlled by diffusion. This suggests that drug molecules predominantly diffuse from the nanoparticle matrix into the surrounding medium. By adhering to the principles of the Higuchi model, the controlled release of drugs from nanoparticles can sustain consistent and prolonged drug levels in the nail, essential for effectively treating nail infections [73].

Furthermore, CLSM findings indicate that the drug effectively penetrates the nail membrane, which is crucial for diagnosing onychomycosis. Based on HET-CAM results, the drug showed reduced irritation potential, shifting from slightly irritating to non irritating when entrapped within the SNEDDS formulation. Correspondingly, antifungal activity conducted in vitro reported a noteworthy reduction in the colony-forming units in presence of the prepared SNEDDS containing GA in comparison to the free drug [74].

Conclusively, the developed SNEDDS proves to be an effective strategy of curing onychomycosis as the nanosized particles are able to easily penetrate the nail barrier providing sustained drug release. Hence, it can be a major breakthrough in the field of modern medicine for treating nail infections.

5. Conclusion

GA acid loaded SNEDDS were prepared using the direct dispersion method, to provide a sustained drug release for the treatment of onychomycosis. The prepared SNEDDS formulation illustrated spherical shapes with a suitable percent entrapment efficiency; zeta potential, PDI and size. Moreover, the prepared GA loaded SNEDDS proved to be optimally stabled as evidenced by the Freeze–Thaw Cycle, heating cooling cycle and centrifugation. When prepared as hydrogel, SNEDDS exhibit excellent spreadability, extrudability and other semi-solid parameters displaying optimal retention for the treatment of onychomycosis. Furthermore, ex vivo investigation evidenced the sustained release impact of the GA loaded SNEDDS hydrogel. The CLSM proved the high accumulation of the SNEDDS across the nail membrane. The HET-CAM study illustrated the perfect irritation potential/tolerability of the prepared formulation show casing its biological safety. Furthermore, the nail clipping study reported the exact penetration of the drug across the nail membrane. At last, the antifungal activity was evaluated against the T. mentagrophytes. These promising results proved that the GA acid loaded SNEDDS hydrogel could be utilized as a possible treatment strategy against onychomycosis. Also, the formulation holds potential as a commercially viable pharmaceutical product due to its scalability, economic feasibility and industrial viability. These factors are crucial for translating research findings into practical applications that benefit both patients and stakeholders in the healthcare industry.

Author contributions

MS Khan, M Fatima, S Wahab and M Khalid wrote the main manuscript and P Kesharwani conceptualized, proofread and supervise during writing of the original manuscript. All authors reviewed the manuscript.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.