Materials

NDFX was acquired as a complimentary sample from Wockhardt Research Centre, located in Aurangabad, India. Soya Phosphatidylcholine-90 was acquired as a complimentary sample from Lipidome Lifesciences, based in Ahmedabad, India. Tween 80 was procured from Hi-Media Laboratories, Pvt. Ltd. (Mumbai, India). Ethanol was procured from S.D. Fine Chem Ltd (Mumbai, India). Carbopol 934 was obtained from LOBA Chemie Pvt. Ltd, located in Mumbai, India. Methanol and chloroform were acquired from Fisher Scientific, India. All remaining chemicals utilized were of analytical grade.

Experimental animal

Wistar rats either male or female weighing from 150 to 200 gm were selected for the study. Before commencing the study, necessary authorization and endorsement were obtained from the Institutional Animal Ethics Committee (IAEC) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), under the Reg. No. 221/Po/Re/S/2000/CPCSEA, during their meeting on 03/12/2022.

MethodsPreparation of NDFX-loaded transethosomes

The cold method was employed for developing NDFX-loaded transethosomes. This method involves mixing of two phases at low temperature (30 °C) in a water bath. In this method aqueous phase (edge activator + water) is added to organic phase (ethanol + phospholipid) with continuous stirring in order to form the vesicles. This method is most widely used for thermo-labile drugs, and it is easily scalable [10].

Soya phosphatidylcholine 90 (phospholipid), Tween 80 (surfactant) and Nadifloxacin (1% w/w) were dissolved in ethanol at a temperature of 30 °C, forming the organic phase. Simultaneously, the aqueous phase (consisting of Millipore water) was heated to 30 °C. The aqueous phase was then added drop by drop to the organic phase, while the mixture was continuously stirred at a speed of 1200 revolutions per minute using a magnetic stirrer (RCT Basic IKA, India). The stirring was continued for 45 min to get the transethosomal dispersions. Thereafter, transethosomal dispersion was ultrasonicated using a probe sonicator (SONICS® VCX 750, USA). The formulation was then refrigerated at 4 °C until further characterization [11].

Optimization of NDFX-loaded transethosomes by Box–Behnken design (BBD)

Based on the literature review, several parameters have been identified that may influence the ideal characteristics of transethosomes for topical delivery and it was found that three variables (factors) such as phospholipid, tween 80 and ethanol concentration might have a direct impact on the critical transethosome characteristics required for successful topical administration. The response surface methodology utilized the Design Expert software to construct a Box–Behnken design consisting of three factors with three levels each (33). This experimental design, generated with the software, comprised a total of 17 runs, including 5 repeated center points, and the resulting outcomes were subsequently measured. By employing the Box–Behnken design, the study aimed to examine the impact of phospholipid, surfactant and ethanol concentrations on dependent variables, namely entrapment efficiency, vesicle size and zeta potential. Table 1 shows the independent and dependent elements chosen for the experimental design. The NDFX-loaded transethosome vesicles were tested for vesicle size, entrapping efficiency and zeta potential [12].

Table 1 Independent factors with their levels and dependent factors with their constraints in Box–Behnken design for the development of NDFX-loaded transethosomesEvaluation of NDFX-loaded transethosomesParticle size, polydispersity index and zeta potential

The Malvern zeta sizer (Malvern Instruments, Worcestershire, UK) was utilized to ascertain the particle size, polydispersity index and zeta potential of the transethosomes. The measurements were taken at a constant angle of 90° and a temperature of 25 ± 2 °C. To facilitate the analysis, the samples were diluted in millipore water and the measurements were taken in triplicate. These parameters provide information regarding the size, homogeneity and stability of the vesicles [13].

Entrapment efficiency

The ultra-centrifugation technique was used for estimating the entrapment efficiency of transethosomal formulations. Two milliliters of each formulation was transferred into a microcentrifuge tube and was subjected to ultracentrifugation at 4 °C by an Ultracentrifuge (Kubota, Japan). Following that, the supernatant was extracted with a micropipette and diluted with methanol to rupture the vesicles. A UV spectrophotometer (Shimadzu -1900, Japan) was used to measure the quantity of drug in the supernatant at 295.0 nm [14].

The following formula was used to compute the % EE.

$$\% } = \frac}\;}\;} - }\;}}}}\;}\;}}} \times 100$$

Surface morphology of optimized transethosomes

Transmission electron microscopy (TEM) was employed to examine the morphology of the prepared transethosomes. To summarize the procedure, a small amount of the diluted transethosomes sample was deposited onto a copper grid and left to air-dry. Once dried, the sample was treated with a 1% w/v solution of phosphotungstic acid for fixation and subsequently subjected to TEM analysis, with accompanying photographs captured [15].

Stability studies of optimized formulation

To determine the stability of the optimized transethosomes, short-term stability studies were conducted in compliance with ICH GCP guidelines. The prepared formulation was stored in glass vials within a humidity-controlled oven maintained at a temperature of 25 ± 2 °C and a relative humidity of 65 ± 5%. Additionally, it was refrigerated at 4 ± 2 °C with a relative humidity of 65 ± 5%. At regular intervals of 0, 15, 30 and 90 days, a sample was extracted for analysis [11].

Preparation of transethosomal gels

The preliminary studies were carried out, the gel was formulated through the dissolution of Carbopol 934 in purified water while stirring continuously; and the pH was adjusted to 6–6.5 by incorporating a 10% solution of triethanolamine. HPMC K4M was dispersed in purified water and left overnight. To create the transethosomal gel, the previously prepared transethosomal dispersion was incorporated into the gel in a 1:1 ratio with adequate stirring. The formulation details for gel bases are provided in Table 2 [16, 17].

Table 2 The composition of different gel basesEvaluation of transethosomal gelsDetermination of pH

In order to ascertain the pH of multiple gels, a digital pH meter was employed. Firstly, 500 mg of pre-prepared transethosomal gels was dissolved in 20 milliliters of distilled water. The resulting mixture was stirred for a duration of 30 min at room temperature using a magnetic stirrer. Subsequently, the pH sensor probe electrode was immersed in the dissolved gel and the pH value of the formulation was recorded from the digital screen [17].

Determination of viscosity

The viscosity of the optimized transethosomal gel formulation was estimated using a Brookfield viscometer. After applying the transethosomal gel formulation, it was allowed to settle for 5 min. After that, spindle number one was revolved at 50 revolutions per minute at a temperature of 25 ± 2 °C [18].

Determination of spreadability

The spreadability of the transethosomal gel was assessed using the glass slide technique. A precisely measured 1.0 g of gel was positioned at the center of a glass slide measuring 10 × 5 cm. Another slide of the same dimensions was placed on top of it. To ensure uniform compression and maintain a consistent thickness, a weight of 100 g was applied to the upper slide for a duration of 5 min. The time required for the glass slide to move 6 cm and separate from the lower glass slide was recorded [19, 20]. The spreadability of the gel was then calculated using the following formula:

where ‘S’ represents Spreadability, ‘M’ represents the weight applied to the upper slide, ‘L’ represents the distance traveled by the slide (6 cm), and ‘T’ represents the time taken in seconds.

Determination of drug content

In a 50-ml volumetric flask, 1 gm of gel was dissolved with 50 ml methanol. The solution underwent sonication in a bath until a transparent solution was achieved. Subsequently, the solution was filtered through a 0.45-µm filter and appropriately diluted using methanol. The drug concentration was determined by measuring absorbance with a UV spectrophotometer at the wavelength of 296 nm, using methanol as the reference solution [19].

$$}\;} = \frac}\;}\;}\;}\;}}}}\;}\;}\;}\;}}} \times 100$$

In vitro drug diffusion study

The Franz diffusion cell was utilized to determine the in vitro diffusion of drugs from different transethosomal gel formulations. To activate the dialysis membrane, it was previously soaked in pH 7.4 buffer. The receptor chamber was then filled with 12 ml of phosphate buffer at pH 7.4, which served as the medium for diffusion in the receptor compartment. The gel formulation, equivalent to 2 mg of the drug, was accurately weighed and thronged in the preactivated dialysis membrane. Throughout the experiment, the receptor medium was maintained at a temperature of 37 ± 2 °C and stirring was consistently performed at 100 rpm. At specific time intervals of 0.5, 1, 2, 4 and 8 h, 0.5 ml samples were withdrawn from the cell and replaced with fresh medium. The quantification of drug release was carried out using a UV spectrophotometer at a wavelength of 291 nm [21].

Ex vivo permeation study

A preliminary study was conducted to examine skin permeation using rat skin and the Franz diffusion cell, which had an effective permeation area of 1.76 cm2. Prior to the permeation study, the rat skin underwent a preparation process. Initially, the hair on the skin was eliminated using an electronic trimmer, followed by the removal of subcutaneous tissue through surgical means. Isopropyl alcohol was used to remove the fat from the dermis side of the skin. Afterward, the skin was washed with PBS and stored at − 20 °C in a deep freezer until it was ready for use. During the experiment, rat skin was affixed onto a diffusion cell with the dermis side facing the receiver compartment and the stratum corneum side facing the donor compartment. The receiver compartment contained a medium of PBS with a pH of 7.4. The temperature in the receiver compartment was maintained at 37 ± 0.5 °C, and throughout the experiment, it was stirred using a magnetic bead at a speed of 100 rpm. At predetermined time intervals of 0.5, 1, 2, 4 and 8 h, 0.5 ml samples were withdrawn and replaced with fresh medium. The collected samples were then analyzed for drug content using a UV spectrophotometer [22].

In vitro anti-bacterial study

Zone of inhibition The cup plate technique was used to investigate the zone of inhibition of prepared NDFX-loaded transethosomal gel against Propionibacterium acne. The Mueller–Hinton agar plates were prepared and sterilized at 121 °C and at a pressure of 15 lb. for 20 min. Subsequently, 25 ml of Mueller–Hinton agar was carefully poured into each Petri dish, allowing it to solidify. Following that, the Petri dishes were streaked with a suspension of Propionibacterium acne. Using a cork borer, four holes, each measuring 6 mm in diameter, were created on every plate. In each of these holes, the following substances were placed: a transethosomal formulation loaded with NDFX at a concentration of 10 µg (test), a commercially available NDFX cream at a concentration of 10 µg (standard), 100 µl of a blank transethosomal formulation (blank) and a negative control consisting solely of the vehicle (ethanol and buffer). The plates were incubated at 37 °C for 48 h in an anaerobic condition. The zone of inhibition was measured, and the antimicrobial activity of each formulation toward the P. acnes was investigated [23,24,25].

Animal studySkin irritation study

A skin irritancy test was conducted on the hairless backs of Wistar rats weighing between 180 and 250 g. The purpose of the test was to determine the irritant or toxic effects of the formulated transethosomal gel on the skin. The animals were segregated into two distinct groups, with each group consisting of six test subjects, as indicated in Table 3. Group I received an application of Optimized NDFX transethosomal gel, whereas Group II received blank transethosomal gel. Prior to the application of gel, the dorsal region of the Wistar rat was trimmed using an electronic trimmer to remove the hair, followed by the application of gel onto the hairless area measuring 5 cm2. A thorough examination was conducted on the rat's skin to identify any signs of erythema and edema. After 1 h, 24 h, 48 h and 7th day of application of test substances, finally, as per the Draize protocol the scoring was given and the skin irritancy potential of the formulated transethosomal gel was determined [26,27,28].

Table 3 Experimental design for in vivo skin irritancy studyAnti-acne studyAnimal model

Wistar albino rats either male or female weighing between 150 and 200 g were employed to examine the potential anti-acne effects of a transethosomal gel loaded with NDFX. These rats were acclimated to the prescribed rehabilitation conditions for a minimum of seven days prior to the experiment.

Bacterial sample

The bacterial strain of Propionibacterium acne was cultivated within an anaerobic gas chamber using brain heart infusion (BHI) broth. The culture was incubated for 48 h at 37 °C, after which the BHI agar plate was used for subsequent culturing bacterial strain. A single colony was selected from the BHI agar plate and introduced into a PBS solution. The turbidity of the suspension was assessed using the 0.5 McFarland (1.5 × 108 CFU/ml) scale. This bacterial suspension is now prepared for injection.

Injection of bacteria

An anesthetized Wistar rat was administered a subcutaneous injection of Propionibacterium acne bacterial suspension, measuring 20 µl, into its right ear. As a vehicle control, phosphate buffer saline was injected into the left ear [29].

Treatment approach

To investigate the anti-acne potential of prepared experimental gel (NDFX-loaded transethosomal gel) in animal models, the Wistar rats were categorized into four groups, with each group consisting of six animals, as depicted in Table 4. In each group, the rats received an injection of the aforementioned bacterial suspension of P. acnes in their left ear, while their right ear was injected with phosphate buffer saline. Initially, no treatment was given to all the groups for the period of seven days (inducing period). Following a period of seven days with no treatment provided to Group I (disease control), Group II was chosen as the benchmark and subjected to treatment using the commercially available anti-acne formulation known as Nadibact cream (containing 1% w/w active ingredient); Group III and Group IV were treated with the prepared NDFX-loaded transethosomal gel and plain NDFX gel, respectively. The treatment was continued till the 14th day for groups II, III, IV, and the measurement was taken using a Vernier Caliper daily, the ear thickness was measured on a predetermined day, and the formula used to calculate the percentage change in ear thickness is as follows

$$\% \Delta }\;} = \frac} \times 100$$

where TAfter represents the thickness of the auricle subsequent to the injection, while TBefore denotes the thickness of the auricle prior to the injection [30].

Table 4 Experimental design for in vivo anti-acne studyHistopathological investigation

Once the anti-acne study was completed successfully on the 14th day, three animals from each group were chosen at random and killed by euthanasia. Prior to sectioning, the ear was surgically removed and immersed in a 10% formalin solution. Subsequently, the sections were treated with hematoxylin and eosin dye for staining. The stained ear slices were then placed on glass slides and examined using an optical microscope [30].

Post-treatment microbiological assay

Upon successful completion of the anti-acne study, the animals from each group were euthanized and the treated area on the right ear was gently wiped with a cotton swab soaked in ethanol before being excised. The excised ear was then divided into small pieces and homogenized in 5 ml of phosphate buffer saline using a tissue homogenizer. Subsequently, the homogenized saline solution was appropriately diluted. A small portion of the homogenate was extracted and evenly spread onto a sterilized cotton swab, which was used to inoculate a Mueller–Hinton agar plate. The plate was then incubated under anaerobic conditions at 37 °C. The number of colony-forming units (CFUs) present on the agar plate was subsequently counted in order to investigate the pharmacodynamic activity of the formulation [25].