All chemicals used in this study were of laboratory-grade. SabiWhite, a standardized curcumin derivative, was procured from Merck, USA. Ethanol and Phospholipon 90G were also sourced from Merck, while the remaining reagents were obtained from certified suppliers in China.

SabiWhite was characterized using multiple analytical techniques to confirm its identity, purity, and physicochemical properties. The capillary method was used to determine the melting point of SabiWhite, a standard approach for assessing purity (Loron et al., 2021). A finely powdered sample of SabiWhite was placed in a sealed glass capillary tube and subjected to controlled heating using a digital melting point apparatus. The temperature at which the substance transitioned from a solid to a liquid state was recorded. A sharp and narrow melting range indicates high purity, whereas a broader or lower melting range suggests the presence of impurities. Fourier-Transform Infrared Spectroscopy (FTIR) was conducted to identify the functional groups and chemical bonds present in SabiWhite (Soury et al., 2023). The sample was prepared by mixing a small quantity of SabiWhite with potassium bromide (KBr) and compressing the mixture into a transparent pellet. The pellet was then analyzed using an FTIR spectrometer, where infrared radiation was passed through the sample. The resulting spectra were examined to detect characteristic absorption peaks corresponding to functional groups such as hydroxyl (-OH), carbonyl (C=O), and aromatic rings. FTIR analysis is essential for verifying the molecular structure of SabiWhite and assessing its compatibility in formulation systems. The crystalline nature and solid-state properties of SabiWhite were evaluated using XRD. A finely ground sample was placed in the sample holder of an X-ray diffractometer, and monochromatic X-rays were directed at the sample under controlled conditions (Soury et al., 2023). The diffracted X-rays produced a pattern of peaks, which were analyzed to determine the crystal lattice structure and degree of crystallinity. Sharp, well-defined peaks in the XRD pattern indicate a highly crystalline material, whereas broad and diffuse peaks suggest an amorphous nature. XRD analysis provided crucial insights into the structural integrity and phase purity of SabiWhite.

SabiWhite-loaded ethosomes were prepared using a modified cold method originally described by Touitou et al (Touitou et al., 2000). Briefly, Phospholipon^® 90G and SabiWhite (5 mg/mL) were dissolved in ethanol and stirred at 700 rpm using a magnetic stirrer (RCT Basic, IKAMAG, IKA, Germany) at 30 ± 1°C in an airtight container. Phosphate-buffered saline (PBS, pH 6.4) was added dropwise (200 µL/min) under continuous stirring at the same temperature. The resulting suspension was stirred for an additional 10 minutes and stored at 4°C overnight for vesicle swelling. To achieve uniform vesicle size, the suspension was probe-sonicated for 5 minutes at 4°C using an ultrasonic sonicator, followed by sequential filtration through sterile 0.45 µm and 0.20 µm syringe filters (10 cycles). The final dispersion was stored at 4°C for further characterization. A 3² factorial design was used for formulation optimization, with phospholipid concentration (X1) and ethanol content (X2) as independent variables, while vesicle size (Y1) and entrapment efficiency (Y2) were selected as dependent variables ( Supplementary Table 1 ). The amount of SabiWhite remained constant across all formulations. Entrapment efficiency (EE%) was determined via ultracentrifugation. A 2 mL aliquot of the ethosomal suspension was centrifuged at 20,000 rpm for 3 hours at 4°C (Kubota Model 7000, Japan). The supernatant was collected, diluted, and analyzed using a UV-visible spectrophotometer (Lab India UV-3200, India) to quantify unencapsulated SabiWhite. EE% was calculated as: % E E = ( T − S T ) X 100 , where T is the total amount of SabiWhite in the formulation, and S is the unencapsulated amount in the supernatant. The optimized formulation was selected based on maximizing entrapment efficiency and achieving a vesicle size within the 100–200 nm range.

The in vitro drug release of SabiWhite from SW-ETH formulations was evaluated using a Franz diffusion cell (diffusion area: 3.14 cm²) with a receptor chamber volume of 20 mL. A dialysis membrane (MWCO 12–14 kDa) was pre-soaked in phosphate-buffered saline (PBS, pH 5.5) overnight at 25 ± 0.5°C before being positioned between the donor and receptor compartments (Salamanca et al., 2018). To simulate non-occluded skin conditions, the donor compartment was left open. The receptor medium was maintained at 37 ± 0.5°C and continuously stirred at 100 rpm using a magnetic stirrer (Remi Equipment Pvt Ltd., India) to ensure uniform mixing. At predetermined time intervals (0.5, 1, 2, 3, 4, 6, 8, and 24 hours), 1 mL aliquots were withdrawn from the receptor compartment and immediately replaced with an equal volume of fresh PBS (pH 5.5) to maintain sink conditions. The withdrawn samples were analyzed for SabiWhite content using a UV-visible spectrophotometer at 284 nm. All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD).

To elucidate the drug release mechanism, the obtained release data were fitted to Zero-Order kinetic models (Alshahrani et al., 2024): (constant release rate independent of drug concentration):C=C₀-K₀t. Where C is the cumulative drug release, C₀ is the initial drug concentration, K₀ is the zero-order rate constant, and t is time. A plot of cumulative drug release versus time was analyzed for linearity. All kinetic models were analyzed by calculating the correlation coefficient (R²), and the best-fitting model was determined based on the highest R² value.

The anti-inflammatory efficacy of SW-ETH was evaluated using a carrageenan-induced paw edema model in Wistar albino rats (Ben Khedir et al., 2016). The study was conducted in compliance with OECD guidelines and received approval from the institutional animal ethics committee. Twelve female, nulliparous, and non-pregnant Wistar rats (200–250 g, aged 8–12 weeks) were randomly divided into four groups (n=3 per group). The control group (Group I) received carrageenan injection without any treatment, while the reference group (Group II) was pre-treated with Diclofenac gel (2 mg/paw equivalent to 10 mg/cm²) 30 minutes before carrageenan injection. Group III was treated with SW-ETH gel (2 mg/paw), and Group IV received a placebo gel. Carrageenan (0.1 mL, 1% w/v) was injected into the plantar region of the right hind paw to induce inflammation, and formulations were applied topically 30 minutes prior to the injection. The paw volume was measured using a digital caliper at specific time points (30, 60-, 90-, 120-, and 150 minutes post-injection).

The percentage inhibition of edema was calculated using the equation:

Percent edema inhibition = [ 1 − ( V T V 0 ) X 100 ] where V_T represents the edema volume in the treated group and V₀ represents the edema volume in the control group.

A skin irritation study was conducted following OECD Guideline 404. Three female Wistar albino rats were used, and their dorsal fur was removed 24 hours before testing. A 0.5 g dose of SW-ETH gel was applied to a 6 cm² area using a gauze patch secured with a semi-occlusive dressing. The patch was removed after four hours, and the test site was observed for erythema and edema at 60 minutes, 24 hours, 48 hours, and 72 hours post-application. Dermal reactions were graded based on OECD-defined criteria, ranging from 0 (no irritation) to 4 (severe irritation) ( Supplementary Table 2 ). The primary irritation index (PII) was calculated as the mean sum of erythema and edema scores. Based on PII values, skin responses were classified as non-irritant (0.0), negligible irritant (0.1–0.4), slight irritant (0.41–1.9), moderate irritant (2.0–4.9), or severe irritant (5.0–8.0).

The stability of the optimized SW-ETH gel was evaluated over 90 days under three different storage conditions: refrigeration (4 ± 2°C), room temperature (25 ± 2°C), and accelerated conditions (37 ± 2°C). The formulation was stored in amber glass containers to minimize light-induced degradation. Samples were analyzed at predetermined intervals for changes in physical appearance, viscosity, pH, drug content, and spreadability (Kirtane et al., 2022). Viscosity measurements were conducted using a Brookfield viscometer (spindle #1, 50 rpm), while pH was determined using a calibrated digital pH meter. Drug content was analyzed via UV-visible spectrophotometry at 284 nm. Spread ability was evaluated using the circular plate method, wherein a 2 cm diameter gel sample was placed between two glass plates, and the final spread diameter was measured. The formulation was considered stable if no significant changes in these parameters were observed over 90 days.

All experiments were performed in triplicate (n=3). Given the exploratory nature of this formulation development study and in accordance with the journal’s statistical guidelines for small sample sizes, non-parametric permutation tests were employed for group comparisons instead of traditional ANOVA. This approach makes no assumptions about data distribution and is specifically recommended for studies with n<5 (DDDT Editorial, June 2023) (Panos and Boeckler, 2023). For all analyses, p-values were generated through 10,000 random permutations of the dataset using R software (version 4.2.1). Effect sizes with 95% confidence intervals are reported where applicable. All data are presented as mean ± SD with permutation-based p-values <0.05 (p(perm)=0.021) considered significant.

The melting point of SabiWhite was determined to be 96°C, aligning closely with previously reported values, thereby confirming the identity and purity of the compound. The structural identity of SabiWhite was further validated through Fourier-transform infrared (FTIR) spectroscopy. The FTIR spectrum ( Figure 1 ) exhibited characteristic absorption peaks at 3421 cm^-1 (O-H stretching), 3200–3000 cm^-1 (C-H stretching), 2933 cm^-1 (OCH₃ stretching), 1601 cm^-1 (C=O stretching), 1457 cm^-1 (C-H bending of methyl groups), 1277 cm^-1 (C-O stretching), and 1032 cm^-1 (C-O-CH₃ stretching). These peaks correspond to functional groups intrinsic to SabiWhite, confirming its molecular structure and chemical integrity.

X-ray diffraction (XRD) analysis of pure SabiWhite ( Figure 2 ) revealed a distinct and sharp diffraction peak at 18.560°, indicative of its crystalline nature. In contrast, the phospholipid exhibited a broad diffraction peak at 21.649°, characteristic of an amorphous structure. The physical mixture of SabiWhite and phospholipid retained the diffraction peak of pure SabiWhite, suggesting that its crystalline structure remained unaltered. However, in the ethosomal formulations, the diffraction peak of SabiWhite was no longer distinctly observed, indicating its integration into the lipid matrix and a transition to an amorphous or disordered state. This structural transition from crystalline to amorphous could potentially enhance the bioavailability of SabiWhite. Amorphous forms of compounds generally exhibit higher solubility compared to their crystalline counterparts, which may improve their dissolution rate and skin permeability. However, this transition may also influence the chemical stability of the compound, as amorphous forms can sometimes be more prone to degradation. Further stability studies would be required to fully assess the impact of this transition on the long-term stability of SabiWhite in its ethosomal formulation.

The formulation of SW-ETH was successfully achieved through the modified cold method. The optimization of vesicle size and entrapment efficiency (EE%) was carried out using a 3² factorial design, with phospholipid concentration (X1) and ethanol content (X2) as independent variables. The resulting formulations exhibited vesicle sizes ranging from 100.6 nm to 271.6 nm and entrapment efficiencies from 77.1% to 94.8%. Table 1 summarizes the vesicle size, entrapment efficiency (EE%), polydispersity index (PDI), and zeta potential (ZP) of all formulations. The formulation SW-ETH 6, with 3% phospholipid and 45% ethanol, achieved the best results in terms of both entrapment efficiency (92.5%) and vesicle size (184.4 nm). This formulation was selected for further optimization and characterization.

Analysis of variance (ANOVA) for both vesicle size and entrapment efficiency was conducted to evaluate the statistical significance of the models. The model F-values were found to be highly significant for both responses (vesicle size and entrapment efficiency), with p-values < 0.0001 for vesicle size and 0.0014 for entrapment efficiency. The results of ANOVA for both responses are shown in Table 2 . Furthermore, regression analysis was conducted for both vesicle size and entrapment efficiency. The quadratic model was found to be the most suitable for both responses, with R² values of 0.9993 for vesicle size and 0.9945 for entrapment efficiency, indicating a strong fit between the observed and predicted data ( Table 3 ).

The effect of phospholipid and ethanol concentration on vesicle size and entrapment efficiency was analyzed using response surface plots, 2D contour plots, and perturbation graphs. For vesicle size, an increase in phospholipid concentration from 2% to 4% resulted in larger vesicles, while a higher ethanol concentration reduced vesicle size, as depicted in the 3D response surface and 2D contour plots ( Figures 3A, B ). Similarly, for entrapment efficiency, both higher phospholipid and ethanol concentrations positively influenced EE%, as illustrated in Figure 3B .

Numerical optimization was employed to identify the optimal formulation with desired attributes. The optimized formulation, SW-ETH 6, exhibited a vesicle size of 184.4 nm and an entrapment efficiency of 92.5%. These results align closely with the predicted values, confirming the accuracy of the model. The optimized formulation was further characterized for PDI and zeta potential, which were found to be 0.25 and -13.50 mV, respectively, indicating a stable and uniform formulation.

The vesicle size of the ethosomal formulation ranged from 120 nm to 220 nm, with the optimized SabiWhite-loaded ethosomal formulation (OPT-SW-ETH) exhibiting a size of 184.40 nm ( Figure 4A ). The polydispersity index (PDI), a key indicator of size uniformity, ranged from 0.10 to 0.27, with OPT-SW-ETH showing a PDI of 0.25, confirming a homogeneous vesicle distribution ( Figure 4A ). A PDI below 0.3 is generally considered acceptable (Danaei et al., 2018), supporting the uniform nature of the formulation. Zeta potential (ZP) analysis was performed to assess the stability of the ethosomal vesicles. The ZP values ranged from -8.80 mV to -14.10 mV, with OPT-SW-ETH displaying a ZP of -13.50 mV ( Figure 4B ). The negative surface charge can be attributed to the presence of ethanol and phospholipid, which contribute to vesicle stability by inducing repulsive forces that prevent aggregation. Higher absolute values of ZP indicate better colloidal stability, reinforcing the integrity of the optimized formulation.

Optical microscopy revealed vesicles with a spherical shape and multi-lamellarity, with no signs of aggregation or fusion ( Figure 5A ). A drop of diluted SW-ETH suspension was placed on a glass slide, covered with a coverslip, and examined under an RXL-4T microscope (20× magnification). The images confirmed the presence of discrete, well-formed vesicles. A drop of the optimized formulation was deposited on a carbon-coated copper grid and left to air-dry, followed by staining with 1% phosphotungstic acid (PTA). The TEM images confirmed the presence of unilamellar vesicles in the nanometer range with slightly irregular shapes ( Figure 5B ). Quantitative vesicle size analysis from TEM images was conducted using ImageJ software, measuring at least 100 vesicles per sample to ensure statistical accuracy.

The in-vitro drug release study was conducted using a Franz diffusion cell to evaluate the release characteristics of SW-ETH formulations in comparison to a pure SabiWhite solution. The cumulative drug release profile was assessed over 24 hours. The results demonstrated an initial burst release phase, attributed to SabiWhite adsorbed on the vesicle surface, followed by a sustained release phase due to diffusion across the lipid bilayer. Among the formulations, SW-ETH 6 exhibited the highest drug release (93.12%), whereas SW-ETH 1 showed the lowest release (76.74%) at 24 hours ( Figure 6 ). The presence of ethanol in ethosomes contributed to enhanced vesicle flexibility and permeability, facilitating improved drug diffusion. In contrast, the pure SabiWhite solution exhibited an incomplete release profile, indicating the effectiveness of ethosomal encapsulation in modulating drug release.

The anti-inflammatory activity of SW-ETH gel, as determined by permutation testing of SW-ETH gel significantly reduced paw edema compared to placebo at all time points (p=0.021 at 150 min, resampled). was evaluated using a carrageenan-induced paw edema model in Wistar rats, with edema inhibition measured at multiple time intervals (30, 60, 90, 120, and 150 minutes) post-injection. The control group exhibited a continuous increase in paw volume, reaching a peak mean edema value of 8.031 ± 0.2895 mm at 150 minutes. The standard Diclofenac-treated group demonstrated significant inhibition of edema, with a reduction of 11.71% at 30 minutes and reaching 41.92% at 150 minutes. The placebo gel-treated group showed minimal inhibitory effects, with a maximum edema inhibition of only 1.93% at 150 minutes. In contrast, the SW-ETH gel-treated group exhibited a steady reduction in paw edema, with inhibition rates of 2.64%, 13.59%, 21.05%, 29.57%, and 36.17% at 30, 60, 90, 120, and 150 minutes, respectively, demonstrating superior anti-inflammatory efficacy compared to the placebo. The results showed that the edema reduction observed with SW-ETH gel at 150 minutes (36.17%) was statistically comparable to the Diclofenac-treated group (41.92%) (p=0.039). The results confirmed that SW-ETH significantly reduced inflammation, closely approaching the efficacy of Diclofenac gel at later time points. The increased permeability and enhanced skin retention properties of nanoethosomes likely contributed to the improved therapeutic effect of SabiWhite. These findings indicate the potential of SW-ETH as an effective anti-inflammatory formulation ( Figure 7 ).

The skin irritation evaluation of SabiWhite-loaded ethosomal gel (SW-ETH) was conducted using Wistar albino rats, following OECD guidelines. Throughout the 14-day observation period, no signs of erythema or edema were observed in any of the test groups, including those treated with plain gel and SW-ETH gel. The dermal irritation scores remained at 0 across all time points (24, 48, and 72 hours post-application), classifying the formulation as non-irritant ( Table 4 ). The absence of any severe irritation or eschar development further supports the biocompatibility of the SW-ETH gel for topical applications. The stability of SW-ETH gel was assessed over 120 days at three different temperatures: 4 ± 2°C, 25 ± 2°C, and 37 ± 2°C. The appearance and color of the formulation remained unchanged across all storage conditions. The pH values exhibited minimal fluctuations, ranging from 5.82 at the start to 5.74 by the end of the study. Drug content remained stable, with a slight reduction (<5%) from 92.77% to 88.41% over time. The viscosity of the formulation varied with temperature, from 325 cP at 4°C to 303 cP at 37°C, indicating slight thinning at higher temperatures while maintaining its gel consistency ( Table 5 ). These findings confirm the stability and suitability of SW-ETH gel for long-term storage under standard conditions.