Formulation and Characterisation of Herbal Ethosomal Gel of Luliconazole and Clove Oil for Modified Drug Diffusion to the Skin

Bhanu Pratap*, Shamim Shamim, Shadab Ali

Department of Pharmacy, IIMT College of Medical Science,

IIMT University, Ganga Nagar, Meerut - 250001, Uttar Pradesh, India.

*Corresponding Author E-mail: bhanuluci666@gmail.com

ABSTRACT:

The creation, characterisation, and optimisation of an herbal ethosomal formulation are all included in this work. The current study aims to create ethosomes filled with drugs and capped with Syzygium Aromaticum L., or clove oil. These ethosomes are then further mixed into Carbopol 934 K, which forms an ethosomal gel. The formulation aims to convey a fungal infection treatment that is successful. The medication (luliconazole), soy lecithin, clove extract, and propylene glycol were used in the formulation of the construct. Six ethosomal formulations with different constituent concentrations were processed in total to identify the best formulation out of the bunch. In addition, a number of assessment criteria, including the percent of entrapment efficiency, the size of the particle, zeta potential, and polydispersity index (PDI), were used to the generated drug-loaded ethosomes. By utilising transmission electron microscopy, the surface morphology was evaluated, while atomic force microscopy was used to get further in-depth information about the surface morphology. In order to study thermal behaviour for luliconazole, soya lecithin, clove extract, physical mixture, and optimised formulation (EF5), a thermal gravimetric analysis graph was produced. The ethosomal gel's viscosity, pH, Spreadability, and extrudability were calculated to assess the formulation's appropriateness for topical application. The entire study concludes that luliconazole can be delivered in an advanced, targeted, and sustained manner using this herbal ethosomal method. Because of its ideal soy lecithin dosage of 400mg and ethanol concentration of 30%, After analysis of the data, the ethosomal formulation EF5 was found to be the most optimal one. This results in a maximum entrapment efficiency of 88.56±0.74%. It was found that the optimal PDI, zeta potential, and vesicle size were 0.186±0.07, − 42.2±0.3mV, and 155.3±1.2nm, respectively. The best stable solutions were found to be ethosomal suspension and lyophilized ethosomal suspension at 4°C/60 ±5 RH. The whole study demonstrates that the combination of ethosomal formulation with luliconazole and clove extract has a synergistic effect and works well to treat fungal infection.

KEYWORDS: Ethosomes, Luliconazole, Clove Oil, Antifungal, Dermal, Transdermal delivery.

INTRODUCTION:

Topical drug delivery is one of the more advanced methods for deliver the drug. It is advantageous because it delivers the medication via the skin at a controlled and predefined rate by passive diffusion, which maximises systemic impact. Drug distribution is regulated using transdermal patches using hydrophilic and lipophilic polymers. For lipid-based systems, ethosomes are essentially new, creative, and intrusive carrier vesicles that were initially described by Touitou et al. (2000). Their sophisticated and improved medicine delivery mechanism is intended for them because of their pliable, soft, and pliable nature. Essentially, these are phospholipid vesicles, and their applications are in transdermal and cutaneous drug delivery systems. It mostly affects ethanol, water, and several, concentric layers of flexible phospholipid; in the case of liposomes, however, cholesterol is included in place of ethanol. It is a bilayer vesicular system (aqueous and lipid) that exhibits attraction for both lipophilic and hydrophobic drugs, hence augmenting their bioavailability. The high percentage of ethanol in it, compared to liposomes, enhances medication administration topically and helps to weaken the lipid bilayer. Consequently, in contrast to liposomal drug delivery systems, it directs the penetration into deep layers of the skin by increasing the permeation through the stratum corneum barrier. This is not simply made possible by the way ethanol acts on the stratum corneum; vesicle permeability and deformability also play a part. Furthermore, its combination with skin lipid enhances skin penetration, leading to a steady state transdermal flow of about 1mg/cm2/h. As of yet, the process by which drugs enter the skin through ethosomes remains unclear. In contrast, a high ethanol content, phospholipid- and skin-lipid-containing vesicles, and high penetration of medication into the deeply embedded layers of skin through the ethosomes transport system are associated with this phenomenon. Ethanol and lipid molecules both improve fluidity in the polar head region, which helps the membrane's permeability rise. Owing to their unique properties, ethosomes are regarded as superior carriers for transdermal medication delivery systems. It is therefore acknowledged as an improved liposome. Previous research has found that ethosomes, such as those for Both in vitro and in vivo, higher skin delivery is shown for ketoprofen, testosterone, cannabidiol, buspirone hydrochloride, erythromycin, benzocaine, fluconazole, finasteride, lamivudine, 5-aminolevulinic acid of medication, and luliconazole. Since it doesn't require any specialised equipment, building up this advanced carrier is rather simple.^1,2,3,4,5,6

As a drug that inhibits the growth of fungus, luliconazole is an imidazole antifungal. The medication essentially has a log P of 4.07, which indicates that it is lipophilic. It has a single chiral centre and is a R enantiomer that is doubly bonded next to the dithiolene group in an E configuration. Pharmacodynamically, the medication destroys the fungus, mostly by altering its cell membrane. Although luliconazole's precise mechanism of action (MOA) is unknown, it is thought to entail the suppression of cytochrome P450 14α-demethylase (P45014M).

The primary component of the fungi cell membrane, ergosterol, is produced by the sterol biosynthesis process, which uses this enzyme. Luliconazole is usually used topically, although clinical research found that after the 1^st treatment, a subject(patient) with Tinea pedia had a Cmax (highest plasma concentration) of 0.400.76 mg/ml (mean±SD). Distribution volume of the drug has not yet been estimated. With over 99% binding, its protein binding is excellent.⁷

The drug's metabolism has not yet been identified. The luliconazole cream that is sold in stores induces allergic responses that include rashes, itching, swelling on the face, tongue, and throat, and breathing difficulties. Therefore, a luliconazole drug-loaded nanosized herbal ethosomal gel was created to counteract the negative effects caused by the cream. Since ethosomal gels are highly effective and have few adverse effects, they will increase the drug's permeability via the skin due to increased transdermal flux, which will boost the drug's bioavailability.^8,9,10

With the use of ethanolic clove extract, an herbal ethosomal system was created for the suggested study that included the medication luliconazole. Then, the drug-loaded ethosomes were added to Carbopol gel, demonstrating the drug's sustained transdermal distribution. In addition, the formulation underwent a number of in-vitro characterization tests, including those measuring the size of the particle, zeta potential, PDI, the percent of entrapment efficiency, TEM, AFM, pH, viscosity, Spreadability, and extrudability, which demonstrated its efficacy and suitability for the treatment.^11,12,13,14

MATERIALS AND METHODS:

Material:

As a gift sample, the medication luliconazole was gathered from Kiyan Chemicals in Gujrat. Chemicals from Aashi Chem (Gujrat), Acuro Organic Limited (New Delhi), Advance Petrochemicals Limited (Gujrat), and Aman Enterprises (Delhi) included soy lecithin, propylene glycol, tri-ethanol amine, and glycerine. Carbopol 934 K was obtained from Shree Chem in Gujrat, India; the remaining components used in the study were pure, high-grade, and analytically graded.¹⁵

Preparation of Extraction of Clove Oil:

Cloves were collected from the market and ensure that the clove buds are clear and free from contamination. Place the clove buds in the distillation flask. Add water to the distillation flask. Heat the water in the distillation flask to produce steam. The steam is carrying the volatile compounds from the cloves. Following passage through a condenser, the steam holding the clove oil vapour cools and condenses back into a liquid. The condensed mixture of water and clove oil is collected in a separatory funnel. The clove oil, being less dense than water, it is float on the surface. It can be collected from the upper layer of the separatory funnel.^16,17,18

Formulation of Luliconazole loaded Ethosomes:

Luliconazole ethosomes were formulated using Touitous's method, weighing necessary ingredients in different concentrations. Luliconazole, ethanolic clove extract, phospholipids and other ingredients in table no.1 was dissolved in ethanol and after that, the mixture was vigorously stirred while covered and kept at 20°C-25°C (room temperature) in a vessel. After that, the resultant mixed solution was warmed on a water bath to 30°C. On other side, in another container, the water is now warmed to 30°C before being poured to the mixture. In a sealed jar, the solution was continuously agitated at 700 round per minute with the help of magnetic stirrer apparatus. The prepared solution was kept at 4°C and subjected to three sonicate with a probe sonicator, lasting roughly 5 minutes each, separated by a 5-minute interval.^19,20

Table no. 1: Composition of luliconazole loaded ethosomes formulation (EF) with soya-lecithin

Ingredients	EF1	EF2	EF3	EF4	EF5	EF6
Luliconazole (mg)	100	100	100	100	100	100
Soya-lecithin (mg)	200	200	300	300	400	400
Ethanolic Clove extract (%)	30	40	30	40	30	40
Propylene glycol (ml)	1	1	1	1	1	1
Water	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.

Luliconazole Ethosomes incorporated into the Gel:

Carbopol 934K, 0.75% w/v, was become a solution in distilled water then stirred by a magnetic stirrer for 1 hour. The polymer swelled, and 20 ml of luliconazole leaded ethosomal suspension was added. The mixture was continuously stirred at 30°C and 700 rpm until a uniform texture resembling ethosomal gel was achieved. Tri-ethanol amine was added to neutralize the pH. Six ethosomal formulations were formulated and used for various evaluation parameters.^21,22,23

Characterization of Luliconazole Loaded Ethosomes:

Determination of the size of Particle, polydispersity index (PDI) and zeta potential:

Determining crucial parameters such as the size of the particle, polydispersity index (PDI), and zeta potential is necessary. These values were analysed using the dynamic light scattering-based Malvern Zeta Sizer Nano ZS. To start the estimate process, one millilitre of the ethosomal mixture was diluted with water for High Performance Liquid Chromatography (HPLC). In addition, measurements were made at a 90° scattering angle, 0.8862 medium viscosity, and 1.36 refractive index.^24,25

Structural study of Luliconazole loaded ethosomes by Transmission electron microscopy (TEM):

The formulation's morphology was determined using Transmission Electron Spectroscopy (TEM), which estimated its internal composition, morphology, and crystallization. 20 millilitres of the formulation were diluted with deionized water for the experiment, and the formulation was then stained for 30 seconds using 2% w/v phosphotungestic. Following that, the specimens were arranged on two copper-laminated grids, one for each specimen, for drying.²⁶

Structural study of Luliconazole loaded ethosomes by Atomic force microscopy (AFM):

Atomic Force Microscopy (AFM) was employed in the study to assess ethosomal suspension. A mica sheet was used to hold the suspension (1 ml), and it was heated up for 5 minutes. After that, unattached ethosomes were removed from the sample by washing it with deionized water. The specimen was allowed to air dry before being subjected to additional scanning with the Advance Integrated Scanning Tool for Nanotechnology (AIST-NT) Model: Smart SPM 1000. Each image was displayed separately, displaying the cantilever's phase, amplitude, and height signals in the trace direction.²⁷

Determination of Drug Entrapment efficiency, EE (%):

The percent of drug clossed in a system of colloidal particles is calculated using the entrapment efficiency (EE). An Eppendroff tube containing a tiny amount of formulation is centrifuged for 15 minutes at 14,000rpm. The supernatant is obtained by using an ultracentrifuge with a TLA-45 rotor. For detecting the drug's concentration at 299 nm, a UV/Visible spectrophotometer is used. The formula is used to determine the total amount of drug trapped in the system of colloidal particles.^28,29

(quantity of incorporated drug – quantity of free drug present in the superanatant)

EE(%) = ----------------------------------------------- × 100

quantity of incorporated drug

Determination of pH, viscosity, Spreadability and extrudability of Gel:

· A digital pH meter was used to determine the pH of prepared ethosomal gel, with the anode and cathode of glass soaked in the ethosomal gel formulation, and the measurements were recorded or exposed to view.

· A Brookfield viscometer was used to measure the viscosity of a formulation by immersing it in a beaker containing ethosomal gel at different speeds, while maintaining room temperature throughout the process.

· After precisely weighing an ethosomal gel, it was sandwiched between two 8 cm-long slides of glass. The time needed to pull the top slide and extend the gel to the lower slide was calculated with various weights applied to the pulley. The reading was taken three times and lastly measurements for Spreadability was determined from the following formula-

S = M* L/T

Where

S = denote the ethosomal gel Spreadability

M = denote weight applied to the top slide (g)

L = indicate the distance slide has moved (cm)

T = indicate the time taken by the top slide to move downwards (sec)

The study involved packing 20g of prepared luliconazole loaded ethosomal gel in a flexible tube, applying pressure, and attaching a clamp to prevent backflow. The gel's amount was then measured and recorded until the pressure remained constant.^30,31

Determination of stability studies:

The ethosomal formulation's stability is influenced by the lipid layers' capacity to drain and accumulate drugs. Stability testing was conducted on ethosomes for two months at different temperatures. Lyophilized ethosomes and ethosomal suspension were stored separately and examined after 7, 15, 30, 60, and 90 days of hoarding. The results highlight the importance of understanding the drug holding capacity of ethosomes.³²

RESULTS:

Determination of the size of Particle, polydispersity index (PDI) and zeta potential:

Based on dynamic light scattering (DLS), the size of particle was estimated, and Table no.2 presents the results for both size of particle and PDI. The range of size of particles in ethosomal formulation is 155.3±1.2 to 196.4±2.4nm, whereas the range of PDI is 0.186± 0.07 to 0.413±0.073.

With an ideal size of particle and PDI of 154.4±1.3 nm and 0.184 ± 0.08, respectively, preparation EF5 has the highest entrapment efficiency when contrasted with the other preparations.

On the other hand, formulation EF6 shows maximum values for PDI and size of particle, which are 196.4±2.4 nm and 0.413±0.073, respectively. When it comes to topical medication administration, vesicle size is quite important. The more adeptly the vesicle transfers the substance into the underlying layers of the skin, the smaller it gets. The size of Particle was ordered as follows: EF5< EF3< EF2< EF1< EF4< EF6.

PDI, on the other hand, was ordered as follows: EF5< EF1< EF3< EF2< EF4< EF6. When assessing whether the ethosomal formulation is stable, zeta potential is taken into account.

According to Table no.2, The values of the zeta potential varied from -22.5±1.6 to -42.2±0.3mV. EF5 exhibits the highest zeta potential value, measuring -42.2±0.03mV, whereas EF3 displays the poorest value, measuring approximately -37.5±0.5mV. Zeta potential can be determined in the following order: EF5< EF3< EF2< EF1< EF4< EF6.^33,34

Structural study of Luliconazole loaded ethosomes by Transmission electron microscopy (TEM):

With the assistance of TEM, the surface morphological concept of the created, optimised EF5 accumulation was expressed in (Fig. no.1). TEM pictures reveal details on the spherical size of the particles in the preparation. In addition to surface morphology. Images captured by TEM revealed that the ethosomes had a spherical form, were smooth, and lacked the drug's crystalline structure.³⁵

Fig. 1: Surface Structural study by TEM

Structural study of Luliconazole loaded ethosomes by Atomic force microscopy (AFM):

AFM pictures can be seen in (Fig. no.2). It is very difficult to extract detailed information from TEM images regarding the morphological characteristics, formulation behaviour, and swelling dynamics that are provided by an AFM image. Furthermore, it provides justification for the outward morphology of the molecule with respect to its height, diameter, and area. The picture makes it quite evident that the diameter and height are within a reasonable range. The diameter, height, and area of the ethosomal particles are determined 26.434 nm, 547.814nm², and 1.067nm, respectively.³⁶

Table no.2: characterization of different ethosomal formulation

Characterization	EF1	EF2	EF3	EF4	EF5	EF6
Size of Particle	177.3 ± 2.5	166.2 ± 3.3	162.7 ± 5.3	178.6 ± 4.1	154.4 ± 1.3	196.4 ± 2.4
PDI	0.252 ± 0.05	0.292 ± 0.05	0.263 ± 0.02	0.298 ± 0.02	0.184 ± 0.08	0.413 ± 0.073
Zeta potential	-29.5 ± 0.6	-32.5 ± 0.8	-37.5 ± 0.5	-27.4 ± 0.5	-42.2 ± 0.3	-22.5 ± 1.6

Fig. 2: Surace Morphology by AFM

Table no.3: pH, Viscosity, Spreadability and Extrudability of different ethosomal preparations

Characterization	EF1	EF2	EF3	EF4	EF5	EF6
pH	6.1	5.7	5.9	5.3	5.2	5.8
Viscosity	6845 ± 0.5	7063 ± 1.3	7521 ± 0.5	8528 ± 0.5	7232 ± 00.5	7652 ± 0.5
Spreadability	5.90 ± 0.8	6.20 ± 1.5	7.83 ± 0.3	8.05 ± 1.5	8.28 ± 0.6	4.8 ± 1.6
Extrudability	++	++	++	++	+++	+++

Determination of pH, viscosity, Spreadability and extrudability:

The range of characteristics for all ethosomal formulations, including pH and viscosity, was determined to be between 6.7 and 5.2 and 8528±2.4 to 6532±1.6 cps, respectively. Table no.3 presents a list of all formulation findings. The optimised formulation (EF5) was found to have a pH of 5.2 and a viscosity of 7232±1.2 cps, respectively. Table no.3 displays an exceptional range of values for the Spreadability and extrudability tests. The optimised formulation, which is EF5, yielded results for Spreadability and extrudability of 8.28±0.6 cm and +++, respectively.^37,38

Calculation of Drug Entrapment efficiency, EE (%):

Drug Entrapment efficiency is a crucial criterion since it provides information on the buildup formulation's delivery capability. Table no. 2 or 3 records the drug entrapment efficiency value of various luliconazole loaded ethosomal formulations. As seen in (fig. no.3), the graph was plotted for each formulation. The graph shows that, at 88.56 ± 0.74%, the formulation EF5 has the highest entrapment efficiency, while EF3 has the lowest, at 61.75 ± 0.63%. In contrast, the entrapment efficiency of formulations EF1, EF2, EF4, and EF6 was 65.23 ± 0.14%, 71.48 ± 0.29%, 75.19 ± 1.63%, and 62.39 ± 2.12%, respectively. For each of the nine ethosomal formulations, the increasing order of drug entrapment efficiency was determined to be EF5> EF4> EF2> EF1> EF6> EF3.^{39,40,41,42,43}

Fig. 3: EE (%) of ethosomal formation EF1 to EF6

Determination of stability studies:

A study of stability was used to find out the ethosomal system's capability to adhere to the medication. For three days, the investigation was conducted constantly at various temperatures. The main problems with ethosomal formulation are drug agglomeration and drainage into/from the lipid bilayer. Stability testing was conducted on the optimised formulation EF5, and the results are presented in Table no. 4, 5, 6 or 7. The lyophilized (non-aqueous) luliconazole loaded ethosome suspension and the un-lyophilized luliconazole loaded ethosome suspension used in this study were both sealed in vials and stored at 4°C/60±5 RH (n = 3) and 25 °C/60 ± 5 RH before conducting the study. Sampling was conducted after 7, 15, 30, 60, and 90 days of hoarding. Following lyophilization, the resulting non-aqueous luliconazole ethosomal sample was redistributed using Deionized water, and further studies were performed to determine the size of the particles, zeta potential, PDI, and percent entrapment efficiency. Following lyophilization, the resulting non-aqueous sample was redistributed using Millipore water, and further works were conducted to find out the size of the particles, zeta potential, PDI, and percent entrapment efficiency. The course of action was run three times to ensure precise findings, and the mean ± SD was used to publish the results.⁴⁴

Table 4: Stability study of optimized luliconazole loaded ethosomes particle suspension (EF5) at 4°C/60±5 RH (n=3)

Time	Microscopic evaluation	% Encapsulation efficiency
Initial	Soft ball-shaped particle	93 ± 2.8
7	Soft ball-shaped particle	95 ± 1.3
15	Soft ball-shaped particle	93 ± 1.1
30	Soft ball-shaped particle	91 ± 2.5
60	Soft ball-shaped particle	87 ± 1.8
90	Rough ball-shaped particle	79 ± 2.2

Table 5: Stability study of optimized luliconazole loaded ethosomes particle suspension (EF5) at 25°C/60 ± 5 RH (n=3)

Time	Microscopic evaluation	% Encapsulation efficiency
Initial	Soft ball-shaped particle	95 ± 1.1
7	Soft ball-shaped particle	91 ± 1.6
15	Soft ball-shaped particle	87 ± 2.1
30	Soft ball-shaped particle	80 ± 2.3
60	Rough ball-shaped particle	66 ± 2.2
90	Clump	60 ± 1.3

Table no. 6: Stability study of optimized Lyophilized luliconazole loaded ethosomes particle suspension (EF5) at 4°C/60 ±5 RH (n=3)

Time	Microscopic evaluation	% Encapsulation efficiency
Initial	Soft ball-shaped particle	95 ± 1.1
7	Soft ball-shaped particle	94 ± 1.7
15	Soft ball-shaped particle	91 ± 1.8
30	Soft ball-shaped particle	87 ± 2.7
60	Rough ball-shaped particle	84 ± 1.4
90	Rough ball-shaped particle	80 ± 3.6

Table no.7: Stability study of optimized Lyophilized luliconazole loaded ethosomes particle suspension (EF5) at 4°C/60 ±5 RH (n=3)

Time	Microscopic evaluation	% Encapsulation efficiency
Initial	Soft ball-shaped particle	93 ± 2.8
7	Soft ball-shaped particle	95 ± 1.3
15	Soft ball-shaped particle	93 ± 1.1
30	Rough ball-shaped particle	89 ± 2.5
60	Rough ball-shaped particle	83 ± 1.6
90	Clump	77 ± 2.0

Discussion:

With the help of ethanolic clove extract (S. aromaticum L.) combined with that of the medication luliconazole, an herbal ethosomal gel was created. Strong antifungal action of clove oil has been seen against opportunistic fungal infections, including Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. Eugenol from cloves is the key component that gives it its antifungal properties. The primary volatile component of the oil derived from buds of clove (S. aromaticum L.). It is utilised in conventional medicine as an anaesthetic, fungicide, bactericide, and other uses is eugenol. It was discovered that, in contrast to bacteria, fungi were the target of its antibacterial activity. In addition to eugenol (49–87%), clove oil also has trace amounts (<1%) of 25–35 other compounds, β-caryophyllene (4–21%), eugenyl acetate (0.5–21%), and lower percent of α-humulene.

To demonstrate the build-up's efficacy and fitness for the therapy, it was put through a series of characterizations. It has been closely examined that the vesicle size is directly correlated with the conce- of soya-lecithin and ethanolic clove oil in the rough calculation of the size of particle and PDI. Increases in soya-lecithin beyond 200mg and ethanolic clove extract concentrations 35%, reveal a sudden enhancement in size of particle and narrow dispensation for PDI. Therefore, it can be inferred that changes in the ratio of lipid to ethanol, either positive or negative, significantly affect the physical characteristics of ethosomes. Because only smaller particles may pass through the skin, the size of the particles and the concentration of ethanolic extract have an impact on the skin's permeability. Conversely, the concentration of ethanol typically has an impact on the drug's permeability. We may conclude that EF5 is entirely appropriate for topical application by taking the mentioned information into consideration. When it comes to zeta potential, research has shown that when a suspension contains high levels of cationic and anionic charge—i.e., a elevated value of either positive or negative zeta potential—the vesicle will encounter repulsive forces from one another and will resist agglomeration. When there is little anionic (-) or cationic (+) charge in the suspension (i.e., a low +ve or -ve zeta potential), there is no force exerted on the particles, which leads to their tendency to clump together. Therefore, an increase in surface charge results in an improvement in ethosome stability.

The ethosomes are characterised by their round shape, smooth appearance, and lack of crystalline structure, as demonstrated by the TEM pictures. On the other hand, deep information on the morphological traits, formulation behaviour, and swelling dynamics with each other is provided by AFM images, which is more difficult to collect from TEM images. Additionally, the photos make it evident that parameters like diameter and height are within a reasonable range, which contributes to the information on morphological traits by expressing the ethosomes' diameter, area, and height. Furthermore, entrapment efficiency also decreases at soya-lecithin concentrations of approximately 200 mg. However, it was found that even while soya-lecithin itself had a 200 mg concentration, the entrapment efficiency had dropped in EF5. This can be the result of a 40% rise in ethanol content. It is generally everyone knowns that ethanolic and lipid vesicles cannot stick together. With the results above, it can be made a final decision that although the concentration of ethanolic and lipid varies, entrapment efficiency and size of particle have a major impact, while the other ingredients remain constant. The formulations' ideal pH and viscosity indicate that they are highly suited for cutaneous therapy, according to the pH and viscosity results. The preparation is suitable and suited to the skin formulation and treatment, as indicated by the ideal range of Spreadability and extrudability. The ethosomal formulation's Spreadability results indicate good gelling properties for topical application.

Thus, it can be made a final decision that the release of drug profile from the preparation is significantly impacted by the alteration of lipid and ethanol. After analysis, the EF5 preparation was recognised as the optimal preparation and suitable for further research on antifungals (Agrawal et al. 2013). Based on study of stability results, it is determined that both lyophilized luliconazole ethosome particle suspension and luliconazole ethosome particle suspension exhibit a decrease in percent entrapment efficiency when stored in well closed container at room temperature, 25 °C/60 ± 5 RH; however, there is no important change in percent entrapment efficiency when stored at 4 °C/60 ± 5 RH (n = 3).

Thus, it was clear that 4 °C/60 ± 5 RH is an acceptable temperature for storing the optimised formulation of EF5 (Kim et al. 2006). Ultimately, it can be said that after taking into account all of the characteristics listed in (Table no. 2 or 3), the ethanol to lipid ratio for formulation EF5 was found to be optimised. It can be said that a high ethanol content is crucial to the formulation.

Conclusion:

Ethosomes demonstrate their superiority as a vehicle for delivering luliconazole, which gets ensnared in the lipid layer, to the skin. Because ethosomes contain a considerable amount of ethanol in their composition, it has been observed that they improve drug permeability through lipid vesicles and offer consistent permeability of skin. Thus, this eventually improves the medication luliconazole's bioavailability. The study's key findings were the vesicle's ideal size, excellent entrapment efficiency, and improved permeability capabilities. These characteristics thus indicate a longer-lasting drug release with increased stability at 4 °C/60 ± 5 RH. Differentiating the formulation's lipid and ethanolic concentrations affects the drug's delivery through ethosomal vesicles. Ultimately, it may be concluded that ethosomes provide effective therapy of antifungal infection by delivering advanced, sustained, and targeted distribution of luliconazole. This kind of cutting-edge technique can help with the development and industry scaling of new formulations in the future. Future research can be conducted based on these findings to advance the ethosomal gel loaded luliconazole formulation towards clinical implementation.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank IIMT College of Medical Science, IIMT University, Meerut for their kind support during this investigation and all other lab studies.

REFERENCES:

1. Ainbinder D, Touitou E. Testosterone ethosomes for enhanced transdermal delivery. Drug Deliv. 2010; 12:297–303.doi: 10.1080/10717540500176910.

2. Ainbinder D, Protok in R, Chaouat M, Touitou E. Effect of honokiol and 5-FU on non-melanoma skin cancer cells. J Drug Deliv SciTech Nol. 2009; 19: 283–287. doi: 10.1016/S1773-2247(09)50053-6.

3. Shumilov M, Touitou E. Buspirone transdermal administration for menopausal syndromes, in vitro and in animal model studies.Int J Pharm. 2010; 387: 26–33. doi: 10.1016/j.ijpharm.2009.11.029.

4. Shumilov M, Bercovich R, Duchi S, Ainbinder D, Touitou E. Ibuprofen transdermal ethosomal gel: characterization and efficiencyin animal models. J Biomed Nanotechnol. 2010; 6: 569–576. doi: 10.1166/jbn.2010.1153.

5. Nethravathi V, Vijaitha V. Effectiveness of Clove oil massage on Lower Back Pain among Post Natal Mothers at Selected Hospitals, Bangalore. Asian J. Nur. Edu. and Research. 2015; 5(4): 467-470.

6. Design, Formulation and in vitro Drug Release from Transdermal Patches containing Nebivolol Hydrochloride as Model Drug. Asian J. Pharm. Res. 2012; 2(4): 136-141.

7. R. S. Kalkotwar, R. B. Saudagar. Design, Synthesis and anti-microbial, anti-inflammatory, Antitubercular activities of some 2,4,5-trisubstituted imidazole derivatives. Asian J. Pharm. Res. 2013; 3(4): 159-165.

8. Rutuja R. Shah, Rohan R. Vakhariya. Formulation and Evaluation of Antifungal Soap of Garlic Oil. Asian J. Pharm. Res. 2020; 10(1):13-16.

9. Pallavi U. Jadhav, Sandhya G. Gujare, Mulchand A. Shende. Ethosomes: A Novel Tool for Vesicular Drug Delivery. Asian Journal of Pharmaceutical Research. 2024; 14(1): 45-2.

10. Prashant S. Wake, M. D. Kshirsagar. Design and Characterization of Solid Lipid Nanoparticle Based Transdermal Drug Delivery System. Asian J. Res. Pharm. Sci. 2017; 7(2): 87-91.

11. Cappelletty D, Eiselstein-McKitrick K. The echinocandins. Pharmacotherapy. 2007; 27: 369–388. doi: 10.1592/phco.27.3.369.

12. Sutton CL, Taylor ZE, Farone MB, Handy ST. Antifungal activity of substituted aurones. Bioorg Med Chem Lett. 2017; 27: 901–903.doi: 10.1016/j.bmcl.2017.01.012.

13. Sheehan DJ, Hitchcock CA, Sibley CM. Current and emerging azole antifungal agents. Clin Microbiol Rev. 1999; 12: 40–79.doi: 10.1128/CMR.12.1.40.

14. Baginski M, Czub J. Amphotericin B and its new derivatives—mode of action. Curr Drug Metab. 2009; 10: 459–469.doi: 10.2174/138920009788898019.

15. Y. Sirisha, Buddarthi Sriram, Ramya Sri S. Formulation Development and Evaluation Nefidipine Loaded Ethosomal Gel for Transdermal Drug Delivery. Asian Journal of Pharmaceutical Research. 2023; 13(2): 77-0.

16. Pandey G, Verma KK, Singh M. Evaluation of phytochemical, antibacterial and free radical scavenging properties of Azadirachtaindica (neem) leaves. Int J Pharm Pharm Sci. 2014; 6(2): 444–447.

17. Rutuja R Shah, Poonam J Patil, Pratibha R Adnaik, Rahul S Adnaik. Biochemical profiling of Antifungal soap activity of betel leaf (Piper betle L.) extract and Garlic oil by In vitro method. Asian J. Pharm. Res. 2020; 10(4): 260-262.

18. Muskan M. Maniyar, Amol S. Deshmukh, Suvarna J. Shelke. Ethosomes: A carrier for Transdermal Drug Delivery System. Asian Journal of Pharmaceutical Research. 2022; 12(3): 225-8.

19. Touitou E, Dayana N, Bergelsonb L, Godina B, Eliaza M. Ethosomes-novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J Control Release. 2000; 65: 403–418. doi: 10.1016/S0168-3659(99)00222-9.

20. Dave V, Sharma S, Yadav RB, Agarwal U. Herbal liposome for the topical delivery of ketoconazole for the effective treatment ofseborrheic dermatitis. Appl Nanosci. 2017; 7: 973–987. doi: 10.1007/s13204-017-0634-3.

21. Anand N, Johnson M, Arulsamy A. Antifungal properties of neem (Azadirachta indica) leaves extract to treatment hair dandruff. E-Int Sci Res J. 2010; 2: 244–252.

22. Paliwal S, Tilaka A, Sharma J, Dave V, Sharma S, Verma K, Tak K, Raghava K, Veera SR. Flurbiprofen-loaded ethanolic liposomeparticles for biomedical applications. J Microbiol. 2019; 161: 18–27.

23. Ch. Nagadev, M. Durga Srinivasa Rao, P. Venkatesh, D. Hepcykalarani, R. Prema. A Review on Transdermal Drug Delivery Systems. Asian J. Res. Pharm. Sci. 2020; 10(2):109-114.

24. Paliwal S, Tilaka A, Sharma J, Dave V, Sharma S, Verma K, Tak K, Raghava K, Veera SR. Flurbiprofen-loaded ethanolic liposomeparticles for biomedical applications. J Microbiol. 2019; 161: 18–27.

25. Paliwal S, Tilak A, Sharma J, Dave V, Sharma S, Yadav RB, Patel S, Verma K, Tak K. Flurbiprofen loaded ethosomes-transdermaldelivery of anti-inflammatory effect in rat model. Lipids Health Dis. 2019; 18: 1–15. doi: 10.1186/s12944-019-1064-x.

26. Pathana IB, Jawarea BP, Shelkeb S, Ambekarc W. Curcumin loaded ethosomes for transdermal application: formulation,optimization, in-vitro and in-vivo study. J Drug Deliv Sci Technol. 2017; 44: 49–57. doi: 10.1016/j.jddst.2017.11.005.

27. Dave V, Yadav RB, Kushwaha K. Hybrid nanoparticles for the topical delivery of norfloxacin for the effective treatment of bacterialinfection produced after burn. J Microencapsul. 2017; 21: 67–77.

28. Ling G, Zhang P, Zhang W, Sun J, Meng Z, Qin Y, Deng Y, He Z. Development of novel self-assembled DS-PLGA hybrid nanoparticles for improving oral bioavailability of vincristine sulphate by P-gp inhibition. J Control Release. 2010; 148: 241–248.doi: 10.1016/j.jconrel.2010.08.010.

29. Shah PP, Desai PR, Singh M. Effect of oleic acid modified polymeric bilayered nanoparticles on percutaneous delivery of spantideII and ketoprofen. J Control Release. 2012; 158: 336–345. doi: 10.1016/j.jconrel.2011.11.016.

30. Mandal B, Bhattacharjee H, Mittal SH, Balabathula P, Thoma LA, Wood GC. Core-shell type lipid-polymer hybrid nanoparticles as adrug delivery platform. Nanomed Nanotechnol Biol Med. 2013; 9: 474–491. doi: 10.1016/j.nano.2012.11.010.

31. Panigrahi L, Ghosal SK, Pattnaik S, Maharana L, Barik BB. Effect of permeation enhancers on the release and permeation kineticsof lincomycin hydrochloride gel formulations though mouse skin. Indian J Pharm Sci. 2006; 68: 205–211. doi: 10.4103/0250-474X.25716.

32. Kim HS, Song IH, Kim JC. In vitro and in vivo gene transferring characteristics of novel cationic lipids, DMKD (O,O 0-dimyristyl-N-lysyl aspartate) and DMKE (O, O0-dimyristyl-N-lysylglutamate) J Control Release. 2006; 11: 234–241.doi: 10.1016/j.jconrel.2006.08.003.

33. Verma DD, Verma S, Blume G, Fah A. Size of particle of liposomes influences dermal delivery of substances into skin. Int J Pharm.2003; 258: 141–151. doi: 10.1016/S0378-5173(03)00183-2.

34. Plessis JD, Ramachandran C, Weiner N, Müller DG. The influence of size of particle of liposomes on the deposition of drug into skin.Int J Pharm. 1994; 103: 277–282. doi: 10.1016/0378-5173(94)90178-3.

35. Bratu I, Borodi G, Kacso I, Moldovana Z, Filip C, Dragan F, Vasilescu M, Simon S. New solid form of Norfloxacin: structuralstudies. J Spectrosc. 2011; 25: 53–62. doi: 10.1155/2011/462913.

36. Muller RH, Radtke M, Wissing SA. Solid lipid nanoparticles and nanostructured lipid carriers in cosmetics and dermatological preparations. Adv Drug Deliv Rev. 2002; 54: 131–155. doi: 10.1016/S0169-409X(02)00118-7.

37. Dave V, Sharma S, Yadav RB, Agarwal U. Herbal liposome for the topical delivery of ketoconazole for the effective treatment of seborrheic dermatitis. Appl Nanosci. 2017; 7: 973–987. doi: 10.1007/s13204-017-0634-3.

38. Panigrahi L, Ghosal SK, Pattnaik S, Maharana L, Barik BB. Effect of permeation enhancers on the release and permeation kinetics of lincomycin hydrochloride gel formulations though mouse skin. Indian J Pharm Sci. 2006; 68: 205–211. doi: 10.4103/0250-474X.25716.

39. Shumilov M, Touitou E. Buspirone transdermal administration for menopausal syndromes, in vitro and in animal model studies.Int J Pharm. 2010; 387: 26–33. doi: 10.1016/j.ijpharm.2009.11.029.

40. Sutton CL, Taylor ZE, Farone MB, Handy ST. Antifungal activity of substituted aurones. Bioorg Med Chem Lett. 2017; 27: 901–903.doi: 10.1016/j.bmcl.2017.01.012.

41. Verma DD, Verma S, Blume G, Fah A. Size of particle of liposomes influences dermal delivery of substances into skin. Int J Pharm.2003; 258:141–151. doi: 10.1016/S0378-5173(03)00183-2.

42. Ling G, Zhang P, Zhang W, Sun J, Meng Z, Qin Y, Deng Y, He Z. Development of novel self-assembled DS-PLGA hybrid nanoparticles for improving oral bioavailability of vincristine sulphate by P-gp inhibition. J Control Release. 2010; 148:241–248.doi: 10.1016/j.jconrel.2010.08.010.

43. Shah PP, Desai PR, Singh M. Effect of oleic acid modified polymeric bilayered nanoparticles on percutaneous delivery of spantide II and ketoprofen. J Control Release. 2012; 158: 336–345. doi: 10.1016/j.jconrel.2011.11.016.

44. Kim HS, Song IH, Kim JC. In vitro and in vivo gene transferring characteristics of novel cationic lipids, DMKD (O, O 0-dimyristyl-N-lysyl aspartate) and DMKE (O, O0-dimyristyl-N-lysylglutamate) J Control Release. 2006; 11: 234–241.doi: 10.1016/j.jconrel.2006.08.003.

Received on 05.06.2024 Revised on 29.10.2024

Accepted on 12.01.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3501-3508.

DOI: 10.52711/0974-360X.2025.00504

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.