Acemetacin-loaded bilosomal gel formulations prepared using different polymers for topical application: Box-Behnken design for bilosomes formulation optimization and in vitro evaluation of the formulations

Abstract

Acemetacin (ACIN) is a poorly water-soluble nonsteroidal anti-inflammatory drug, which limits its effectiveness in topical therapeutic applications. This study aimed to enhance the delivery potential and dermal applicability of ACIN through the development of bilosome-loaded hydrogel formulations. Bilosomes were prepared using the thin-film hydration method and optimized using Box-Behnken Design (BBD). The amount of phosphotidylcholine (lecithin), cholesterol and sodium taurocholate were chosen as the formulation parameters and their effects were evaluated on the resulting vesicle size, zeta potential and entrapemnet efficiency percentage (EE%). The optimized formulation displayed a vesicle size of 137.3 nm, a zeta potential of −30.1 mV, a polydispersity index (PDI) of 0.384 and EE% of 84.5%. Bilosomes were incorporated into hydrogel bases containing hydroxypropyl methylcellulose (HPMC) or Carbopol. HPMC-based gels exhibited a favorable pH (~4) for skin application and were selected for further evaluation. These gels provided sustained drug release for up to eight hours. Cytocompatibility testing on L929 fibroblasts using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated cell viability above 90% within the tested concentration range (0.05–2 µg/mL), indicating good biocompatibility. The bilosome-loaded HPMC gel formulation exhibited desirable physicochemical properties, sustained drug release, and excellent cytocompatibility, making it a promising vehicle for topical delivery of ACIN. Further anti-inflammatory and in vivo studies are recommended to confirm its potential for topical or anti-inflammatory applications.

Graphical Abstract

Introduction

Acemetacin (ACIN), chemically known as 1-(4-chlorobenzoyl)−5-methoxy-2-methyl-1 H-indol-3-yl acetic acid carboxymethyl ester, is a glycolic acid ester derivative of indomethacin with antipyretic, anti-inflammatory, and analgesic properties. It has been widely used for the treatment of inflammatory and degenerative disorders [1, 2]. According to the European Pharmacopoeia (EP 10), ACIN is practically insoluble in water [3]. However, it exhibits good solubility in certain organic solvents, such as dimethylformamide (≈ 25 mg/mL), dimethyl sulfoxide (≈ 25 mg/mL), and ethanol (≈ 3 mg/mL). Its solubility in phosphate-buffered saline (PBS, pH 7.2) is approximately 0.5 mg/mL [4]. ACIN’s solubility varies with pH; it is more soluble in alkaline environments and less soluble in acidic ones. Because the drug’s solubility in the vehicle and at the skin’s surface may significantly impact its release and penetration profile, this feature is especially crucial for topical preparations. ACIN acts as a prodrug and undergoes hepatic first-pass metabolism to yield indomethacin, a non-selective cyclooxygenase-2 (COX-2) inhibitor, thereby exerting anti-inflammatory effects. Compared with indomethacin, ACIN is generally assumed to cause less gastric mucosal damage; however, this assumption is supported only by a few small-scale, short-term endoscopic studies, and sufficient clinical evidence is lacking [5, 6].

Our study aimed to design and optimize ACIN-loaded bilosomal gel formulations intended for topical drug delivery, with a focus on improving formulation stability and physicochemical characteristics. First, we utilized a Box-Behnken Design (BBD) to prepare and optimize ACIN-loaded bilosomes. This design was chosen for its efficiency, reduced number of experimental runs, and avoidance of extreme axial points. Replicated center points were incorporated to provide a reliable estimate of experimental error and to strengthen model robustness. Thin film hydration method was used to prepare bilosomes formulations. Then, the optimum formulation was incorporated into a gel prepared using Carbopol or hydroxypropyl methylcellulose (HPMC) and in vitro characterization studies were performed. According to Chávez-Piña et al., ACIN may have gastric-sparing effects since it lessens the induction of leukocyte adhesion. In contrast to indomethacin, which is known to increase mucosal damage through greater leukocyte adherence and subsequent inflammatory responses, ACIN drastically reduced leukocyte–endothelial contact in experimental settings. ACIN’s enhanced gastrointestinal tolerability may be attributed to a less inflammatory insult to the stomach mucosa, as seen by the lower levels of leukocyte activation and adhesion seen with the medication [7].

Despite its therapeutic potential, topical delivery of ACIN is limited by poor aqueous solubility, low permeability across the stratum corneum, and chemical instability in conventional formulations. These limitations often result in inadequate local bioavailability and reduced pharmacological efficacy, highlighting the need for novel formulation strategies to enhance solubility, stability, and skin penetration. Several studies have addressed these challenges: ACIN nanosuspensions have achieved approximately a ten-fold increase in saturated solubility compared with raw ACIN, demonstrating that conventional forms are insufficient for dermal application [8]. Self-emulsifying microemulsions of ACIN have also been developed to improve dissolution and skin permeation, with optimized formulations showing significantly higher ex vivo permeation than ACIN suspensions [9]. Furthermore, interactions of ACIN with phospholipid bilayers were shown to depend on its ionization state, suggesting that passive diffusion through lipid domains may be unfavorable under certain conditions [10]. Finally, reviews of nanosuspension delivery systems emphasize that poorly soluble drugs, such as ACIN (BCS Class II), often require advanced strategies to overcome dissolution and stability barriers in both topical and systemic applications [11].

Bilosomes have recently emerged as promising carriers for improving the dermal and transdermal delivery of poorly soluble drugs. They are bile salt-stabilized lipid vesicles that exhibit high deformability and enhanced interaction with biological membranes [12]. The incorporation of bile salts improves both the solubilization of hydrophobic drugs and the elasticity of vesicular bilayers, facilitating drug permeation through the skin. Bilosomes also provide advantages such as improved stability, sustained drug release, and reduced systemic side effects [13].

Hydrogels, on the other hand, are widely used as topical bases due to their biocompatibility, hydration capacity, and ability to provide controlled drug release. Polymers such as HPMC and Carbopol^® are commonly employed to prepare transparent, stable, and spreadable hydrogels suitable for dermal application. HPMC is a hydrophilic polymer frequently used in modified-release formulations because of its gelling, thickening, and swelling capabilities. It can form clear and stable hydrogels suitable for topical applications [14]. Carbopol^®, a cross-linked polyacrylic acid polymer with high molecular weight, is commonly employed as a suspending, thickening, and stabilizing agent in pharmaceutical and cosmetic products. It is valued for its high efficiency, moderate electrolyte tolerance, and ease of use. Various types of Carbopol^® (e.g., Ultrez (U), ETD, EZ, 940, 941, 980) are available, each characterized by distinct rheological properties. For instance, Carbopol^® 940 and 941 are known for their long wetting times and poor transparency [15]. These polymers can swell up to 1000 times their original volume, forming a mucus-like dispersion [16]. Upon neutralization with agents such as sodium hydroxide or triethanolamine, the ionization of carboxylic acid groups induces negative charges, promoting cross-linking among the swollen polymer chains and enhancing gel strength [17].

This study aimed to develop and optimize bilosome-loaded hydrogel formulations of ACIN and to evaluate their physicochemical characteristics and in vitro performance for topical delivery. To achieve this, ACIN-loaded bilosomes were prepared and optimized using a BBD via the thin-film hydration method. The optimized bilosome dispersion was subsequently incorporated into hydrogels formulated with Carbopol or HPMC, and the developed formulations were subjected to comprehensive in vitro characterization studies.

Materials and methods

Materials

ACIN, cholesterol, and sodium taurocholate were purchased from Sigma-Aldrich (USA). Lecithin was obtained from Shankar (India). Carbopol^® Ultrez^™ 10 was procured from BF Goodrich (USA), and HPMC was supplied by Drogsan (Turkey, Batch No: Y210000957). Dulbecco’s Modified Eagle Medium (DMEM) High Glucose, fetal bovine serum (FBS) and 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) was purchased form Sigma-Aldrich (USA). All other chemicals and solvents used were of analytical grade.

Methods

Selection of independent variables

Selection of the formulation variables was carried out after a comprehensive literature review to identify the primary components with a documented influence on the physicochemical properties of bilosomes, including vesicle size, entrapment efficiency percentage (EE%), and zeta potential. Based on this evaluation, Lecithin (X1), Sodium taurocholate (X2), and Cholesterol (X3) were selected as the Critical Material Attributes (CMAs). Lecithin acts as the primary phospholipid forming the bilayer and determining hydrophobic packing. Sodium taurocholate, an anionic bile salt, ehnances membrane elasticity and interfacial behavior, contributing to vesicle deformability and surface charge. Cholesterol adjusts bilayer rigidity, minimizes structural defects and hence decreases leakage of encapsulated agents. These three components are considered as the principal formulation-dependent determinants of the critical quality attributes (CQAs) of bilosomes i.e. vesicle size, EE%, and zeta potential. The level ranges selected for the three formulation variables were based on established concentrations reported in previous bilosome studies; however, the ranges were deliberately expanded in the present work to allow a more comprehensive assessment of factor influence across a wider design space [18]. In contrast, critical process parameters (CPPs), including hydration volume, temperature, and sonication time, were held constant throughout the study. Their optimal levels were adopted from previously optimized protocols in the bilosome literature and subsequently verified during initial trial preparations to ensure reproducible film hydration and consistent vesicle formation under our specific experimental conditions. By controlling these process parameters, the experimental design was focused exclusively on elucidating the relationships between the formulation’s composition and its resulting physicochemical characteristics.

Box–Behnken experimental design and optimization

A three-factor, three-level BBD was constructed using Design-Expert^® Version 13 (Stat-Ease Inc., USA) to statistically optimize the composition of ACIN-loaded bilosomes. Lecithin (X1), sodium taurocholate (X2), and cholesterol (X3) were evaluated at low, medium, and high levels, while vesicle size (Y1), zeta potential (Y2), and EE% (Y3) were selected as the CQAs. The structured design framework for the optimization is summarized in Table 1.

Table 1.

Independent variables (CMAs), their experimental levels, and dependent responses (CQAs) used in the BBD for optimization of ACIN-loaded bilosomes

Independent variables (CMAs)	Units	Level
		Low	Medium	High
A: Lecithin (X1)	Mg	100	300	500
B: Sodium Taurocholate (X2)	Mg	10	17.5	25
C: Cholesterol (X3)	Mg	15	22.5	30
Dependent Variables (CQAs)	Units	Optimization target
Y1: Vesicle Size	Nm	Minimize
Y2: Zeta Potential	mV	Optimize
Y3: EE	%	Maximize

All responses were analyzed by Analysis of Variance (ANOVA), and the most appropriate polynomial model for each response was identified based on statistical criteria: significant model terms (p < 0.05), non-significant lack-of-fit (p > 0.05), and acceptable values of R², and adequate precision. Response surfaces were generated to visualize individual and interaction effects across the design space. Following model validation, numerical optimization using the desirability function was performed, complemented by graphical optimization to confirm the feasible region. The formulation presenting the highest composite desirability value was selected as the optimized ACIN-loaded bilosome and carried forward for full physicochemical characterization and incorporation into the gel matrix [19, 20].

Preparation of ACIN-loaded bilosomes

First, we prepared ACIN-loaded bilosomes using the thin-film hydration method under fixed process conditions and evaluated in triplicate [21]. Briefly, lecithin, cholesterol, and ACIN (60 mg) were dissolved in ethanol. Ethanol was removed using a rotary evaporator (IKA, RV 3 eco, Germany) at 40 °C under reduced pressure to form a thin lipid film in a round-bottomed flask. The formed lipid film was then hydrated with 20 mL of distilled water containing sodium taurocholate (as a surfactant). The dispersion was sonicated in a bath sonicator (Ultrasonic cleaner WUC-A, Germany) for 10 min to reduce vesicle size. The bilosomal dispersions were stored in a refrigerator (4 °C) until further studies. Blank bilosomes were prepared the same procedure without ACIN.

Preparation of blank bilosomes or ACIN-loaded bilosomal gel formulations

Bilosomal gel formulations were prepared using two different gelling agents (Carbopol^® Ultrez 10 or HPMC). Bilosomes containing Carbopol gels were prepared according to the previously described method [22]. Bilosomal gels were prepared using Carbopol^® Ultrez 10 and HPMC at concentrations of 2.5% and 5% (w/v). Carbopol Ultrez 10 was gradually dispersed in warm distilled water and mixed at room temperature using a mechanical stirrer at 500 rpm until a uniform gel base was formed. HPMC was hydrated separately under the same stirring conditions until a viscous colloidal polymer solution was obtained. After polymer hydration, each gel base was left undisturbed overnight to eliminate air bubbles and prevent air entrapment in the final formulation. Subsequently, 20 mL of the optimized ACIN-loaded bilosome formulation (Formulation No. 15; Table 2) or blank bilosomes was added to the respective polymer bases, and mixing was continued until smooth and homogeneous Carbopol or HPMC bilosomal gels were obtained. Carbopol gels were neutralized with triethanolamine to achieve proper gelation, ensuring adequate viscosity and a skin-compatible pH.

Table 2.

BBD and the obtained responses

	Independent Variables (CMAs)			Dependent Variables (CQAs)
Run	A: Lecithin	B: Sodium taurocholate	C: Cholesterol	Vesicle size (n = 3)	Y2 = Zeta potential (n = 3)	Y3 = EE (n = 3)	Y4 = PDI (n = 3)
	Mg	mg	mg	Nm	mV	%
1	500	17.5	30	181.9 ± 0.11	−28.9 ± 0.02	50.4 ± 0.11	0.340 ± 0.03
2	300	17.5	22.5	169.5 ± 0.12	−45.9 ± 0.02	64.8 ± 0.0.09	0.265 ± 0.01
3	300	10	30	174 ± 0.11	−41.4 ± 0.01	67 ± 0.12	0.257 ± 0.01
4	500	17.5	15	145.5 ± 0.12	−32.7 ± 0.01	62.8 ± 0.10	0.315 ± 0.08
5	100	17.5	15	560 ± 0.13	−55.3 ± 0.02	49.5 ± 0.09	0.318 ± 0.03
6	500	25	22.5	174.7 ± 0.12	−39 ± 0.02	78.9 ± 0.09	0.262 ± 0.02
7	300	17.5	22.5	116.9 ± 0.12	−49.1 ± 0.01	63.2 ± 0.10	0.228 ± 0.01
8	300	25	15	105.5 ± 0.11	−43.9 ± 0.02	69.8 ± 0.12	0.252 ± 0.05
9	300	17.5	22.5	141.4 ± 0.11	−50.2 ± 0.02	68.1 ± 0.11	0.241 ± 0.01
10	100	17.5	30	231 ± 0.12	−54.9 ± 0.01	49.6 ± 0.12	0.365 ± 0.01
11	300	17.5	22.5	182 ± 0.11	−35.2 ± 0.01	63.5 ± 0.11	0.264 ± 0.05
12	300	25	30	127.4 ± 0.12	−37.6 ± 0.02	82.7 ± 0.10	0.369 ± 0.09
13	100	10	22.5	227 ± 0.12	−56.7 ± 0.01	67.8 ± 0.11	0.207 ± 0.01
14	300	17.5	22.5	214.9 ± 0.11	−55.1 ± 0.02	52.3 ± 0.09	0.258 ± 0.02
15	500	10	22.5	137.3 ± 0.12	−30.1 ± 0.02	84.5 ± 0.10	0.384 ± 0.01
16	100	25	22.5	373.7 ± 0.12	−58.6 ± 0.02	80.1 ± 0.11	0.342 ± 0.04
17	300	10	15	159.7 ± 0.11	−64.4 ± 0.01	60.5 ± 0.12	0.367 ± 0.02

In vitro characterization

Vesicle size, polydispersity index (PDI) and zeta potential values and morphological properties of bilosomal formulations.

The vesicle size, PDI and zeta potential measurements for bilosomes formulations were carried out via dynamic light scattering (DLS) technique (Zetasizer Nano ZS; Malvern Instruments, ZEN3600, UK) at room temperature for samples diluted 1:10. We determined the zeta potential values of appropriately diluted samples (1:10) using Zetasizer Nano ZS at room temperature.

The morphological features of the fabricated bilosomal formulations, including blank bilosomes and the optimized ACIN-loaded formulation (Formulation No. 15), were examined using Transmission Electron Microscopy (TEM). The analysis was performed using a Hitachi (Tokyo, Japan) TEM operated at an accelerating voltage of 100 kV. For sample preparation, a drop of the respective bilosomal dispersion was carefully placed onto a carbon-coated copper grid and allowed to air dry at room temperature. Images were subsequently acquired at appropriate magnifications to ensure accurate visualization of vesicle morphology [23].

Texture analysis, pH and viscosity measurement for bilosomal gel formulations

We used a Texture Analyser (TexturePro CT V1.9 Build 35, Brookfield Engineering Labs. Inc.) equipped with a 1.5 kg load cell to determine the texture properties (adhesiveness, cohesiveness, and hardness) of the bilosomal gel formulations. Briefly, a 10-mm (diameter) cylindrical probe was compressed into the gel (10 g) for a distance of 10 mm (at a speed of 2 mm/sec) and redrawn. Three measurements were made at 25 ± 0.5 °C for each sample.

The pH measurements for ACIN-loaded bilosome and bilosomal gel formulations were performed via a pH meter (Mettler Toledo FiveGo, Switzerland). Three measurements were made at 25 ± 1 °C for each sample [24]. The viscosities of the hydrogels were measured at room temperature (25±2 °C) with a Brookfield DV3T rheometer–viscometer (Anadolu University AÜBİBAM Central Research Laboratory, Spindle No 3, 200 rpm).

EE% values for bilosomal formulations and bilosomal gel formulations

To determine the percentage of EE% for ACIN-loaded bilosomes or ACIN-loaded bilosomal gel formulation, the method described by Akaki et al. was used with modifications [25]. ACIN-loaded bilosomes (0.5 g) or ACIN-loaded bilosomes-based gel formulation (0.5 g) were mixed with 100 mL of mobile phase (ACN:%1 formic acid; 60:40) and mixed for 1 h on magnetic stirrer (500 rpm) at room temperature. Then, the mixture was filtered through a membran filter (pore size: 0.45 μm) and analyzed in high performance liquid chromatography (HPLC). The experiments were made triplicate. The EE% was calculated by using the following equation= (Amount of ACIN in the formulation/Total amount of ACIN added)x100.

In vitro release study

The prepared ACIN-loaded bilosomes or ACIN-loaded bilosomes-based gel formulations (1 g) were filled into the dialysis bags (cut off: 12–14 kDa). Then, they were put into 100 mL of release medium (PBS-pH 7.4 and ethanol; 70:30 v/v; for sink condition) and regularly stirred at 150 rpm at 32 ± 1 °C. Samples (1 mL) are collected at predetermined time intervals (0, 0.25, 0.5, 1, 2, 3, 4, 6, 8 h) and and replaced with same volume fresh release medium. The samples were filtered through a membran filter (pore size: 0.45 μm) and analyzed in HPLC. For the quantification of ACIN, a validated HPLC method was developed and employed. The analysis was performed using an Agilent 1260 Infinity II system equipped with a C18 column (5 μm, 15 × 4.6 mm). The mobile phase consisted of acetonitrile and 1% formic acid (60:40, v/v), delivered at a flow rate of 1.0 mL/min. The column temperature was maintained at 30 °C, and the detection wavelength was set at 254 nm. The injection volume was 20 µL. Linearity was evaluated in the concentration range of 1–100 µg/mL at nine concentration levels. Method validation parameters, including linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and selectivity, were determined in accordance with the ICH Q2 (R1) guidelines [26]. The addition of organic co-solvents to the dissolution/release medium can increase drug solubility. There are studies in the literature on the use of co-solvents to increase drug solubility. In these studies, ethanol was used at various rates (10%, 40%, etc.) to ensure that sink condition was maintained [27].

In vitro drug release kinetic modeling

The in vitro release data of ACIN from the optimized bilosomes and bilosomal HPMC gels were fitted to four kinetic models of zero-order, first-order, Higuchi, and Korsmeyer–Peppas to characterize the underlying release mechanism. Linearized plots were constructed following standard transformations: cumulative drug release versus time (zero order), log % drug remaining versus time (first order), cumulative release versus the square root of time (Higuchi), and log cumulative release versus log time (Korsmeyer–Peppas). For the Peppas model, the release exponent (n) was determined to differentiate Fickian from anomalous diffusion. Linear regression was performed for each model, and the coefficient of determination (R²) was used to identify the best-fit kinetic model. The model showing the highest R² value for each formulation was considered most representative of the release behavior [28].

Cytotoxicity assay

In this study, L929 mouse fibroblast cell line (NCTC clone 929 ATCC, USA), a beneficial model for assessing toxicity from dermal exposure, was used. The cells were cultured in high-glucose DMEM supplemented with 10% FBS, 1% non-essential amino acids, and 1% penicillin-streptomycin. When the cells reached 90% confluence in culture flasks, they were passaged using Trypsin-EDTA.

Cytotoxicity of formulations was determined with the MTT assay. Briefly, L929 cells were seeded into 96-well microplates (10,000 cells/well). After overnight incubation at 37 °C, the growth medium was replaced with fresh medium containing from 0 (blank control group), 0.05, 0.1, 0.25, 0.5, 1, and 2 µg/mL blank bilosomes, ACIN-loaded bilosomes, blank bilosomal gel formulation prepared using HPMC (2.5%) (B-bilosomal HPMC 2.5 gel), and ACIN-loaded bilosomal gel formulation prepared using HPMC (2.5%) (ACIN-bilosomal HPMC 2.5 gel in 96 well plates. The cells were incubated for additional 24 h. The final concentration of dimethyl sülfoxide (DMSO) in solvent control and dilutions was 0.1%. MTT was then added to a final concentration of 0.5 mg/mL and the cells incubated at 37 °C for 3 h. The medium was removed, and formazan crystals were dissolved in DMSO (Sigma Aldrich, USA). The optical density of the solution in each well was measured at 570 nm (the formazan absorption peak) by a microplate spectrophotometer (Varioskan LUX Multimode Microplate Reader, Thermo Fisher). The viability of cells was determined by comparing formazan concentrations of the treated cells with those of untreated control cells and expressed as a percentage relative to the control group [29]. Each treatment group consisted of six replicate wells. The experiments were repeated three times.

Fourier transform infrared (FTIR) spectroscopy

FTIR spectra were obtained using a Shimadzu IRAffinity-1 S spectrophotometer (Shimadzu, Japan) equipped with an Attenuated Total Reflectance (ATR) accessory. In the ATR technique, IR radiation undergoes internal reflection within a high-refractive-index crystal, generating an evanescent wave that interacts with the sample surface; attenuation of this wave produces the characteristic absorption pattern. Approximately 1–2 mg of each sample was placed directly onto the ATR crystal under uniform pressure. Spectra were recorded in % transmittance mode over the 4000–400 cm⁻¹ range, with 30 scans, 4 cm⁻¹ resolution, and Happ–Genzel apodization. The analysed samples included pure ACIN, lecithin, cholesterol, sodium taurocholate, HPMC, blank bilosomes, ACIN-loaded bilosomes, and the ACIN-loaded bilosomal HPMC gel.

Statistical analysis

Data were analyzed using GraphPad Prism software (version 10.2.3). Following the Shapiro-Wilk normality test, one-way ANOVA was performed, followed by Tukey’s post hoc test. p-value of less than 0.05 was considered statistically significant.

Stability testing

Short-term stability studies were conducted for both the optimized ACIN-loaded bilosome formulation and the corresponding HPMC gel. The samples were stored at 4 °C, 25 °C, and 40 °C for 1 month, and were periodically evaluated for particle size, zeta potential, pH, and drug content to assess their physical and chemical stability.

Results and discussion

Experimental design and statistical analysis

The BBD generated a total of 17 ACIN-loaded bilosome formulations, each representing a varying amount of lecithin, sodium taurocholate, and cholesterol. The resulting formulations displayed marked variability in their physicochemical attributes. The measured responses including vesicle size, zeta potential, PDI, and EE% are presented in Table2. The PDI values across all formulations were consistently within the acceptable range, with minimal variability, and therefore were not included as an optimization parameter in the design. These data provided the basis for subsequent model fitting, ANOVA interpretation, and identification of the optimum as well as the formulation variables exerting the most significant effects on the CQAs of the bilosomal system.

Statistical evaluation of the BBD revealed distinct model behaviors for the three responses. Vesicle size was best fit by a quadratic model, demonstrated by a significant overall model fit and a non-significant lack-of-fit, confirming that the polynomial structure adequately represented the experimental data. Lecithin (A), the A×C interaction, and the quadratic effect of A² emerged as the major determinants of size variation, indicating strong nonlinear and synergistic influences of phospholipid concentration and cholesterol on vesicle architecture. In contrast, zeta potential followed a linear relationship, with a highly significant model fit and an acceptable lack-of-fit, showing that only lecithin exerted a statistically meaningful effect within the tested formulation domain. EE % was also best explained by a quadratic model, supported by significant model statistics and an adequate lack-of-fit. Here, the nonlinear contributions of sodium taurocholate (B²) and cholesterol (C²) were the primary drivers of encapsulation efficiency. The final equations (coded factors) for all three responses reflect these distinct linear and nonlinear factor contributions and they are given below:

A BBD output analysis of the model type, statistical significance, and influential terms is provided in Table 3.

Table 3.

Statistical summary of Box–Behnken models for ACIN-Loaded bilosomes

Response	Best Fit Model	p-Value	Lack of Fit p-Value	R²	Significant Terms
Vesicle Size	Quadratic	0.0444	0.0516	0.8322	A, AC, A²
Zeta Potential	Linear	0.0013	0.6803	0.6876	A
EE	Quadratic	0.0327	0.2979	0.8485	B², C²

Effect of independent variables on vesicular size

Vesicle size is a critical determinant of skin penetration efficiency, making it a fundamental parameter in transdermal drug delivery. In this study, the vesicle size demonstrated considerable variation across the 17 experimental runs detailed in Table 2, ranging from 105.5 nm to 560 nm. Among the formulation variables, lecithin concentration emerged as the most influential factor affecting vesicle size. A distinct inverse relationship was observed, where increasing lecithin concentration consistently produced smaller vesicles. This trend is clearly visualized in the 3D response surface plot of Fig. 1, which examines the combined effects of lecithin and sodium taurocholate. The plot reveals a steep decline in vesicle size along the lecithin axis, while the sodium taurocholate axis shows minimal variation, underscoring the dominant role of lecithin in determining vesicle dimensions. Moreover, a significant interactive effect between lecithin and cholesterol was observed and illustrated in Fig. 2. The response surface demonstrates that at low lecithin concentrations, increasing cholesterol content substantially reduced vesicle size. However, this cholesterol-mediated size reduction became progressively less pronounced as the lecithin concentration increased. In contrast to the pronounced effects of lecithin, sodium taurocholate exhibited minimal individual impact on vesicle size within the investigated range. The combined influence of sodium taurocholate and cholesterol, as depicted in Fig. 3, was similarly modest. At constant high lecithin concentration, increasing sodium taurocholate resulted in a slight reduction in vesicle size, while increasing cholesterol produced a marginal size increase.

Fig. 1.

3D Surface showing the effect of amount of lecithin and sodium taurocholate on the vesicular size of ACIN-loaded bilosome formulations

Fig. 2.

3D Surface showing the effect of amount lecithin and cholesterol on the vesicular size of ACIN-loaded bilosome formulations

Fig. 3.

3D Surface showing the effect of amount of sodium taurocholate and cholesterol on the vesicular size of ACIN-loaded bilosome formulations

 

Effect of independent variables on zeta potential

The zeta potential of the prepared formulations as detailed in Table 2, demonstrated a broad range from −28.9 mV to −64.4 mV, confirming that lipid composition significantly influences surface charge. Analysis of the data identified a clear and direct relationship between lecithin concentration and zeta potential. As the lecithin content increased the zeta potential values systematically became less negative, shifting from a highly negative range (e.g., −55.3 mV to −64.4 mV at 100 mg lecithin) towards a moderately negative range (e.g., −28.9 mV to −39 mV at 500 mg lecithin). The 3D response surface plots in Figs. 4, 5 and 6 illustrate the modifying effects of the other components. Sodium taurocholate was observed to intensify the negative surface charge, as evidenced by comparing runs with different bile salt levels at constant lecithin. In contrast, cholesterol consistently demonstrated a neutralizing effect, reducing the magnitude of the zeta potential. Despite these modulations, all formulations maintained a zeta potential at or beyond the ±30 mV threshold, indicating a colloidally stable of prepared ACIN bilosomes.

Fig. 4.

3D Surface showing the effect of amount of lecithin and cholesterol on zeta potential values of ACIN-loaded bilosome formulations

Fig. 5.

3D Surface showing the effect of amount of lecithin and sodium taurocholate on zeta potential values of ACIN-loaded bilosome formulations

Fig. 6.

3D Surface showing the effect of amount of sodium taurocholate and cholesterol on zeta potential values of ACIN-loaded bilosome formulations

Effect of independent variables on EE%

EE% is a crucial parameter that directly determines the drug loading capacity and therapeutic activity of the delivery system. The EE% of ACIN-loaded bilosomes demonstrated considerable variation across the formulations, ranging from approximately 50% to 85% as shown in Table 2. While the main effects of the individual components (lecithin, sodium taurocholate, and cholesterol) were not statistically significant, strong non-linear relationships were identified through the significant quadratic terms for both sodium taurocholate (B², p = 0.0016) and cholesterol (C², p = 0.0313). The 3D plots (Figs. 7, 8 and 9) show that EE% rises with increasing lecithin and optimal bile salt concentrations, whereas higher cholesterol levels flatten the surface and decrease EE%, reflecting a curved response surface characteristic of nonlinear bilayer behavior. Figure 7 demonstrates that increasing lecithin concentration consistently improved EE%, while sodium taurocholate modulated the efficiency depending on its level, suggesting a balance between bilayer fluidization and stabilization. Figure 8 further reveals that cholesterol concentration follows a non-linear trend, where EE% reaches a maximum at moderate levels (around 22.5 mg) before declining slightly at higher concentrations, consistent with cholesterol-induced bilayer rigidity. Figure 9 shows that at high lecithin loading (500 mg), combinations of elevated cholesterol and either low or high sodium taurocholate concentrations yield the highest ACIN encapsulation into bilosomes.

Fig. 7.

3D Surface showing the effects of amounts of sodium taurocholate and lecithin on EE%

Fig. 8.

3D Surface showing the effects of amounts of lecithin and cholesterol on EE%

Fig. 9.

3D Surface showing the effects of amounts of sodium taurocholate and cholesterol on EE%

Formulation optimization

The optimization of ACIN bilosomes was carried out using the point prediction and desirability function (0–1) approach in Design-Expert software to achieve an optimal balance among vesicle size, zeta potential, and EE%. Based on this analysis, Formulation No. 15 (Table 2) was identified as the optimal formulation. The optimization targeted the simultaneous minimization of vesicle size, optimization of zeta potential, and maximization of EE%, yielding the highest overall desirability within the design space. The optimized formulation exhibited a vesicle size of 137.3 ± 0.12 nm, PDI of 0.384 ± 0.01, zeta potential of −30.1 ± 0.02 mV, and EE% of 84.5%, demonstrating excellent physicochemical balance among the CQAs. In comparison, the corresponding blank bilosomal formulation showed a vesicle size of 160 ± 10.12 nm, PDI of 0.365 ± 0.05, and zeta potential of −24.5 ± 0.12 mV (n = 3).

The multi-response optimization yielded an overall combined desirability score of 0.704, indicating a robust and statistically optimized formulation within the experimental design space. As shown in the desirability plot (Fig. 10), all three independent variables attained individual desirability values of 1.0, confirming that their optimized levels successfully met the predefined goals. The responses also exhibited high desirability for vesicle size (0.9999) and zeta potential (1.0), corresponding desirable particle size and an optimal surface charge of approximately −30 mV, ensuring good colloidal stability and suitability for transdermal application. Although the EE% desirability (0.54) was comparatively lower, this result highlights a well-balanced optimization outcome, where enhanced vesicle stability and reduced particle size were successfully achieved without compromising satisfactory drug loading efficiency.

Fig. 10.

Desirability value of the independent vraibles and the responses

The overlay plot (Fig. 11) further delineated the design space where all responses simultaneously met the optimization criteria. The yellow region represented the optimal zone, indicating that the most desirable combination was obtained at 500 mg lecithin, 10 mg sodium taurocholate, and 22.5 mg cholesterol, yielding vesicles with an average size of 151 nm, zeta potential of −35.6 mV, and EE% of 84.28%. The use of the point prediction method demonstrated close agreement between software-predicted and experimental results, validating the precision and reliability of the developed models.

Fig. 11.

Overlay plot of the optimum levels of the responses: vesicular size, zeta potential and EE%

Morphological evaluation of the optimum bilosome formulations

TEM images were obtained to evaluate the morphology of the optimum bilosome formulations (Fig. 12). In Fig. 12A, the blank bilosomes appeared as nanoscale nearly spherical vesicles with smooth surfaces. The optimized ACIN-loaded bilosome formulation (Formulation No. 15, Fig. 12B1 and B2) was nano-sized, spherical and had distinct boundaries. The observed vesicle sizes of ACIN-loaded bilosome formulation in TEM images typically ranged from 130 to 265 nm.

Fig. 12.

TEM images of optimum blank bilosomes (A) and ACIN-loaded bilosomes (Formulation no: 15) (B1 and B2)

Texture analysis and pH measurement for bilosomal gel formulations

In this study, gel formulations containing the optimum bilosomal formulation were also prepared using Carbopol^® Ultrez^™ 10 or HPMC and in vitro characterized. Firstly, the mechanical properties such as adhesiveness, cohesiveness, and hardness of the ACIN-loaded bilosomal gel formulations prepared using Carbopol or HPMC (2.5 or 5%) were investigated using texture profile analysis (Table 4).

Table 4.

The mechanical properties of ACIN-loaded bilosomal gel formulations prepared using carbopol or HPMC (2.5 or 5%) (Mean±SD; n = 3)

	Carbopol 2.5 gel	Carbopol 5 gel	HPMC 2.5 gel	HPMC 5 gel
Hardness (N)	0.180 ± 0.010	1.250 ± 0.078∗	0.030 ± 0.000	0.047 ± 0.006
Adhesiveness (mJ)	0.993 ± 0.068	3.490 ± 0.010∗	0.057 ± 0.006	0.220 ± 0.026∗
Cohesiveness	0.873 ± 0.015	0.770 ± 0.017	0.667 ± 0.095	0.517 ± 0.119

Carbopol 2.5 gel and Carbopol 5 gel: ACIN-loaded bilosomal gel formulations prepared using Carbopol (2.5 or 5%); HPMC 2.5 gel and HPMC 5 gel: ACIN-loaded bilosomal gel formulations prepared using HPMC (2.5 or 5% (^∗: p < 0.05)

In addition, the pH values were determined for ACIN-loaded bilosomes (the optimum formulation) and also ACIN-loaded bilosomal gel formulations prepared using Carbopol or HPMC (2.5 or 5%). The pH values are given in Table 5. As shown in Table 6, ACIN incorporation markedly increased the viscosity of the bilosome dispersion, while dilution with hydrogel reduced the apparent viscosity due to polymer network relaxation.

Table 5.

The pH values of ACIN-loaded bilosome formulation and ACIN-loaded bilosomal gel formulations prepared using carbopol or HPMC (2.5 or 5%) (Mean±SD; n = 3)

Formulation	pH
ACIN-loaded bilosomes	3.46 ± 0.015
Carbopol 2.5 gel	3.76 ± 0.053
Carbopol 5 gel	3.81 ± 0.045
HPMC 2.5 gel	4.07 ± 0.026
HPMC 5 gel	4.08 ± 0.031

ACIN-loaded bilosomes: ACIN-loaded bilosome formulation (optimum formulation); Carbopol 2.5 and Carbopol 5: ACIN-loaded bilosomal gel formulations prepared using Carbopol (2.5 or 5%); HPMC 2.5 and HPMC 5: ACIN-loaded bilosomal gel formulations prepared using HPMC (2.5 or 5%)

Table 6.

Viscosity of bilosome dispersions and bilosome-loaded hydrogels after 1:1 (v/v) dilution (Mean±SD, n = 3)

Formulation	Polymer	Concentration (%)	Individual values (cP)	Viscosity (cP)
Blank bilosome dispersion	—	—	18.72, 18.88, 18.88	18.83 ± 0.09
ACIN bilosome dispersion	—	—	55.20, 54.88, 57.60	55.89 ± 1.49
ACIN bilosome-loaded hydrogel (1:1 v/v)	Carbopol	2.5%	9.12, 9.18, 9.68	9.33 ± 0.31
ACIN bilosome-loaded hydrogel (1:1 v/v)	Carbopol	5%	18.14, 18.89, 18.56	18.53 ± 0.38
ACIN bilosome-loaded hydrogel (1:1 v/v)	HPMC	2.5%	81.44, 80.00, 79.68	80.37 ± 0.91
ACIN bilosome-loaded hydrogel (1:1 v/v)	HPMC	5%	144.30, 144.80, 144.50	144.53 ± 0.25

EE% values for ACIN-loaded bilosomal gel formulations and in vitro release studies

Moreover, EE% values were determined for ACIN-loaded bilosomal formulations prepared using Carbopol or HPMC (2.5 or 5%). While the EE% values for ACIN-loaded bilosomal gel formulation prepared using Carbopol (2.5 or 5%) were 86 ± 0.12% and 86 ± 0.15 (n = 3), ACIN-loaded bilosomal gel formulation prepared using HPMC (2.5 or 5%) were determined as % 89 ± 0.13% and 88 ± 0.14 (n = 3), respectively.

In our study, the in vitro release studies were also performed for the optimum bilosome formulation and the bilosomal gel formulations prepared using HPMC (2.5 or 5%) in PBS-pH 7.4 and ethanol (70:30 v/v) mixture. The results of the release studies are presented in Fig. 13A and 13B.

Fig. 13.

The dissolution profile for pure ACIN (A) and the in vitro release profiles of the optimum bilosome formulation containing ACIN (A) and ACIN-loaded bilosomal gel formulations [HPMC 2.5 and HPMC 5: ACIN-loaded bilosomal gel formulations prepared using HPMC (2.5 or 5%)] (B)

The polymer concentration was varied between 2.5% and 5%, a range selected based on in vitro drug release profiles. Concentrations below 2.5% yielded gels with insufficient viscosity and rapid drug release, whereas concentrations above 5% resulted in highly viscous gels that could impede drug diffusion. This range was therefore chosen to achieve an optimal balance between sustained drug release and ease of application, ensuring both effective delivery and patient comfort.

In vitro kinetic modeling

In vitro release data were fitted to zero-order, first-order, Higuchi, and Korsmeyer–Peppas models in order to elucidate the ACIN release mechanism from the bilosomal formulations. The optimized ACIN-loaded bilosomes showed the best fit with the Korsmeyer–Peppas model (R² = 0.839), with an exponent n = 0.438, indicating a diffusion-dominated, anomalous transport process. The 2.5% HPMC bilosomal gel also followed the Korsmeyer–Peppas model (R² = 0.949, n = 0.352), consistent with Fickian diffusion through a hydrated polymer matrix. In contrast, the 5% HPMC bilosomal gel exhibited the highest correlation with the Higuchi model (R² = 0.984), demonstrating that drug release was mainly governed by matrix-controlled diffusion in the thicker gel network.

The results of cytotoxicity assay

Cell viability values obtained after 24 h exposure of cells (n = 6) to the test substance at specified concentrations (0.05, 0.1, 0.25, 0.5, 1, and 2 µg/mL) are presented in the Fig. 14. The percentage of viability of cells treated with the formulations was calculated by comparing it with that of control cells whose viability was taken as 100%.

Fig. 14.

The viability of cells treated withthe bilosomes and bilosomal HPMC 2.5 gel formulations (after 24 h incubation) (Control: Untreated cells; B-bilosome: Blank bilosomes; B-bilosomal HPMC: Blank bilosomal gel prepared using HPMC; ACIN-bilosomes: ACIN-loaded bilosomes; ACIN-bilosomal HPMC: ACIN-loaded bilosomal gel prepared using HPMC

The viability of cells treated with the above-mentioned formulations ranged from 87.76% to 106.9% (Fig. 14). The viability of cells was about 90% and above in all tested samples, indicating that both blank or ACIN-loaded bilosomes and bilosomal HPMC 2.5 gel formulations had no cytotoxic effect in the indicated concentration range. Although slight decreases in cell viability were observed at 2 µg/mL in ACIN-loaded bilosomal HPMC 2.5 gel group, the difference were not statistically significant (p > 0.05).

FTIR results

The FTIR spectrum of ACIN (ACIN, A) exhibited distinct absorption bands confirming the intact chemical structure of the drug. Characteristic C = O stretching of carboxylic and amide groups appeared at 1710–1680 cm⁻¹, aromatic C = C stretching was observed at 1600–1500 cm⁻¹, and C–O stretching vibrations were identified around 1250–1200 cm⁻¹. Lecithin (B) exhibited typical lipid-associated absorptions, including C–H stretching at 2920 and 2850 cm⁻¹, P = O asymmetric stretching at 1240–1250 cm⁻¹, and C–O–P vibrations between 1060 and 1080 cm⁻¹, representing its phospholipid backbone. Sodium taurocholate (C) showed strong S = O stretching of the sulfonate group in the 1210–1150 cm⁻¹ region, along with characteristic O–H/N–H stretching and C–O vibrations, consistent with its bile salt structure. Cholesterol (D) presented a broad O–H stretching band near 3400 cm⁻¹, C–H stretching at 2930–2860 cm⁻¹, and C–O stretching around 1050–1020 cm⁻¹, all of which correspond to its sterol ring and hydroxyl functionalities. The HPMC (E) spectrum displayed a broad O–H stretching band between 3300 and 3400 cm⁻¹, C–H stretching near 2920 cm⁻¹, and intense C–O–C stretching bands in the 1050–1150 cm⁻¹ range. The FTIR spectra of the blank bilosomes (F), ACIN bilosomes (G), and ACIN bilosomal HPMC gel (H) exhibited nearly identical profiles (Fig. 15).

Fig. 15.

FTIR spectra of (A) ACIN, B Lecithin, C Cholesterol, D Sodium taurocholate, E HPMC, F Blank bilosomes, G ACIN-loaded bilosomes, and H ACIN-loaded bilosomal HPMC gel

Stability testing results

No significant changes were observed in particle size, zeta potential, pH, or drug content of the ACIN-loaded bilosomes and HPMC gel during the one-month storage period at 4 °C, 25 °C, and 40 °C, confirming the physical and chemical stability of the formulations under the tested conditions.

Discussion

BBD was employed to optimize ACIN-loaded bilosomes by varying lecithin, sodium taurocholate, and cholesterol concentrations, and evaluating their effects on vesicle size, zeta potential, and EE%. Each of these responses is a critical determinant of bilosomal stability, performance, and transdermal drug delivery efficiency.

Vesicle size, a key determinant of bilosomal stability and skin permeation, showed a clear inverse relationship with lecithin concentration. This contrasts with conventional liposomes, where higher phospholipid levels typically yield larger vesicles. Similar findings were reported by Narayanan et al. [30], where high lecithin-to-bile salt ratios produced smaller vesicles. The observed reduction in size with increasing lecithin concentration can be explained by enhanced phospholipid packing and reduced interfacial free energy. At higher lecithin levels, phosphatidylcholine molecules align more tightly through van der Waals and hydrophobic interactions, leading to reduced curvature and the formation of smaller, compact vesicles. Cholesterol further stabilizes this structure by inserting between acyl chains, decreasing membrane fluidity and preventing aggregation. Meanwhile, sodium taurocholate introduces localized curvature through its surfactant-like properties.

Zeta potential determines electrostatic stabilization of bilosomal dispersions. The negative potential values arise from the anionic phosphate and bile salt groups on the vesicle surface. Increasing lecithin concentration partially shields these groups via zwitterionic choline headgroups, thus lowering the magnitude of surface charge. Cholesterol contributes to charge condensation by tightening lipid packing, while sodium taurocholate introduces additional negative charges via its sulfonate group, counterbalancing the reduction induced by lecithin. An optimized zeta potential near −30 mV provides a stable electrostatic barrier against vesicle coalescence.

EE% reflects the bilosomal capacity to retain the hydrophobic ACIN within the lipid matrix. Mechanistically, higher lecithin content increases the hydrophobic volume and reduces bilayer permeability, enhancing drug encapsulation. Cholesterol contributes to this by ordering the phospholipid tails, reducing leakage pathways and improving bilayer cohesion. Sodium taurocholate exhibits a dual effect: at low concentrations, it facilitates drug partitioning into the bilayer, whereas excessive amounts disrupt membrane integrity due to excessive fluidization.

Additionally, in this study, the gel formulations containing the optimum bilosomal formulation were prepared using Carbopol^® Ultrez^™ 10 or HPMC and evaluated in vitro. Firstly, the mechanical properties such as adhesiveness, cohesiveness, and hardness of the ACIN-loaded bilosomal gel formulations were evaluated using texture profile analysis. The absence of new peaks or notable shifts in the FTIR spectra indicates that no chemical interactions or degradation occurred during formulation. The observed minor peak attenuation and broadening of ACIN in the bilosome spectrum can be attributed to physical encapsulation, and drug incorporation and gel formulations did not induce any chemical interaction or structural modification. A limitation of the present study is the absence of solid-state characterization techniques such as differential scanning calorimetry (DSC) or X-ray diffraction (XRD). These analyses could provide additional insight into the physical state of ACIN and its interaction with the bilosomal and hydrogel matrices. Such investigations are planned as part of future studies to further elucidate the structural characteristics of the developed formulations. Hardness, defined as the ability of the gel formulation to be removed from the container, was determined in our study (Table 2). A low hardness value indicates easy removal and easy application of the formulation, while on the other hand, it indicates that the retention time at the application site may be shortened [31]. The molecular weight and concentration of the polymer have a significant effect on the hardness of the gel formulation. Sezer et al. reported that the hardness value ​​of hydrogel increased significantly (5 times) due to the increase in the concentration of chitosan (from 1.5% to 2%) [32]. In another study, the hardness values ​​of chitosan or polycarbophil gels increased four- and seven-fold, respectively, as the polymer concentration increased from 2% to 3% (for chitosan) and from 2% to 4% (for polycarbophil) [33]. In our study, an increase in the hardness value of ACIN-loaded bilosomal gel formulations was obtained with the increase in polymer concentration (especially for bilosomal Carbopol gel) (Table 2).

The higher cohesiveness, which is a significant parameter for determining the reconstruction ability of the gel after application, the better the structural recovery is generally observed. Thus, product performance at the administration site can be improved [31]. The increase in polymer concentration has not generally provided a significant increase in the cohesiveness of the bilosomal gel formulations (Table 2; p > 0.05).

For an effective treatment, gels should retain in the application area for the desired period of time. Therefore, the adhesiveness is another important parameter to be determined for gels. Sezer et al. reported that the concentration and molecular weight of the polymer used to prepare the gel affected the adhesiveness of the gel, and that adhesiveness increased as the polymer concentration and molecular weight increased [32]. In our study, there was an increase in the adhesiveness of ACIN-loaded bilosomal gel formulations with increasing polymer (Carbopol or HPMC) concentration (Table 2).

In addition, the pH values were determined the gel formulations. The pH of human skin, which is generally acidic, can vary greatly between 4.0 and 7.0 [34]. There is a general consensus that topical products should have an acidic pH and that their pH value should typically be in the range of 4–6 [35]. Therefore, the pH values of the bilosomal gel formulations prepared using HPMC were found to be more suitable for skin application (Table 5), and further studies were conducted on the bilosomal gel formulations prepared using HPMC. The viscosity of the bilosome formulations was strongly dependent on polymer type and concentration after 1:1 (v/v) dilution. ACIN loading increased the viscosity of the bilosome dispersion compared to the blank formulation, indicating enhanced internal structural organization. Among the hydrogels, Carbopol-based systems exhibited relatively low viscosity values, even at higher polymer concentrations, reflecting their dilution-sensitive rheological behavior. In contrast, HPMC-based hydrogels showed a marked, concentration-dependent increase in viscosity, suggesting the formation of a more cohesive polymeric network. These results emphasize that polymer selection is a key determinant of the rheological properties of bilosome-loaded hydrogels and is likely to influence their spreadability and dermal residence time.

Moreover, we assessed the dissolution of pure ACIN and the release of ACIN from the optimum ACIN-loaded bilosomal formulation or ACIN-loaded bilosomal gel formulations prepared using HPMC (2.5 or 5%) (Fig. 13A). Approximately 90% of pure ACIN dissolved within 3 h. However, approximately 15%, 32%, 46% and 55% of ACIN were released from the optimum ACIN-loaded bilosomal formulation in 0.5 h, 1 h, 3 h and 8 h, respectively (Fig. 13A). The bilosomal formulation exhibited biphasic release profile with initial burst release (15%, 0.5 h; due to the release of ACIN on the surface of vesicle) followed by sustained release (55%, 8 h; due to the release of ACIN encapsulated within vesicle) (Fig. 13A).

Ahmed ve ark. reported that bilosomal systems have biphasic release profile (initial burst release and later sustained release). They emphasized that lornoxicam on the surface of the bilosomal system is responsible for the initial burst phase, while the sustained phase is due to the high affinity of lornoxicam (lipophilic drug) to the bilosomal system [36]. Zafar et al. prepared luteolin-loaded bilosomal or pegylated bilosomal formulations and stated that both formulations exhibited biphasic release with initial fast release followed by sustained release, that the initial fast release could be due to the release of luteolin from the surface of the vesicle, and that the slow release of luteolin from the formulations was due to cholesterol reducing membrane fluidity [37].

In addition, about 29%, 42%, 64% and 77% of ACIN were released from the ACIN-loaded bilosomal gel formulation prepared using HPMC (2.5%) in 0.5 h, 1 h, 3 h and 8 h, respectively (Fig. 13B).

The high swellability of HPMC when in contact with water or biological fluid provides a faster drug release [38]. However, approximately 13%, 20%, 40% and 58% of ACIN were released from the ACIN-loaded bilosomal gel formulation prepared using HPMC (5 g) in 0.5 h, 1 h, 3 h and 8 h, respectively (Fig. 13B). When the amount of HPMC was increased in the formulation, the release of ACIN from the bilosomal gel was slowed.

Pan et al. prepared HPMC hydrogels for topical application and reported that when the HPMC concentration was decreased from 13% to 12%, the drug release increased due to the decreased viscosity [14].

Moreover, MTT assay method was used to estimate the cell viability. In our study, the viability of cells was about 90% and above both optimum bilosome and bilosomal HPMC 2.5 gel formulations. Therefore, both prepared formulations are biocompatible.

Watroba et al. reported that according to ISO 10,993, materials that provide over 70% cell viability are biocompatible, while those that reduce viability greater than 30% are cytotoxic [39].

Conclusion

In this study, a BBD was used to prepare and optimize ACIN-loaded bilosome formulations. The optimized ACIN-loaded bilosome formulation had nano-size (137.3 nm), PDI of 0.384, EE% above 80% and negative zeta potential value (−30.1 mV; this value is sufficient for the physical stability of the colloidal dispersions). ACIN-loaded bilosomal gel formulations were also prepared using Carbopol or HPMC. Since the pH values (about 4) of ACIN-loaded bilosomal gel formulations prepared using HPMC (2.5 or 5%) were suitable for topical application, they were selected for further studies. The optimum ACIN-loaded bilosomes and ACIN-loaded bilosomal gel prepared using HPMC were able to sustain the ACIN release over eight hours. For cytotoxicity assay, only ACIN-loaded bilosomal gel prepared using HPMC (2.5%) was evaluated in L929 cell line. This formulation did not cause a significant decrease in cell viability at different concentrations and was considered to be biocompatible.

Abbreviations

ACIN

Acemetacin

ANOVA

Analysis of variance

ATR

Attenuated total reflectance

BBD

Box–Behnken design

CMAs

Critical material attributes

CPPs

Critical process parameters

CQAs

Critical quality attributes

COX-2

Cyclooxygenase-2

DLS

Dynamic light scattering

DMSO

Dimethyl sulfoxide

DMEM

Dulbecco’s Modified Eagle Medium

DSC

Differential scanning calorimetry

EE%

Entrapment efficiency percentage

FBS

Fetal bovine serum

FTIR

Fourier transform infrared spectroscopy

HPLC

High-performance liquid chromatography

HPMC

Hydroxypropyl methylcellulose

LOD

Limit of detection

LOQ

Limit of quantification

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

PDI

Polydispersity index

TEM

Transmission electron microscopy

XRD

X-ray diffraction

Author contributions

The authors confirm their contributions to the paper as follows: study conception and design by ED, MC and MSK; data collection, analysis and interpretation of the results by ED, TÇ, ABÖ and LB; manuscript drafting, revision, and/or correction by ED, MC and MSK; literature survey by ED, TÇ, ABÖ, LB, MC and MSK. All authors reviewed the results and approved the final version of the manuscript.

Funding

The authors declare that no funds, grants, or other financial support were received during the preparation of this manuscript.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to institutional policy restrictions but are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study did not involve any experiments on human or animal subjects; therefore, ethical approval from an institutional review board or animal ethics committee was not required.

Consent for publication

Not applicable. This manuscript does not contain any individual person’s data in any form (including images, videos, or personal details). Therefore, consent for publication is not required.

Competing interests

The authors declare no competing interests.