Novel ethosomal gel formulation for enhanced transdermal delivery of curcumin and cyclosporine: a preclinical approach to rheumatoid arthritis management

Abstract

Vesicular systems have demonstrated efficacy in the management of Rheumatoid Arthritis (RA). This study explores the synergistic effect of edge-activated ethosomal gel to enhance the transdermal delivery of Curcumin (CUR) and Cyclosporine (CYC). Ethosomal vesicles prepared via the ethanol injection method were incorporated into a gel, with the optimized formulation exhibiting an average particle size of 93.3 ± 1.17 nm and a zeta potential of −29.2 ± 0.17 mV. Ex vivo diffusion studies on porcine ear skin demonstrated 97.115 ± 0.40% CUR and 98.331 ± 1.08% CYC release over 18 hours, exhibiting Hixson-Crowell diffusion mechanisms. The steady-state flux and permeability coefficients were 0.095 µg/cm²/hr and 0.0095 cm/hr for CUR, and 0.0804 µg/cm²/hr and 0.01608 cm/hr for CYC respectively. In anti-inflammatory tests on lipopolysaccharide (LPS)-induced RAW 264.7 cells, the gel significantly increased IL-10 levels (p < 0.001), inhibited prostaglandin-E2, and reduced IL-6 and TNF-α levels (p < 0.001). Moreover, the ethosomal gel demonstrated nonirritating properties and exhibited significant reduction in arthritic symptoms in the Complete Freund’s Adjuvant induced 28-day rat model, surpassing the effects of marketed and conventional gel. These findings highlight the synergistic benefits of combining CUR and CYC in an ethosomal gel, offering a promising alternative for RA management. Future clinical investigations are warranted to validate its safety and efficacy in humans and facilitate potential therapeutic integration.

GRAPHICAL ABSTRACT

1. Introduction

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that predominantly affects synovial joints, leading to progressive disability, increased mortality rates, and significant socioeconomic burdens (Negi et al. 2024). Clinically, RA is characterized by symmetrical joint involvement manifested as hyperplasia, arthralgia, edema, erythema, and impaired mobility (Smolen et al. 2023). Systemic manifestations, including infection, glucocorticoid-induced osteoporosis (GIOP), and the involvement of extra-articular organs such as the lungs, heart, nervous system, and musculoskeletal system, further complicate disease progression (Wu et al. 2022). Women are disproportionately affected, being three times more likely to develop RA compared to men. Globally, RA affects an estimated 18 million individuals, as reported by the Global Burden of Disease (GBD) study in 2019 (Vos et al. 2020; Cai et al. 2023). In the United States, the prevalence ranges between 0.6–1.0%, whereas in India, it is approximately 0.75% (Malaviya et al. 1993; Xu and Wu 2021). Diagnosis typically occurs within three months to two years after symptom onset, with disease progression often resulting in irreversible joint deformities and significant impairment in daily functioning (Bullock et al. 2018). Notably, over 50% of RA patients in developed nations reportedly leave the workforce within a decade of diagnosis (Kirkeskov and Bray 2023).

The pathogenesis of RA involves a complex interplay of genetic, immunological, and inflammatory mechanisms (Siouti and Andreakos 2019). Genetic predisposition is linked to specific human leukocyte antigen (HLA) alleles, while the immune response targets self-antigens, leading to the production of autoantibodies such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) (Van Delft and Huizinga 2020). These autoantibodies recognize citrullinated antigens, generated via peptidylarginine deaminase-mediated post-translational modifications, triggering inflammation. Infiltration of immune cells into the synovium and subsequent cytokine release activate inflammatory pathways, with key mediators such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) exacerbating synovial inflammation and systemic effects (Jang et al. 2022). Chronic inflammation results in pannus formation, a pathological structure comprising immune cells, fibroblasts, and blood vessels, which invades adjacent tissues and promotes cartilage degradation, bone erosion, and joint deformities. Concurrently, systemic inflammation contributes to complications such as cardiovascular disorders, pulmonary issues, and a generalized inflammatory response (Panagopoulos and Lambrou 2018; Nandakumar et al. 2023). Although disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and monoclonal antibodies effectively alleviate symptoms and slow disease progression in RA, these therapies are often inadequate, leaving a substantial unmet need for more effective treatments (Radu and Bungau 2021).

Cyclosporine (CYC), initially approved as an immunosuppressant to prevent organ rejection, has demonstrated efficacy in RA patients unresponsive to conventional treatments such as methotrexate and NSAIDs/DMARDs (Tapia et al. 2023). CYC suppresses immune activity by inhibiting T-cell activation, which is a critical mediator of the inflammatory response in RA (Liddicoat and Lavelle 2019). However, oral formulations of CYC, such as Sandimmune^® Soft Gelatin Capsules and Oral Solution, exhibit erratic bioavailability (approximately 34%), attributed to incomplete gastrointestinal absorption and extensive first-pass metabolism (Schuetz et al. 2005; Ershad et al. 2023). Furthermore, CYC administration is often associated with nephrotoxicity, limiting its clinical utility (Steinmuller 1989; Wu et al. 2018). Interestingly, curcumin (CUR), a polyphenolic compound with potent anti-inflammatory and antioxidative properties, has shown potential in mitigating CYC-induced nephrotoxicity while alleviating RA-associated inflammation and pain (Huang et al. 2018; Kadhim et al. 2021).

CUR, derived from the rhizomes of Curcuma longa, has demonstrated efficacy in reducing oxidative stress and chronic inflammation, making it a promising candidate for RA therapy. CUR modulates TNF-α expression to attenuate cartilage breakdown and inflammation (Sivani et al. 2022; Kunnumakkara et al. 2023). However, its therapeutic potential is hampered by low solubility, chemical instability, and limited bioavailability due to rapid hepatic metabolism into water-soluble conjugates like curcumin-glucuronide and curcumin-sulfonate (Sohn et al. 2021; El-Saadony et al. 2023). Addressing these limitations, this study explores a transdermal delivery system for CUR in combination with CYC, bypassing first-pass metabolism. Despite their potential, poor aqueous solubility and low permeability hinder the transdermal delivery of both CYC and CUR (El Hosary et al. 2024; Elhabal et al. 2024). While previous studies have explored the transdermal delivery of curcumin (CUR) and cyclosporine (CYC) individually using carriers such as liposomes (Pierre and dos Santos Miranda Costa 2011), solid lipid nanoparticles (Liu et al. 2020), and nanostructured lipid carriers (Khan et al. 2023), many of these systems do not comply with the Food and Drug Administration’s (FDA) Inactive Ingredient Database (IIG) limits. This is primarily due to the incorporation of high concentrations of surfactants, which may result in skin irritation or systemic toxicity. Moreover, several reported formulations do not account for the stability of CYC during processing, often involving harsh solvents or aggressive manufacturing conditions that could degrade the active compounds.

In our previously published study, a nanoemulsion-based gel system was developed for the co-delivery of CUR and CYC for the management of RA (Gharat et al., 2024). While the nanoemulsion approach improved drug solubilization and provided enhanced permeation via nanosized emulsion droplets, the current study explores an alternative vesicular strategy using ethosomes. Ethosomal gels offer a unique advantage due to their high ethanol content, which not only fluidizes the stratum corneum lipids but also enhances vesicle deformability, facilitating deeper transdermal penetration. This distinct mechanism positions ethosomes as a promising carrier for the effective delivery of both CUR and CYC through the skin, addressing the limitations associated with conventional transdermal systems. Ethosomes, phospholipid vesicles enriched with ethanol, offer a promising solution by enhancing solubility and permeability. Their unique structure enables deeper skin penetration, improved drug delivery, and increased therapeutic efficacy. Furthermore, smaller particle sizes enhance absorption while reducing aggregation and coalescence. Soya lecithin and cholesterol are widely utilized for ethosomal formulation due to their ability to stabilize lipid bilayers and enhance vesicular flexibility for effective skin penetration. Soya lecithin, rich in phosphatidylcholine, forms bilayers encapsulating drugs, while cholesterol maintains optimal bilayer fluidity and structural stability (Jadhav et al. 2024). Edge-activated ethosomes, a specialized form of ethosomes that incorporate edge activators such as Tween 80, Span 80, and sodium cholate, further enhance deformability and skin penetration. These surfactants disrupt phospholipid packing, increasing vesicle flexibility and preventing aggregation, thereby ensuring a stable and uniform formulation (Kumar Sarwa et al. 2015).

The objective of the present study is to develop and evaluate a transdermal delivery system for the co-delivery of CUR and CYC using ethosomal vesicles, with the aim of overcoming the limitations associated with previously reported carriers. The proposed system emphasizes the use of biocompatible excipients, minimal surfactants, and mild processing techniques to enhance drug solubility, permeability, and therapeutic efficacy. Specifically, edge-activated ethosomes encapsulating CUR and CYC were developed via the ethanol injection method and incorporated into a gel using Carbopol^® Ultrez 10 NF. The final ethosomal gel was subjected to a series of evaluations including cell line studies, in vitro and ex vivo permeation analyses, and preclinical assessments using a Complete Freund’s Adjuvant (CFA)-induced arthritic rat model to establish its potential for rheumatoid arthritis management.

2. Materials and methods

2.1. Materials

Curcumin (99%) was purchased from Otto chemie Pvt. Ltd, Mumbai, India. Cyclosporine was provided as a gift sample by Concord Biotech Ltd, India. Tween 80 was procured from S.D. Fine chemicals Ltd., Mumbai, India. Soya lecithin (97%) and Cholesterol (99%) were purchased from Otto chemie. Pvt. Ltd, Mumbai, India. Absolute ethanol (99.9%), Polyvinyl alcohol (Molecular weight- 85,000 Da to 124,000 Da), Potassium dihydrogen phosphate (purified), and Disodium hydrogen phosphate (LR Grade) were obtained from S.D. Fine-Chem Limited, India. Carbopol^® Ultrez 10 NF was generously provided by Lubrizol India Pvt Ltd, Mumbai, India. The RAW 264.7 cells were obtained from National Center for Cell Science, Pune, India. Enzyme-linked immunoassay (ELISA) kits for mouse interleukin-10 (IL-10), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were purchased from Ray Biotech Labs, Norcross, Georgia. All excipients used in the formulation are ‘Generally Recognized as Safe’ listed.

2.2. Methods

2.2.1. Formulation of CUR-CYC loaded edge-activated ethosomes (CUR+CYC-etho)

The ethanol injection method is a preferred technique for preparing small unilamellar vesicular systems due to its simplicity and industrial scalability. The CUR+CYC loaded edge-activated ethosomes (CUR+CYC-Etho) were developed using ethanol injection method (cold method). Phospholipid (soya lecithin) was selected for ethosome preparation due to its ability to form stable, flexible vesicular bilayers essential for effective drug encapsulation. In combination with ethanol, it imparts ultra-deformability to the vesicles, enhancing skin penetration and improving the solubility and entrapment of poorly water-soluble drugs like CUR and CYC, thereby supporting an effective and stable delivery system for ­rheumatoid arthritis management (Jadhav et al. 2024). Furthermore, Cholesterol was used to enhance the structural integrity and stability of the ethosomal vesicles by modulating membrane fluidity. Briefly, phospholipid (soya lecithin) and cholesterol in stoichiometric ratio of 1:0.526, followed by CUR and CYC were dispersed and dissolved in ethanol in a covered vessel with vigorous stirring using overhead stirrer (Remi Stirrer RQ 124 A/D 20 L, India) at 400 rpm ± 100 rpm at room temperature. Separately, Polyvinyl alcohol (PVA) and Tween 80 was dissolved in phosphate buffer solution (PBS) pH 6.8 under stirring using overhead stirrer (Remi Stirrer RQ 124 A/D 20 L, India) at 400 rpm ± 100 rpm at room temperature. The ethanolic phase was further added to the aqueous phase with an addition rate of 0.5 ml/min and was stirred for 2.5 hours using overhead stirrer (Remi Stirrer RQ 124 A/D 20 L, India) at 400 rpm ± 100 rpm at room temperature to form a clear, transparent yellow ethosomal dispersion. Ethosomal batches (F1 to F9) were prepared by varying the stoichiometric ratio, % of PVA and % of Ethanol as shown in Table 1 (Kumar Sarwa et al. 2015).

Table 1.

Composition of various edge-activated ethosomal batches (CUR+CYC-etho).

Batch	CUR (mg)	CYC (mg)	Soya lecithin (mg)	Cholesterol (mg)	Stiochiometric ratio (soya lecithin: Cholesterol)	Ethanol (%)	PVA (%)	Tween 80 (%)
F1	50	25	10	10	1:1.75	10	0.1	5
F2	50	25	10	20	1:3.507	20	0.2	5
F3	50	25	10	30	1:5.260	40	0.3	5
F4	50	25	50	10	1:0.351	10	0.1	5
F5	50	25	50	20	1:0.701	20	0.2	5
F6	50	25	50	30	1:1.052	40	0.3	5
F7	50	25	100	10	1:0.175	10	0.1	5
F8	50	25	100	20	1:0.351	20	0.2	5
F9	50	25	100	30	1:0.526	40	0.3	5

Abbreviations: CUR – Curcumin, CYC– Cyclosporine, PVA – Polyvinyl alcohol, mg – milligram.

2.2.2. Characterization of the developed ethosomes (CUR+CYC-etho)

The evaluation of the ethosomal dispersion included assessments of visual appearance, such as color, uniformity, and clarity. Surface morphology and structural characteristics of the ethosomal vesicles were analyzed using Transmission Electron Microscopy (TEM) with a Tecnai G2 Spirit Biotwin LaB6 microscope. The samples were prepared by negative staining with 1% phosphotungstinic acid (PTA) to enhance contrast. Imaging was performed using an OSIS Veleta CCD Camera, controlled via the Tecnai Imaging Analysis (TIA) software. The instrument operated with a lanthanum hexaboride (LaB₆) electron gun at an accelerating voltage range of 20–120 kV, and images were captured at magnifications ranging from 22 kX to 300 kX. The TEM analysis confirmed the formation of well-defined, spherical, and uniformly distributed ethosomal vesicles, supporting successful formulation. The particle size and zeta potential of the ethosomal formulation were determined using a Zetasizer Nano-ZS (Malvern zetasizer Nano ZS). Prior to analysis, the samples were diluted with Millipore water in a 1:100 ratio to ensure optimal scattering intensity. Measurements were carried out in triplicate at 25 °C with a fixed scattering angle of 90°, and the results were reported as mean values ± standard deviation. Zeta potential was assessed to evaluate the surface charge and colloidal stability of the vesicles, as higher absolute zeta potential values indicate strong repulsive forces between particles, thereby minimizing aggregation and ensuring formulation stability (Paliwal et al. 2019; Martihandini et al. 2021).

2.2.3. Entrapment efficiency of (CUR+CYC-etho)

To determine the drug loading, 1 gram of the ethosomal dispersion (CUR+CYC-Etho) was transferred into a centrifuge tube of 2 ml capacity (Eppendorf^®) and was centrifuged using an Eppendorf SE Centrifuge 5430 R at 30,000 × g for 30 minutes (Fan et al. 2013). The settled ethosomal layer were carefully separated, then diluted with 10 ml of methanol and was vortexed for 15 minutes. Further dilutions were made using the mobile phase- Acetonitrile: Methanol: water (70:20:10). This facilitated the determination of CUR and CYC concentrations through Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis (Desai et al. 2019).

2.2.4. Incorporation of optimized ethosomal dispersion in a gel base (CUR+CYC-etho-gel)

Polymers like Carbopol^® Ultrez 10 NF, Pemulen^™ TR-1 and Carbopol^® 974 NF were screened as potential gelling polymers. Based on considerations of consistency and appearance, it was determined that 0.75% Carbopol^® Ultrez 10 NF exhibited optimal suitability for the ethosomal gel. The procedure for preparing ethosomal gel involved addition of Carbopol^® Ultrez 10 NF in the ethosomal dispersion, followed by stirring using an overhead stirrer for 10 minutes at a speed of 100 to 200 rpm. To achieve the desired viscosity and ensure homogeneity of the gel, a 10% aqueous solution of meglumine was added to adjust the pH between 6.0 to 6.5 (Price 2003).

2.3. Evaluation and characterization of ethosomal gel (CUR+CYC-etho-gel)

2.3.1. pH measurement

The pH evaluation of the CUR+CYC-Etho-gel was carried out by diluting 5.0 g of the gel with 50 ml of distilled water (Gadad et al. 2020). The pH was measured at 25 °C, using a calibrated pH meter (Labman pH meter).

2.3.2. Drug content

Reversed phase High Performance Liquid Chromatography (HPLC) (Agilent Infinity 1260) was employed to quantify the amount of CUR and CYC in CUR+CYC-Etho-gel. Briefly, 1.0 g of CUR+CYC-Etho-gel was dispersed in 10 mL of methanol and sonicated for 15 minutes to ensure complete drug extraction. Subsequent dilutions were carried out using mobile phase followed by HPLC analysis. The analysis was carried out using a mobile phase consisting of acetonitrile: methanol: water in the ratio of 70:20:10 (v/v/v) at a flow rate of 0.5 mL/min. Detection was performed at a wavelength of 214 nm with a total run time of 10 minutes. The retention time of cyclosporine was observed at 6.9 minutes. These optimized chromatographic conditions ensured accurate, reproducible, and sensitive estimation of drug content in the formulation (Gharat et al., 2024).

2.3.3. Spreadability

The evaluation of the spreadability of the developed CUR+CYC-Etho-gel utilized the ‘drag-and-slip’ method. The ‘drag-and-slip’ method is a widely used technique for evaluating the spreadability of semi-solid formulations. The time taken or distance moved by the upper slide under a known weight gives an indication of the spreadability of the gel. Shorter times or longer distances typically reflect better spreadability, which is essential for uniform application and patient compliance. This method helps assess how easily the formulation can be applied to the skin surface, an important parameter in transdermal drug delivery systems. Briefly, a wooden block connected to a pulley, along with a glass slide, was used for measuring the spreadability. The top slide was attached to a pan through a pulley and 2.0 grams of the gel was sandwiched between the glass slides. Incremental weights were added in the pan added until detachment of the top slide from the gel surface. The time taken for the upper slide to traverse 10 centimeters was recorded, enabling the calculation of spreadability (Gharat et al., 2024). The spreadability was calculated using following formula:

$$\text{S}=\left(\text{M x L}\right)/\mathrm{T}$$

where, S = Spreadability (g·cm/s), M = Weight tied to the upper slide (g), L = Length the slide moves (cm) and T = Time taken to slip or move that distance (s)

2.4. In vitro and ex vivo diffusion from CUR+CYC-etho-gel

The in vitro drug diffusion from CUR+CYC-Etho-gel was performed using a vertical Franz diffusion cell apparatus with a donor and receptor compartments. A dialysis membrane (molecular weight cutoff of 150 Daltons) was placed between both the cell compartments (Vlachou et al. 1992). The receptor compartment was filled with a diffusion medium composed of phosphate buffer at pH 6.8 and ethanol in a 2:3 ratio. The experiment was carried out at 32 °C ± 0.5 °C, with continuous stirring at 100 rpm. Aliquots were withdrawn from the receptor compartment at fixed intervals and quantified using the developed RP-HPLC method. Upon withdrawal of the aliquots, fresh diffusion media was replenished in the receptor compartment. Same procedure was employed for conducting the ex vivo diffusion using porcine ear skin. Porcine ear skin, closely resembling human skin in structure and permeability, was used as a reliable ex vivo model that mimic skin barrier properties, ideal for assessing drug permeation in transdermal formulations (Hwang et al. 2021; Brighenti et al. 2025). The porcine skin used in the study was obtained from Annasaheb Vartak Municipal Market, a government-approved abattoir located in Vile Parle (West), Mumbai, India. It was collected following the routine slaughter process in compliance with regulatory standards, as a by-product of a legally approved commercial slaughtering process, not specifically sacrificed for research purposes; therefore, no ethical clearance was required (Cristiano et al. 2021). The permeation kinetics was determined by measuring the concentration of the drug that permeated through a circular porcine ear skin sample with an area of 4.9062 cm² over time. The steady-state flux (Jss) was determined by examining the gradient of the linear segment of the plot via linear regression analysis (Mehavarshini et al. 2023).

2.5. Skin irritation study by hen’s egg test – chorioallantoic membrane (HET-CAM)

The HET-CAM assay is a rapid and sensitive technique used to predict skin irritancy. This assay evaluates changes in the chorioallantoic membrane of fertilized eggs. The chorioallantoic membrane comprises a complex lamina vascular system consisting of arteries, veins, and capillaries (Kundeková et al. 2021). This system is susceptible to the effects of harmful substances, leading to the initiation of an inflammatory response (de Silva et al. 1992). Ethical clearance is not required for HET-CAM studies because the embryo is used before day 14 of development, prior to the formation of a functional nervous system and the ability to perceive pain (Sarogni et al. 2021). The test protocol recommended by ‘Interagency Coordinating Committee on the Validation of Alternative Methods’ (ICCVAM) for assessing the irritation potential of the CUR+CYC-Etho-gel by HET-CAM assay was followed (Protocol 2010). The Central Poultry Development Organization, Mumbai provided 9-day old fertile White Leghorn chicken eggs, weighing between 55 ± 5 g. For fertilization, the eggs were incubated at 37.5 ± 0.5 °C and 62.5 ± 7.5% relative humidity, in an automatic rotating machine at the poultry. Validation of the test protocol was done using untreated (control), 0.9% NaCl (positive control), 0.1 N NaOH (negative control), and 0.75% w/w Ultrez-30 NF (gelling polymer) groups. Visual inspection for irritation potential was carried out for 5 minutes post-application of 0.3 mL of the test solutions to the CAM. Subsequently, the irritation score (IS) was determined.

$$\mathit{\text{IS}}=\frac{\left(301–\mathit{\text{tH}}\right)\ast 5}{300}+\frac{\left(301–\mathit{\text{tL}}\right)\ast 7}{300}+\frac{\left(301–\mathit{\text{tC}}\right)\ast 9}{300}$$

where, tH, tL, and tC denotes time required in seconds for the occurrence of hemolysis, lysis, and coagulation, respectively. The IS score of the CUR+CYC-Etho-gel batch was compared with Placebo-Etho-gel, CUR-Etho-gel, CYC-Etho-gel, and Trexjoy^® gel (1% w/w Methotrexate gel). The test samples were categorized based on the IS scores as follows: IS < 0.9 denoted nonirritating nature of the test sample, 1.0 ≤ IS ≤ 4.9 denoted mildly irritating nature of the test sample, 5.0 ≤ IS ≤ 8.9 denoted moderately irritating nature of the test sample, and 9.0 ≤ IS ≤ 21.0 denoted severely irritating nature of the test sample. To ensure the accuracy and reliability of the results, the experimentation was repeated three times.

2.6. Stability study

3 months stability studies of the developed CUR+CYC-Etho-gel was carried out at various stability conditions as per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use – Quality Guideline 1 A, Revision 2 (ICH Q1A (R2)) guidelines, i.e. 4 °C ± 3 °C, 25 °C ± 2 °C/60% RH ± 5% RH and 40 °C ± 2 °C/75% RH ± 5% RH. The ICH Q1A (R2) guideline - Stability Testing of New Drug Substances and Products, provides a standardized framework for conducting stability studies to assess how the quality of a drug substance or product changes over time under various environmental conditions such as temperature and humidity (Guideline 2003). During the stability testing period the formulation was evaluated for assay, physical appearance, particle size, zeta potential, pH, viscosity, spreadablility and assay (González-González et al. 2022).

2.7. Studies on RAW 264.7 cell line

RAW 264.7 cells, derived from mouse monocytes and transformed with the Abelson leukemia virus, serve as a widely employed model for investigating anti-inflammatory activity. Its utility often necessitates the presence of lipopolysaccharide (LPS) for complete activation, facilitating the study of inflammatory responses and potential therapeutic interventions (Hwang et al. 2019). The RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) High Glucose media supplemented with 10% Fetal Bovine Serum, 1% antibiotic-antimycotic solution, and 1% L-Glutamine (200 mM). Cultures were maintained in a controlled environment with 5% Carbon dioxide (CO₂) and 18–20% Oxygen (O₂) at 37 °C in a CO₂ incubator (Forma^™ Steri-Cycle^™ i250 CR CO₂ Incubator, Thermo Fisher Scientific India Pvt. Ltd. Mumbai, India). Sub-culturing was performed every 2 days to ensure optimal cell health and viability. For the current study, cells at passage number 39 were utilized.

2.7.1. Cytotoxicity study of CUR+CYC-etho on RAW 264.7 cell line

The cytotoxicity of an optimized formulation on RAW 264.7 cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The 96 well microtiter plates (Sigma, Germany) were seeded with 2 × 10⁴ RAW 264.7 cells per well and were incubated overnight at 37 °C in a 5% CO₂ atmosphere with 200 μL of complete DMEM medium. Subsequently, cells were treated with varying concentrations of Methotrexate, CUR, CYC, CUR+CYC, Ethosome (CUR+CYC-Etho), and Placebo (concentrations ranged from 6.25 µg/ml to 200 µg/ml) for 24 hours. After treatment, MTT solution (0.5 mg/ml) was added, and cells were incubated for 3 hours at 37 °C. Further, 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the purple-colored formazan crystals formed, followed by measuring the absorbance of the resulting solution at λ max = 570 nm and 630 nm using an ELISA microplate reader (BioTek Synergy H1 Multimode Reader, USA). The % cell viability was calculated using below formula:

$$\%\text{cell viability}=\frac{\text{Absorbance of the treated cells}}{\text{Absorbance of the untreated cells}}\times 100$$

Furthermore, the cytotoxicity of LPS in combination with the test compounds was assessed on RAW 264.7 cells. The 96 well microtiter plates were seeded with 2 × 10⁴ RAW 264.7 cells per well and were incubated overnight at 37 °C in a 5% CO₂ atmosphere with 200 μL of complete DMEM medium. Cells were further treated with 1 μg/ml of LPS for 2 hours to induce inflammatory conditions within the cells followed by addition of 10 μg/ml of test compounds- Methotrexate, CUR, CYC, CUR+CYC, Ethosome (CUR+CYC-Etho), and Placebo. The cells were allowed to react with the test compounds for 24 hours at 37 °C in a 5% CO₂ in an incubator. After the treatment, MTT solution (0.5 mg/ml) was added to each well and was incubated again for 3 hours at 37 °C. Further, 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the purple-colored formazan crystals formed, followed by measuring the absorbance of the resulting solution at λ max = 570 nm and 630 nm using an ELISA microplate reader (BioTek Synergy H1 Multimode Reader, USA) (Soni et al. 2021; Suresh et al. 2022). The % cell viability was calculated using the aforementioned formula.

2.7.2. LPS-induced anti-inflammatory study on RAW 264.7 cells

The expression levels of both pro-inflammatory cytokines (IL-6 and TNF-α) and the anti-inflammatory cytokine (IL-10), which play pivotal roles in mediating the inflammatory response, were examined. The concentrations of cytokines IL-10, IL-6, and TNF-α was assessed using the enzyme-linked immunosorbent assay (ELISA) method. The 12 well microtiter plates (Sigma, Germany) were seeded with 0.5 × 10⁶ RAW 264.7 cells per well and were incubated overnight at 37 °C in a 5% CO₂ atmosphere with 200 μL of complete DMEM medium. Cells were further treated with 1 μg/mL of LPS for 2 hours to induce inflammatory conditions, followed by the addition of 10 μg/mL of test compounds—Methotrexate, CUR, CYC, CUR+CYC, Ethosome (CUR+CYC-Etho), and Placebo (Choo et al. 2019). The test compounds were used at a concentration of 10 μg/mL, determined after preliminary cytotoxicity screening (MTT assay) to ensure cell viability remained above 80%, indicating nontoxic levels. This concentration enabled effective comparison of anti-inflammatory responses while maintaining cellular integrity. The cells were allowed to react with the test compounds for 24 hours at 37 °C in a 5% CO₂ in an incubator. Samples and standard (Methotrexate) were added into respective wells, which led to binding of IL-10, IL-6, and TNF-α to the immobilized antibodies. After thorough washing, biotinylated anti-mouse IL-10, IL-6, TNF-α antibodies, HRP-conjugated streptavidin, TMB (3,3′,5,5′-Tetramethylbenzidine) substrate solution was added sequentially. TMB substrate solution is a chromogenic reagent used in ELISA assays to detect enzyme activity, producing a color change upon reaction with horseradish peroxidase (HRP). It was specifically used in this study to quantify cytokine levels by measuring absorbance, indicating the extent of inflammatory response (Amsen et al. 2009). The color measurement was performed using a microplate reader (ELX-800, BioTek, USA) at 450 nm (Qiu et al. 2014; Chung et al. 2018).

2.7.3. Prostaglandin E2 (PGE2) inhibition assay

PGE2 arises from the metabolism of arachidonic acid that occurs within the cells. It is not stored but rather synthesized de novo and released upon cellular activation or when arachidonate is provided. In vivo, PGE2 rapidly metabolizes into an inactive metabolite via the PG 15-dehydrogenase pathway, exhibiting a short circulatory half-life of approximately 30 seconds. The measurement of PGE2 typically involves measuring its metabolites due to its rapid metabolism. The assay relies on the competitive interaction between PGE2 and PGE2 Tracer, along with a PGE2 Monoclonal Antibody, resulting in a yellow product with absorption at 412 nm, indicative of PGE2 concentration inversely. The 12 well microtiter plates (Sigma, Germany) were seeded with 0.5 × 106 RAW 264.7 cells per well and were incubated overnight at 37 °C in a 5% CO₂ atmosphere with 200 μL of complete DMEM medium. Cells were further treated with 1 μg/ml of LPS for 2 hours to induce inflammatory conditions within the cells followed by addition of 10 μg/ml of test compounds- Methotrexate, CUR, CYC, CUR+CYC, Ethosome (CUR+CYC-Etho), and Placebo. The cells were allowed to react with the test compounds for 24 hours at 37 °C in a 5% CO₂ in an incubator. The culture medium alone acted as a blank control for all the ELISA studies. Upon treatment completion, the particulate matter and cell debris was separated from the cell culture supernatant by centrifugation at 1,000 × g for 15 mins. PGE2 was quantified using an enzyme immunoassay kit (Cayman Chemical, USA), as per the instructions provided on the ELISA Kit. The resulting color intensity, subsequent to the addition of Ellman’s reagent, was measured using a microplate reader (ELX-800, BioTek, USA) at 405 nm (Yang et al. 2012; Baek et al. 2020).

2.8. Preclinical evaluation by complete freund’s adjuvant-induced arthritic rat model

Wistar albino rats weighing 150–200 g were obtained from the National Institute of Biosciences, Pune, India. Upon procurement, the rats were housed under standard conditions in the animal facility at Biocyte Institute of Research and Development, India, with controlled temperature and humidity conditions. The animals were given unrestricted access to water and provided with a standard commercial pelleted diet. The experimental protocol for this study was reviewed and approved by the Institutional Animal Ethical Committee, Biocyte Institute of Research and Development, under research project proposal number IAEC/Sangli/2023-24/13, in accordance with the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Before the study, the animals underwent a one-week acclimatization period in the animal house. After random allocation, the animals were distributed into eight groups: Control (Group I), Negative control (Group II), CUR+CYC-Etho-gel (Group III), CUR-Etho-gel (Group IV), CYC-Etho-gel (Group V), Placebo-Etho-gel (Group VI), Trexjoy^® gel (Group VII), and Conventional gel (Group VIII). Rheumatoid arthritis was induced by injecting 0.1 mL of Complete Freund’s Adjuvant (CFA) into the left hind paw of rats via intraplantar region. Each ml of CFA contained 10 mg of heat-killed Mycobacterium tuberculosis (H37Rv strain) suspended in sterile paraffin oil. 0.5 g of ethosomal gel was topically applied once daily to the dorsal surface of the hind paw at a fixed time each day, over a 28-day treatment period following disease induction. The administered doses of CYC and CUR were 6.25 mg/kg/day and 12.5 mg/kg/day, respectively. The application site was left uncovered after application to mimic standard topical administration. Body weights and paw volumes of animals were observed at days 0, 4, 7, 14, 21, and 28 post-adjuvant administration (Cristiano et al. 2021). Each group consisted of three animals. At the study’s end (day 28), euthanasia was performed via CO₂ asphyxiation. Blood samples were collected in an EDTA-containing tubes for analysis. The levels of red blood cells (RBCs), white blood cells (WBCs), and hemoglobin (Hb) were measured using an Automated Hematology Analyzer (Nihon Kohden, Model: MEk-6510). Cytokine levels of IL-6, IL-10 and TNF-α in plasma were determined using ELISA kits. Histopathological examination involved excising and preserving kidneys and proximal interphalangeal joints in 10% formalin. Sections were stained with eosin-hematoxylin and observed under 100x magnification. X-rays of hind legs assessed soft tissue swelling, bony erosions, and joint space narrowing (Ye et al. 2021; Adin et al. 2023). All histopathological and X-ray evaluations were conducted by a certified Toxicopathologist.

2.9. Statistical analysis

Values in the text and figures represent the mean ± standard error of the mean (SEM), with each experiment conducted in triplicate. Multiple regression analysis was employed to assess the effect of independent variables on the dependent variable using Microsoft Excel (Office 2021). Statistical analysis involved utilizing one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test to assess any significant differences, while the body weight and paw volume measurements were subjected to statistical analysis by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Tukey’s multiple comparison test identified significant differences between group means after ANOVA while controlling for type I error, ensuring statistical validity. GraphPad Prism version 10.0.3 (GraphPad Software, San Diego, CA, USA) was utilized for all statistical analyses to ensure clear visualization and accurate interpretation of the results.

3. Results

3.1. Formulation development

Nine batches of edge-activated ethosomes (F1 to F9) were systematically developed by varying independent variables, including the stoichiometric ratio of soya lecithin to cholesterol, the percentage of polyvinyl alcohol (PVA), and ethanol concentration. The influence of these independent variables on key dependent parameters such as particle size, zeta potential, and entrapment efficiency were evaluated. The concentration of edge activators was found to significantly affect ethosomal properties and performance. Various edge activators, including Tween 20, Tween 80, and Span 80, were screened during the preformulation studies to evaluate their solubility-enhancing potential for CUR and CYC. Among them, Tween 80 demonstrated the highest solubility, with 29.92 mg/g for CUR and 15.22 mg/g for CYC, and was therefore selected for further formulation development. Preliminary feasibility trials were conducted using 3% and 5% Tween 80 in the absence of polyvinyl alcohol (PVA). The trial with 3% Tween 80 yielded ethosomes with a zeta potential of −5.97 ± 0.494 mV, while the 5% Tween 80 formulation showed a zeta potential of 14.71 ± 2.95 mV. These relatively low zeta potential values indicated insufficient colloidal stability of the ethosomes. Further increase in Tween 80 concentration led to excessive foam generation during manufacturing, making it impractical. Consequently, various concentrations of PVA (0.1%, 0.2%, and 0.3%) were evaluated in combination with 5% Tween 80 to improve system stability, as detailed in Table 1. Consequently, based on the outcomes of these preliminary studies, the concentration of Tween 80 was standardized at 5% across all nine batches.

The manufacturing conditions, including stirring speed and duration, were optimized to 400 ± 100 rpm for 2.5 hours at room temperature to minimize air entrapment. Among the tested formulations, batch F9 emerged as the optimized batch, prepared with a stoichiometric ratio of 1:0.526 (soya lecithin: cholesterol), 40% ethanol, and 0.3% PVA. This batch produced a clear yellow ethosomal dispersion with an average particle size of 93.31 ± 1.17 nm, a narrow polydispersity index (PDI) of 0.254 ± 0.021, and a zeta potential of −29.2 ± 0.17 mV, suggesting that the dispersion exhibited acceptable colloidal stability, minimizing the risk of aggregation during storage and application, as shown in Figure 1. The drug entrapment efficiency of batch F9 was the highest among all formulations, achieving values of 97.22 ± 0.74% for curcumin (CUR) and 95.53 ± 1.31% for cyclosporine (CYC), as detailed in Table 2. Transmission electron microscopy (TEM) analysis of batch F9 confirmed the presence of spherical ethosomal nanoparticles with smooth surfaces and uniform dispersion, as depicted in Figure 2.

Figure 1.

(a) Particle size and (b) zeta potential of optimized CUR+CYC-etho batch measured using malvern zetasizer nano ZS.

Prior to analysis, samples were diluted with millipore water in a 1:100 ratio to ensure optimal scattering intensity. Measurements were carried out in triplicate at 25 °C with a fixed scattering angle of 90°. results are reported as mean ± standard deviation (SD).

Table 2.

Characterization of various ethosomal batches.

Batch	Z average diameter (nm)	PDI	Zeta potential (mV)	% Entrapment
				CUR (%)	CYC (%)
F1	126.8 ± 1.14	0.256 ± 0.011	−9.82 ± 0.21	47.19 ± 0.27	41.01 ± 1.72
F2	104.8 ± 1.39	0.258 ± 0.020	−11.9 ± 1.42	52.81 ± 1.18	49.97 ± 1.01
F3	60.34 ± 1.87	0.152 ± 0.062	−20.5 ± 0.59	57.60 ± 1.02	54.22 ± 0.02
F4	141.1 ± 1.23	0.368 ± 0.027	−7.67 ± 0.33	62.31 ± 0.41	59.19 ± 1.27
F5	127.8 ± 0.11	0.233 ± 0.056	−14.4 ± 1.91	69.02 ± 1.31	62.99 ± 1.52
F6	77.64 ± 1.94	0.297 ± 0.064	−22.3 ± 1.47	71.43 ± 0.81	75.72 ± 1.28
F7	151.3 ± 1.33	0.305 ± 0.048	−10.7 ± 0.24	85.14 ± 1.51	80.51 ± 0.19
F8	134.3 ± 0.42	0.276 ± 0.016	−18.1 ± 1.32	95.57 ± 1.07	91.07 ± 0.17
F9	93.31 ± 1.17	0.254 ± 0.021	−29.2 ± 0.17	97.22 ± 0.74	95.53 ± 1.31

All measurements were carried out in triplicates (n = 3). Data are presented as mean ± standard Deviation (SD).

Abbreviations: CUR – Curcumin, CYC– Cyclosporine, PDI – Polydispersity Index, nm – nanometer, mV – millivolt.

Figure 2.

Transmission electron microscopy (TEM) images of the optimized CUR+CYC-etho batch.

TEM images of the optimized CUR+CYC-etho batch, captured using a tecnai G2 spirit biotwin LaB₆ microscope. Samples were negatively stained with 1% phosphotungstinic acid (PTA) for contrast enhancement. Imaging was performed with a LaB6 electron gun (20–120 kV) and an OSIS veleta CCD camera, with analysis via tecnai imaging analysis (TIA) software at 22 kX to 300 kX magnification.

3.2. Incorporation of ethosomal dispersion into gel

The optimized ethosomal dispersion was incorporated into a Carbopol^® Ultrez 10 NF gel based on preliminary formulation studies. A specific quantity of Carbopol^® Ultrez 10 NF was added to the ethosomal dispersion and stirred at 100–200 rpm for 10 min using an overhead stirrer to achieve uniform polymer dispersion. The pH of the resulting mixture was adjusted with a 10% meglumine solution, yielding a yellow, transparent gel.

3.3. Evaluation of CUR+CYC-etho-gel

The optimized CUR+CYC-Etho-gel displayed a smooth, grit-free texture, a clear yellow appearance, and excellent transparency. Assay results showed high drug content, with 98.52 ± 0.31% for CUR and 97.16 ± 0.61% for CYC. Extrudability studies confirmed the gel’s suitability for dispensing, as it exhibited smooth and consistent flow under nominal pressure. The pH of the ethosomal gel was determined to be 6.22 ± 0.11, indicating compatibility with skin application and minimal risk of irritation. Viscosity measurements revealed a value of 6781 ± 1.20 cps (57.2 ± 1.13% torque), obtained using spindle number 18 at 10 rpm for 1 min, indicating optimal gel consistency. Additionally, the gel demonstrated excellent spreadability with a value of 18 ± 1.25 g·cm/s, suggesting ease of application without requiring excessive force.

3.4. Drug diffusion studies

The in vitro drug diffusion studies showed the release of 98.573 ± 1.22% of CUR after 7 hours and 99.169 ± 1.73% of CYC after 8 hours from the ethosomal dispersion (CUR+CYC-Etho). Similarly, in vitro drug diffusion from the CUR+CYC-Etho-gel resulted in 98.845 ± 1.65% CUR and 99.299 ± 0.13% CYC release at the 8-hour mark. The ex vivo diffusion studies conducted on porcine ear skin demonstrated cumulative drug release of 97.115 ± 0.40% for CUR and 98.331 ± 1.08% for CYC over 18 hours, as illustrated in Figure 3. During these studies, CUR exhibited a steady-state flux of 0.095 µg/cm²/hr with a permeability coefficient of 0.0095 cm/hr. In comparison, CYC showed a steady-state flux of 0.0804 µg/cm²/hr and a permeability coefficient of 0.01608 cm/hr. Curve-fitting analysis revealed that the drug release followed Hixson-Crowell diffusion kinetics, with R² values of 0.9468 and 0.9553 for CUR and CYC, respectively. These findings suggest that the release of both drugs from the ethosomal gel is governed by surface area changes and particle diameter reduction over time, consistent with the Hixson-Crowell mechanism (Abbas et al. 2013; Tapdiqov 2020).

Figure 3.

Comparative results of diffusion studies (a) In vitro diffusion from CUR+CYC-etho, (b) In vitro diffusion from CUR+CYC-etho-gel, (c) ex vivo diffusion from CUR+CYC-etho-gel.

For in vitro diffusion, a dialysis membrane (molecular weight cutoff 150 Da) was used, while porcine ear skin was employed for ex vivo diffusion. The diffusion medium consisted of phosphate buffer (pH 6.8) and ethanol in a 2:3 ratio. Experiments were conducted at 32 ± 0.5 °C with continuous stirring at 100 rpm. All measurements were carried out in triplicates (n = 3). Data are presented as mean ± standard Deviation (SD).

3.5. HET-CAM method for assessing skin irritation

The optimized CUR+CYC-Etho-gel demonstrated an irritation score (IS) of 0.023 ± 0.001, indicating no skin irritation or sensitizing effects, compared to the positive control group, which had an IS of 9.36 ± 0.014, indicating high irritation potential, as illustrated in Figure 4.

Figure 4.

Estimation of skin irritation potential of the formulation using the HET-CAM (hen’s egg test–chorioallantoic membrane) method, conducted as per the test protocol recommended by the interagency coordinating committee on the validation of alternative methods (ICCVAM).

All measurements were carried out in triplicates (n = 3). Abbreviations: CUR – Curcumin, CYC– Cyclosporine, CUR+CYC-Etho-gel – Ethosomal gel, Placebo-Etho-gel – Placebo Ethosomal gel, CUR+Etho-gel – Curcumin Ethosomal gel, CYC+Etho-gel – Cyclosporine Ethosomal gel.

3.6. Stability study

The developed ethosomal gel formulation utilizing the F9 batch underwent a 3-month stability assessment, during which it was evaluated for organoleptic properties, particle size, zeta potential, viscosity, spreadability and drug assay. Results demonstrate the stability of the developed formulation throughout the stability conditions, as depicted in Table 3.

Table 3.

Results of stability studies of CUR+CYC-etho-gel.

Stability parameter	Results (day 0)	Results (after 3 months)
		4 °C ± 3 °C	25 °C ± 2 °C/ 60% RH ± 5% RH	40 °C ± 2 °C/ 75% RH ± 5% RH
Physical appearance	Yellow colored clear and transparent gel with no grittiness	Same as day 0	Same as day 0	Same as day 0
pH	6.22 ± 0.11	6.32 ± 0.32	6.28 ± 0.64	6.16 ± 0.24
Particle size (nm)	93.31 ± 1.17	90.53 ± 1.31	91.39 ± 1.26	91.74 ± 1.49
Zeta potential (mV)	−29.2 ± 0.17	−29.2 ± 0.23	−28.4 ± 0.35	−26.7 ± 0.28
Viscosity (cPs)	6781 ± 1.20	6752 ± 1.52	6782 ± 1.63	6634 ± 1.28
Spreadablility (g cm/s)	18 ± 1.25	18 ± 1.83	18 ± 1.37	18 ± 1.81
Assay
1) CUR	98.52 ± 0.31%	98.38 ± 0.41%	99.14 ± 1.73%	100.01 ± 1.25%
2) CYC	97.16 ± 0.61%	97.22 ± 0.62%	98.73 ± 0.44%	99.18 ± 1.82 %

All measurements were carried out in triplicates (n = 3). Data are presented as mean ± standard Deviation (SD).

Abbreviations: CUR – Curcumin, CYC – Cyclosporine, PDI – Polydispersity Index, nm – nanometer, mV – millivolt, RH -Relative Humidity, cPs – centipoise, g.cm/s – Gram Centimeter Per Second.

3.7. Cytotoxicity study of test compounds on RAW 264.7 cells

The results of cytotoxicity study indicate following IC50 values for CUR (29.76 ± 3.02 µg/ml), CYC (123.34 ± 17.45 µg/ml), CUR+CYC (23.62 ± 1.03 µg/ml), Ethosome (43.89 ± 2.48 µg/ml) and Placebo (85.53 ± 0.28 µg/ml) on RAW 264.7 cells without LPS treatment as illustrated in Figure 5a. As a result of these findings, the optimum concentration was determined to be 10 µg/ml, since all test compounds presented cell viability more than 80%, suggesting their potential for anti-inflammatory properties and justifying further investigation. The % cell viability of 10 µg/mL test compounds on RAW 264.7 cells co-treated with 1 µg/mL LPS is illustrated in Figure 5b. Microscopic images of RAW 264.7 cells treated with various compounds, as shown in Figure 5c, indicate that the cells maintained normal morphology. These findings confirm that the tested concentrations are non-cytotoxic and can be safely employed in subsequent experiments for cytokine estimation.

Figure 5.

(a) IC50 Values of test compounds (b) percentage cell viability of test compounds in combination with LPS on RAW 264.7 cells (c) Microscopic images of RAW 264.7 cells treated with various compounds.

Data was analyzed in GraphPad prism 10.0.3, using one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test (Note: ns- non significant (p > 0.05), *- significant (p ≤ 0.02), **- very significant (p ≤ 0.002), ***-highly significant (p ≤ 0.001)). All measurements were carried out in triplicates (n = 3). Abbreviations: CUR – Curcumin, CYC– Cyclosporine, LPS – Lipopolysaccharide, IC 50 – Half-maximal inhibitory concentration, µg/mL – micrograms per milliliter, RAW 264.7 – Monocyte/macrophage cell line.

3.8. Anti-inflammatory effect on RAW 264.7 cells treated with LPS

The impact of various test compounds on pro- and anti-inflammatory cytokines was evaluated using an LPS-induced RAW 264.7 cell model. As shown in Figure 6a and b, IL-6 and TNF-α levels were significantly elevated in the LPS-alone group and were progressively reduced upon treatment with both standard and test compounds. IL-6 levels were reduced by approximately 3.56-fold in the Methotrexate-treated group and by 3.01-fold in the ethosome-treated group compared to the LPS control. Regression analysis confirmed significant suppression of IL-6 in the LPS+CUR, LPS+CYC, LPS+(CUR+CYC), and LPS+Ethosome groups (F(1.08, 2.15) = 2561, p < 0.001, R² = 0.999), indicating a robust anti-inflammatory effect. A high F-value (2561) suggested a highly significant effect. 1.08 is the numerator degrees of freedom (df1), which corresponds to the number of groups being compared minus one. 2.15 is the denominator degrees of freedom (df2), which corresponds to the residual or error degrees of freedom, reflecting the variability within the groups. p < 0.001, indicated that the results are statistically highly significant, i.e. the likelihood that the observed effect is due to random chance is less than 0.1%. The coefficient of determination (R²) indicated that 99.9% of the variance in IL-6 levels. Similarly, TNF-α levels were reduced by 2.23-fold in the Methotrexate group and 1.82-fold in the ethosome group compared to LPS control. This reduction was statistically significant (F(1.00, 2.00) = 1688, p < 0.001, R² = 0.999), supporting the anti-inflammatory efficacy of these treatments. The placebo-treated group showed a marginal reduction in pro-inflammatory cytokines, suggesting a limited effect. In contrast, IL-10 levels, an anti-inflammatory marker, increased by approximately 2.81-fold in the Methotrexate group and 2.82-fold in the ethosome group relative to the LPS control. Groups treated with CUR, CYC, CUR+CYC, and ethosomes exhibited a significant upregulation of IL-10 (F(1.00, 2.00) = 2715, p < 0.001, R² = 0.999), as illustrated in Figure 6c. While CYC showed moderate efficacy, CUR, CUR+CYC, and ethosome treatments demonstrated pronounced anti-inflammatory activity through enhanced IL-10 expression.

Figure 6.

Investigation of anti-inflammatory activity by cell culture studies: (a) interleukin 6 levels (b) tumor necrosis factor-α levels (c) interleukin 10 levels (d) prostaglandin E2 inhibition.

Data was analyzed in GraphPad prism 10.0.3, using one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test (Note: ns- non significant (p > 0.05), *- significant (p ≤ 0.02), **- very significant (p ≤ 0.002), ***-highly significant (p ≤ 0.001)). All measurements were carried out in triplicates (n = 3). Data are presented as mean ± Standard Error of Mean (SEM). Abbreviations: CUR – Curcumin, CYC– Cyclosporine, LPS – Lipopolysaccharide, IL-6 – Interleukin 6, IL-10 – Interleukin 10, TNF-α – Tumor necrosis factor-α, pg/mL – picograms per milliliter, RAW 264.7 – Monocyte/macrophage cell line.

3.9. Prostaglandin E2 inhibition assay

The anti-inflammatory potential of the test compounds was further evaluated by measuring PGE₂ levels in an LPS-induced RAW 264.7 cell model. As expected, the LPS-alone group exhibited a marked increase in PGE₂ concentrations, indicative of an inflammatory response. Treatment with the test compounds significantly reduced PGE2 levels (F(1.00, 2.00) = 3045, p < 0.001, R² = 0.999), demonstrating strong anti-inflammatory activity. Specifically, PGE₂ levels decreased by approximately 1.84-fold in the Methotrexate-treated group and by 2.73-fold in the ethosome-treated group, relative to the LPS control. Among the treatments, the LPS+CUR+CYC-Ethosome group exhibited the most pronounced inhibition of PGE₂, as illustrated in Figure 6d.

3.10. Preclinical studies on CFA induced arthritis rat model

Wistar rats were subjected to the induction of RA through the injection of 0.1 ml of CFA into the left hind paw on Day 0. IL-6, IL-10, and PGE2 levels, along with body weight and paw width, were measured at regular intervals following induction. Hematological parameters (RBC, WBC and HB levels) were measured on 28^th day after the treatment period. On Day 28, the animals were euthanized for X-ray and histopathological analysis of hind paws and kidneys. Throughout the 28-day treatment period, no visible signs of erythema, edema, or skin irritation were observed at the site of gel application in any of the treatment groups. These observations are supported by the paw images presented in Figure 8, which show normal skin appearance post-application. Additionally, the animals did not exhibit any abnormal behaviors indicative of discomfort, such as excessive grooming, licking, or aversive reactions, suggesting good tolerability of the formulations.

Figure 8.

Effect of 4-week treatment on paw volume, measured on 0th, 4^th, 7^th, 14^th, 21^st and 28^th day.

Data was analyzed in GraphPad prism 10.0.3, using two-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test; significance is denoted by @p < 0.001 when compared to the negative control group. #p < 0.001 when compared to the placebo group. All measurements were carried out in triplicates (n = 3). Data are presented as mean ± Standard Error of Mean (SEM). Abbreviations: CUR – Curcumin, CYC– Cyclosporine, CUR+CYC-Etho-gel – Ethosomal gel, Placebo-Etho-gel – Placebo Ethosomal gel, CUR+Etho-gel – Curcumin Ethosomal gel, CYC+Etho-gel – Cyclosporine Ethosomal gel, mm³ – Cubic Millimeter.

3.10.1. Haematological parameters

As compared to the control group, significant changes in the hematological parameters were observed in the negative control group on the 28^th day of treatment. Notably, hemoglobin (F (1.41, 2.83) = 51.4, p < 0.05, R² = 0.963) and RBC levels (F (1.41, 2.83) = 206, p < 0.05, R² = 0.990) decreased significantly, while WBC levels (F (2, 4) = 39.3, p < 0.05, R² = 0.952) increased significantly indicating successful induction of arthritic conditions. Inflammatory conditions lead to a shortened lifespan of RBCs by altering their morphology, enhancing their adherence to the endothelium of blood vessels, and subsequent elimination from circulation, consequently lowering hemoglobin levels (Straat et al. 2012). Elevated IL-6 levels in RA stimulate WBC production, accounting for increased WBC count. After treatment, significant increases in hemoglobin and RBC levels were observed in the CUR+CYC-Etho-gel group compared to the negative control, placebo, Trexjoy^® gel, and conventional gel groups, indicating the effectiveness of the formulation. No significant increase in RBC and hemoglobin counts were observed in the CUR-Etho-gel and CYC-Etho-gel groups as compared to the CUR+CYC-Etho-gel, indicating a synergistic effect of both drugs. Moreover, a notable reduction in WBC levels was noted in the group treated with CUR+CYC-Etho-gel compared to the negative control, placebo, and conventional gel groups after treatment, attributed to reduced IL-6 levels in plasma. Notably, a significant difference in WBC levels was observed between the CUR+CYC-Etho-gel and Trexjoy^® gel groups, indicating the efficacy of the developed ethosomal gel. The CUR-Etho-gel and CYC-Etho-gel groups did not exhibit substantial decreases in WBC count compared to the CUR+CYC-Etho-gel group, further suggesting a synergism by combining CUR with CYC as shown in Figure 7b.

Figure 7.

Impact of 28-days treatment on (a) red blood cell levels, (b) white blood cell levels, and (c) hemoglobin levels.

Data was analyzed in GraphPad prism 10.0.3, using one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test (Note: ns- non significant (p > 0.05), *- significant (p ≤ 0.02), **- very significant (p ≤ 0.002), ***-highly significant (p ≤ 0.001)). All measurements were carried out in triplicates (n = 3). Data are presented as mean ± Standard Error of Mean (SEM). Abbreviations: CUR – Curcumin, CYC– Cyclosporine, CUR+CYC-Etho-gel – Ethosomal gel, Placebo-Etho-gel – Placebo Ethosomal gel, CUR+Etho-gel – Curcumin Ethosomal gel, CYC+Etho-gel – Cyclosporine Ethosomal gel, RBC – Red blood cells, WBC – White blood cells, Hb – Hemoglobin, gm/dl – grams per deciliter.

3.10.2. Impact on paw edema and body weight

Paw edema and weight of rats was monitored on days 0, 4, 7, 14, 21, and 28 of the study. Treatment with CUR+CYC-Etho-gel resulted in a progressive augmentation in body weight starting from the 7th day, as compared to other groups as shown in Table 4. Furthermore, a significant reduction in edema was observed on in animals treated with CUR+CYC-Etho-gel compared to the other groups, as demonstrated in Figure 8, underscoring the effectiveness of the developed formulation.

Table 4.

Impact of 28-days treatment on body weight measured on 0^th, 4^th, 7^th, 14^th, 21^st and 28^th day.

Groups	Day 0	Day 4	Day 7	Day 14	Day 21	Day 28
	Avg. wt. (gm) ± SEM	Avg. wt. (gm) ± SEM	Avg. wt. (gm) ± SEM	Avg. wt. (gm) ± SEM	Avg. wt. (gm) ± SEM	Avg. wt. (gm) ± SEM
Control	180.974 ± 0.275	181.930 ± 0.304	181.332 ± 0.001	181.066 ± 0.318	180.629 ± 0.363	181.066 ± 0.318
Negative Control	193.967 ± 0.988	192.035 ± 0.542	187.167 ± 0.333	185.520 ± 0.650	182.302 ± 0.262	182.290 ± 0.500
CYC+CYC-Etho-gel	172.649 ± 0.887	158.943 ± 0.494	157.500 ± 0.236	162.110 ± 0.336	164.511 ± 1.522	172.301 ± 0.827
CUR-Etho-gel	182.110 ± 3.100	179.551 ± 3.001	175.167 ± 0.367	169.070 ± 2.838	169.966 ± 3.954	173.186 ± 3.089
CYC-Etho-gel	171.074 ± 4.049	158.665 ± 0.764	152.000 ± 0.249	142.630 ± 0.356	147.037 ± 0.545	152.563 ± 1.717
Placebo-Etho-gel	185.412 ± 0.760	181.082 ± 2.710	181.500 ± 0.312	180.449 ± 0.587	176.504 ± 0.574	172.628 ± 0.041
Trexjoy® gel	171.712 ± 3.669	165.804 ± 2.831	152.000 ± 0.667	153.083 ± 0.291	154.144 ± 0.250	154.382 ± 0.853
Conventional gel	164.356 ± 2.445	160.284 ± 3.622	152.833 ± 1.000	154.680 ± 2.236	155.248 ± 1.942	153.730 ± 0.262

All measurements were carried out in triplicates (n = 3). Data are presented as mean ± standard error of mean (SEM).

Abbreviations: CUR – Curcumin, CYC– Cyclosporine, SEM- Standard Error of Mean, CUR+CYC-Etho-gel – Ethosomal gel, Placebo-Etho-gel – Placebo Ethosomal gel, CUR+Etho-gel – Curcumin Ethosomal gel, CYC+Etho-gel – Cyclosporine Ethosomal gel, Avg. wt. – Average weight.

3.10.3. Measurement of IL-6, IL-10 and TNF-α in plasma

Following treatment with the developed CUR+CYC-Etho-gel formulation, there was a notable decrease in the levels of inflammatory cytokines IL-6 (F (1.771, 3.541) = 118.5, p < 0.05, R² = 0.990) and TNF-α (F (1.777, 3.554) = 26.62, p < 0.05, R² = 0.995) compared to other groups. No significant reduction in IL-6 and TNF-α levels was observed in groups treated with CUR-Etho-gel and CYC-Etho-gel as compared to the CUR+CYC-Etho-gel group, suggesting a synergistic effect between both the drugs as depicted in Figure 9a and c. Comparable pattern was noted with the anti-inflammatory cytokine IL-10. In the group treated with CUR+CYC-Etho-gel, IL-10 levels were significantly increased (F (1.486, 2.973) = 56.96, p < 0.001, R² = 0.992), indicating a reduction in inflammation compared to the other groups. Notably, the CUR+CYC-Etho-gel exhibited no significant difference when compared with the marketed Trexjoy^® gel as depicted in Figure 9b.

Figure 9.

Concentrations of cytokines in plasma of CFA induced rats observed on 0^th, 7^th, 21^st and 28^th day of treatment: a) Plasma concentrations of IL-6, b) Plasma concentrations of IL-10, c) Plasma concentrations of TNF-α.

Data was analyzed in GraphPad prism 10.0.3, using one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test (Note: ns- non significant (p > 0.05), *- significant (p ≤ 0.033), **- very significant (p ≤ 0.002), ***-highly significant (p ≤ 0.001)). All measurements were carried out in triplicates (n = 3). Data are presented as mean ± Standard Error of Mean (SEM). Abbreviations: CUR – Curcumin, CYC– Cyclosporine, CUR+CYC-Etho-gel – Ethosomal gel, Placebo-Etho-gel – Placebo Ethosomal gel, CUR+Etho-gel – Curcumin Ethosomal gel, CYC+Etho-gel – Cyclosporine Ethosomal gel, IL-6 – Interleukin 6, IL-10 – Interleukin 10, TNF-α – Tumor necrosis factor-α, pg/mL – picograms per milliliter.

3.10.4. Radiographic and histopathological evaluations

X-Ray assessment of the hind paw in the control group revealed a healthy joint with no signs of distension and normal radio density in the joint space (Figure 10a). On the contrary, the negative control group displayed arthritic alterations, such as increased joint radio density and reduced joint space, as depicted in Figure 10b. As illustrated in Figure 10c, the CUR+CYC-Etho-gel group exhibited joint features similar to those of the marketed Trexjoy^® gel group, suggesting ­effective anti-inflammatory and anti-arthritic properties. Nevertheless, other treatment groups demonstrated varying degrees of joint damage, as depicted in Figure 10. Microscopic examination of the paw tissue further elucidated these findings. The control group displayed normal bone tissue, while the negative control and placebo-etho-gel groups exhibited inflammatory changes such as inflammatory cell infiltration, synovial epithelium hyperplasia, and bone erosion compared to the positive control group. In contrast, animals treated with CUR+CYC-Etho-gel and Trexjoy^® gel displayed reduced severity of inflammatory cells and synovial epithelium hyperplasia, indicating anti-arthritic effects. Histopathological assessment of rat kidneys revealed normal glomerular shape with no signs of congestion or tubular degeneration in the CUR+CYC-Etho-gel group, suggesting a protective effect against CYC-induced nephrotoxicity, as illustrated in Figure 10c.

Figure 10.

Radiographic and histopathological evaluations after the treatment period for following animal groups (a) control, (b) negative control, (c) CUR+CYC-etho-gel, (d) CUR-etho-gel, (e) CYC-etho-gel, (f) Placebo-etho-gel, (g) trexjoy^® gel, and (h) conventional gel.

Abbreviations: CUR – curcumin, CYC– Cyclosporine, CUR+CYC-etho-gel – ethosomal gel, Placebo-etho-gel – Placebo ethosomal gel, CUR+etho-gel – curcumin ethosomal gel, CYC+etho-gel – Cyclosporine ethosomal gel.

4. Discussion

Cyclosporine (CYC), an immunosuppressant used in rheumatoid arthritis (RA), has shown efficacy in patients unresponsive to methotrexate and NSAIDs/DMARDs. However, its poor oral bioavailability and nephrotoxicity limit clinical use. Curcumin (CUR), a natural anti-inflammatory and antioxidant, not only alleviates RA symptoms but also mitigates CYC-induced nephrotoxicity. Both compounds face challenges such as low solubility, instability, and limited bioavailability. To address these issues, this study aimed to develop and evaluate a novel edge-activated ethosomal gel loaded with cyclosporine (CYC) and curcumin (CUR) for enhanced transdermal therapy of rheumatoid arthritis (RA). Optimization of the drug-loaded edge-activated ethosomes was performed by varying independent variables, including the stoichiometric ratio of soya lecithin to cholesterol, ethanol concentration, and polyvinyl alcohol (PVA) concentration. The effects of these variables on dependent outcomes such as particle size, zeta potential, and entrapment efficiency were analyzed using multiple regression models. The stoichiometric ratio significantly influenced the particle size of ethosomes (F (1, 7) = 6.802, p = 0.0350, R² = 0.928), which could be attributed to alterations in the lipid bilayer’s fluidity and packing (Abdulbaqi et al. 2016). The ratio also markedly affected entrapment efficiency (F (1, 7) = 22.491, p = 0.002, R² = 0.962 for CUR; F (1, 7) = 29.806, p = 0.0009, R² = 0.998 for CYC), likely due to changes in the partition coefficient of drugs within the ethosomal lipid matrix. These findings are summarized in Table 2. However, no significant effect on zeta potential was observed (F (1, 7) = 0.446, p = 0.525, R² = 0.059), indicating minimal influence of the stoichiometric ratio on the surface charge of ethosomes.

Ethanol concentration was another critical factor. Increasing ethanol levels from 10% to 40% significantly reduced particle size (F (1, 7) = 32.388, p = 0.0007, R² = 0.9822), likely due to ethanol’s ability to disrupt lipid bilayer packing and enhance vesicle curvature (Ingólfsson and Andersen 2011; Galindo et al. 2024). Ethanol also increased entrapment efficiency (F (1, 7) = 40.629, p = 0.0441, R² = 0.954 for CUR; F (1, 7) = 48.815, p = 0.0037, R² = 0.9118 for CYC) and had a significant impact on zeta potential (F (1, 7) = 34.089, p = 0.0006, R² = 0.982). These results align with earlier studies demonstrating ethanol’s role as a penetration enhancer and lipid bilayer fluidizer, which modifies surface charge and stabilizes ethosomes (Patra et al. 2006; Pathan 2016). Further molecular-level investigations could elucidate the consistency of these effects across different lipid compositions and drugs. PVA was incorporated to stabilize ethosomal nanoparticles and prevent aggregation. Variations in PVA concentration (0.1% to 0.3%) significantly influenced particle size, with a notable reduction observed (F (1, 7) = 24.049, p = 0.0017, R² = 0.9745). PVA’s surfactant properties likely reduce interfacial tension, yielding smaller and more uniform nanoparticles (Heinz et al. 2017). Additionally, increasing PVA concentration decreased zeta potential (F (1, 7) = 30.379, p = 0.0008, R² = 0.9812) due to the hydrophilic nature of PVA shielding surface charges. These findings reinforce PVA’s dual role in enhancing size uniformity and stabilizing ethosomal systems (Quintanar-Guerrero et al. 2005; Hernández-Giottonini et al. 2020). Batch F9, identified as the optimized formulation, demonstrated a particle size of 93.31 ± 1.17 nm, PDI of 0.254 ± 0.021, and zeta potential of −29.2 ± 0.17 mV, confirming the stabilizing effects of PVA.

The ethosomal gel exhibited stable, spherical ethosomes with smooth surfaces, transparent and smooth-textured gel properties, and optimal viscosity, ensuring ease of application. The viscosity, spreadability and pH of the ethosomal gel significantly enhance patient compliance, as these properties ensure easy application of the formulation on the affected areas. Moreover, the gel’s stability guarantees prolonged efficacy, maintaining its therapeutic performance over time, which further contributes to patient acceptance and consistent adherence to the treatment regimen. Additionally, the gel demonstrated optimum viscosity, allowing smooth extrusion from the tube under pressure, highlighting its excellent extrudability and ease of application.

Analysis of release kinetics suggested a Hixson-Crowell ­diffusion mechanism for both drugs, indicating a change in ethosome surface area over time. This suggests a strong correlation between the experimental data and the kinetic model, supporting the hypothesis that the release process is primarily governed by changes in surface area and ­particle diameter. However, further extrapolation of dermatopharmacokinetics is necessary for a better understanding of the formulation. HET-CAM studies confirmed the gel’s nonirritant nature, while anti-inflammatory assays revealed a significant reduction in pro-inflammatory markers (e.g. IL-6, TNF-α, PGE2) and an increase in IL-10 in LPS-treated RAW 264.7 cells. These effects were comparable to those of Methotrexate, highlighting the gel’s potential in inflammatory disorders.

As compared to previously reported ethosomal and liposomal formulations for RA, the CYC+CUR-loaded ethosomal gel exhibited significantly enhanced therapeutic efficacy, anti-inflammatory activity, and safety. Ethosomes are more efficient than other lipidic nanoformulations due to their ethanol content, which enhances vesicle deformability and disrupts stratum corneum lipids, improving skin permeability and allowing deeper penetration into inflamed skin and synovial tissues. In contrast, other lipidic formulations like ethosomes, solid lipid nanoparticles (SLNs) and Nano lipid carriers (NLCs) lack ethanol, reducing flexibility and penetration. The ethosomal gel demonstrated superior permeability, higher steady-state flux, and faster drug release in in vitro and ex vivo studies compared to our previously published cyclosporine-curcumin nanoemulgel. Additionally, it showed comparable anti-inflammatory effects on LPS-stimulated RAW 264.7 macrophage cells, with similar suppression of pro-inflammatory mediators (Gharat et al., 2024). Compared to other nanocarrier systems loaded with NSAIDs or curcumin, the ethosomal gel achieved greater reduction in pro-inflammatory cytokines (e.g. TNF-α, IL-6). This enhanced efficacy is attributed to the immunomodulatory effects of CYC and its synergistic effect with CUR, highlighting its potential as a superior therapeutic option for RA management.

In vivo evaluations in a CFA-induced rat model of RA demonstrated the gel’s therapeutic efficacy, reflected in improved hematological, biochemical, and histological parameters. The CUR+CYC-Etho-gel demonstrated a synergistic effect by restoring hemoglobin and RBC levels to normal, reducing elevated WBC counts associated with RA, and effectively alleviating systemic inflammation (Williams et al. 2000). These effects were absent in gels containing CUR or CYC alone, emphasizing the importance of their combined formulation. Paw edema reduction, weight normalization, and radiographic preservation of joint architecture further substantiated the gel’s efficacy, comparable to the marketed Trexjoy^® gel. Importantly, the developed ethosomal gel formulation mitigated nephrotoxicity typically associated with CYC, likely due to CUR’s antioxidant and anti-inflammatory properties (Huang et al. 2018; Kadhim et al. 2021). Therefore, the potential clinical implications of this approach may improve the quality of life for patients with rheumatoid arthritis and renal impairments by providing a safer, more effective treatment option. Furthermore, a study on the suppression of rat collagen-induced arthritis and inhibition of macrophage-derived mediator release by two intravenous liposomal methotrexate formulations—one using egg lecithin and the other conjugated with polyethylene glycol—demonstrated that while liposomes were potent inhibitors of pro-inflammatory mediators in vitro, liposomes with prolonged circulation times did not exhibit significant therapeutic potential for treating arthritis in vivo (Williams et al. 2000). In contrast, our in vivo studies of the CUR+CYC-loaded ethosomal gel demonstrated comparable therapeutic efficacy to the methotrexate gel. Furthermore, the developed ethosomal gel demonstrated 89.27% inhibition of inflammation, indicating significantly enhanced anti-inflammatory efficacy. This result surpasses that of other RA formulations, such as methotrexate-loaded ultra-deformable liposomal gel, which exhibited only 40% inhibition of inflammation (Zeb et al. 2017), and transethosomal formulations of naproxen-sulfapyridine, which showed a reduction of approximately 84.63% in inflammation (Babasahib et al. 2022). Additionally, a study on methotrexate-loaded liposomal gels with penetration enhancers reported 80% inhibition (Sadarani et al. 2019). The superior inflammation inhibition observed in the developed ethosomal gel formulation highlights its greater potential for more effective rheumatoid arthritis management compared to other formulations.

Considering the structural and manufacturing similarities between ethosomes and liposomes, ethosomes are positioned as a good candidate for large-scale manufacturing and commercialization. Insights gained from the successful commercial scalability of Doxil^® (doxorubicin HCl liposome injection) provide a foundation for understanding the industrial feasibility of ethosome-based systems (U.S. Food and Drug Administration n.d). Additionally, the preparation of ethosomes involved precise control of various process parameters, such as stirring speed and ethanol addition rate. However, these parameters are likely to be influenced by changes in batch size during scale-up processes. As the batch size increases, factors such as stirrer tip speed and mixing efficiency may vary, necessitating adjustments to maintain consistent ethosome characteristics. Specifically, the ethanol addition rate, which plays a critical role in determining ethosome size, needs to be carefully controlled during scale-up trials. It is therefore crucial to conduct scale-up studies with a detailed understanding of tip speed calculations and to optimize stirring rates to ensure uniform ethosomal dispersion and consistent drug encapsulation across different production scales. In conclusion, the developed CUR+CYC-loaded ethosomal gel exhibited a robust therapeutic profile, offering a safe, effective, and innovative approach for RA management. These findings warrant further clinical evaluation to explore its broader applicability in inflammatory disorders.

5. Conclusion

The development and evaluation of a CUR+CYC-loaded edge-activated ethosomal gel represent a significant advancement in transdermal drug delivery systems for rheumatoid arthritis. The optimized ethosomal formulation demonstrated enhanced stability, superior drug entrapment, and excellent anti-inflammatory properties, supported by preclinical findings in both in vitro and in vivo models. Notably, the synergistic combination of cyclosporine and curcumin not only enhanced therapeutic efficacy but also mitigated nephrotoxicity concerns associated with cyclosporine. The study highlights the potential of ethosomal technology to address complex therapeutic challenges, offering a robust platform for localized and sustained drug delivery with minimal systemic side effects.

6. Future perspectives

Building on the promising preclinical results, further investigations should focus on detailed dermatopharmacokinetics and long-term toxicity studies to validate the safety and efficacy profile of the CUR+CYC-loaded ethosomal gel. Future studies should focus on evaluating biochemical markers of renal function, such as serum creatinine and blood urea nitrogen levels, to complement histopathological findings and provide a more comprehensive understanding of the nephroprotective effects of CUR when used in combination with CYC. Clinical trials will be essential to establish its therapeutic potential and compare its performance against existing treatments. Additionally, exploring the scalability of ethosomal manufacturing using liposome-based production infrastructure could facilitate the transition from lab-scale development to commercial viability. Further studies on the molecular interactions within ethosomal systems and their impact on drug stability and release kinetics may provide deeper insights into optimizing this technology for other inflammatory disorders and chronic conditions.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Ethical approval

Animal studies were conducted to evaluate the efficacy of the developed ethosomal gel formulation. The authors confirm that all in vivo experiments complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines to ensure scientific rigor, reproducibility, and ethical compliance. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) under approval number IAEC/Sangli/2023-24/13. A total of 24 Wistar rats were used in the study. The animals were housed under standard laboratory conditions at the Biocyte Institute of Research and Development, India, with a controlled temperature and relative humidity, following a 12-hour light/dark cycle. They were provided with a standard commercial pelleted diet and had ad libitum access to water. Environmental enrichment, including nesting material and shelters, was provided to promote natural behaviors and reduce stress. All procedures were designed to minimize animal suffering, and continuous monitoring of their well-being was conducted by trained personnel. At the end of the experimental period (Day 28), euthanasia was performed using CO₂ asphyxiation in accordance with institutional and regulatory guidelines to minimize distress.

Consent for publication

All authors have given consent for this publication.

Disclosure statement

Dr. Abdelwahab Omri is a member of the editorial board of Drug Delivery. This role had no influence on the editorial process or the peer review of this manuscript. The authors declare no other competing interests.

The author declares that there are no relevant financial or non-financial competing interests to disclose in relation to the content of this article.

Data availability statement

The authors confirm that the data supporting the findings of this study are accessible within the article.