Development of Brimonidine-Loaded Ethosomes for Glaucoma: Investigation of Intraocular Pressure-Lowering Potential In Vivo

Abstract

Background/Objectives: Brimonidine tartrate (BRT), a selective α2-adrenergic receptor agonist, is commonly used in the treatment of glaucoma. However, conventional eye drop formulations suffer from poor ocular bioavailability and rapid elimination. This study aimed to develop and evaluate BRT-loaded ethosomes as a nanocarrier-based alternative to enhance intraocular delivery and therapeutic efficacy. Methods: Ethosomes were prepared using the thin-film hydration method and optimized via central composite design. The optimized formulation was subjected to physicochemical characterization, in vitro release testing, and ocular irritation assessment using the Hen egg test—chorioallantoic membrane (HET-CAM) model. Additionally, the intraocular pressure (IOP)-lowering efficacy of the formulation was evaluated in a rat glaucoma model. Results: The optimized ethosomal formulation exhibited favorable physicochemical properties, including a mean particle size of 122.6 ± 0.7 nm, zeta potential of −1.8 ± 0.9 mV, and encapsulation efficiency of 87.33 ± 0.04%. In vitro release data followed Higuchi kinetics. HET-CAM analysis indicated non-irritancy. In vivo, the ethosomal BRT formulation achieved comparable IOP-lowering effects to the marketed eye drops at one-third of the dose. Conclusions: The developed BRT-loaded ethosomal system demonstrated promising physicochemical stability, sustained release, and therapeutic potential. These findings suggest that ethosomes may offer a safe and effective strategy for enhancing the ocular delivery of BRT in glaucoma therapy.

1. Introduction

Glaucoma is a chronic, progressive optic neuropathy characterized by retinal ganglion cell (RGC) death and excavation of the optic nerve head, leading to irreversible visual field loss [1,2]. The only clinically validated modifiable risk factor for the progression of glaucoma is elevated intraocular pressure (IOP), and as such, IOP-lowering therapies remain the cornerstone of glaucoma management [3].

Among the available pharmacological treatments, brimonidine tartrate (BRT), a selective α2-adrenergic receptor agonist, is widely used because it lowers IOP by both decreasing aqueous humor production and increasing uveoscleral outflow [3,4]. Despite its efficacy, Conventional eye drop formulations of BRT face substantial challenges, including rapid precorneal elimination, poor corneal permeability, and low ocular bioavailability (<5%) due to tear film dilution, blinking, and nasolacrimal drainage [5]. These barriers necessitate frequent dosing, which compromises patient compliance and therapeutic outcomes.

To overcome such limitations, nanotechnology-based ocular drug delivery systems have emerged as promising platforms to improve drug residence time, permeation, and targeted delivery to intraocular tissues [6,7]. In this context, vesicular systems such as liposomes, niosomes, transfersomes, and ethosomes have shown significant potential for ophthalmic drug delivery, especially in the treatment of chronic diseases like glaucoma [8,9,10].

Liposomes, composed of biocompatible phospholipid bilayers, can encapsulate both hydrophilic and lipophilic agents and provide sustained drug release; however, their instability and limited penetration through corneal barriers have restricted their clinical utility [9]. Niosomes, formed by non-ionic surfactants and cholesterol, have shown improved chemical stability, affordability, and encapsulation efficiency, making them suitable alternatives to liposomes [9]. Transfersomes are ultra-deformable lipid vesicles incorporating edge activators (e.g., sodium cholate or Tween 80) that allow them to traverse the tight junctions of the corneal epithelium, enhancing transcorneal permeation [9].

Among these systems, ethosomes, which are phospholipid vesicles containing ethanol, are particularly notable for their high deformability and ability to alter lipid structures in biological membranes, thus enhancing corneal and scleral penetration [9,10]. The ethanol content (20–45%) in ethosomes improves drug solubility and enhances membrane fluidity, thereby increasing intraocular drug delivery. Prior research has shown that ethosomal formulations of ophthalmic drugs such as timolol, ketoconazole, and Amphotericin B result in improved therapeutic efficacy and prolonged action compared to their conventional counterparts [10,11,12].

Despite the promise of vesicular systems, BRT-loaded ethosomal formulations have been relatively underexplored in the literature, representing a potential opportunity for innovation. Given BRT’s short half-life and the need for frequent administration, incorporating it into an ethosomal system may enhance ocular retention, bioavailability, and therapeutic efficacy.

In the present study, BRT-loaded ethosomes were formulated and characterized to address the limitations of conventional ocular formulations. Soybean phosphatidylcholine (SPC), previously demonstrated biocompatibility and membrane-forming ability in vesicular delivery systems, has primarily been studied in dermal and transdermal applications, with data on its ocular applications being quite limited [9,13]. In this study, SPC was used for the first time to produce BRT-loaded ocular ethosomes. Their IOP-lowering efficacy was evaluated in vivo using a rat glaucoma model and compared with that of a commercially available BRT eye drop. It is hypothesized that the ethosomal nanocarrier system may provide enhanced therapeutic efficacy and improved patient adherence by prolonging ocular residence time and increasing bioavailability.

2. Materials and Methods

2.1. Materials

Brimonidine tartrate (BRT) was generously supplied by World Medicine (Istanbul, Turkey). α-Phosphatidylcholine from soybean (SPC) and cholesterol were obtained from Sigma-Aldrich (Munich, Germany). Sodium dihydrogen phosphate and disodium hydrogen phosphate, employed in the preparation of phosphate buffer solutions, were sourced from IsoLab (Berlin, Germany). Ethanol (EtOH, HPLC grade), acetonitrile (ACN, HPLC grade), and trifluoroacetic acid (HPLC grade) were purchased from VWR Chemicals (Vienna, Austria). Ultrapure water was prepared using a Milli-Q purification system equipped with a 0.22 μm membrane filter (Millipore, Bedford, MA, USA). Unless otherwise specified, all other chemicals and reagents were of analytical grade and used without further purification.

2.2. Methods

2.2.1. Preparation of Formulations

Ethosomal formulations were prepared by adapting previously reported methods with minor modifications [10,14]. Briefly, predetermined amounts of BRT, cholesterol, and soybean phosphatidylcholine (SPC) were accurately weighed based on the experimental design and transferred into a round-bottom flask. Cholesterol was included at a low fixed concentration (0.1%, w/v) in order to support membrane integrity while preserving the high bilayer fluidity characteristic of ethosomal systems, while BRT was maintained at a constant concentration of 0.2% (w/v) in all formulations. Then, 10 mL of chloroform was added to the mixture. The components were dissolved by sonication in an ultrasonic bath for 2 min. The organic solvent was subsequently removed under reduced pressure using a rotary evaporator at 40 °C for 1 h, yielding a thin lipid film. This film was hydrated with 10 mL of phosphate-buffered saline (PBS, pH 6.8) containing ethanol at varying concentrations. The dispersion was sonicated in an ultrasonic bath for 15 min to ensure complete hydration. Finally, the obtained ethosomal dispersions were passed 11 times through a mini extruder (Avanti Polar Lipids, Alabaster, AL, USA) equipped with a 0.20 µm membrane filter (Whatman, Kent, UK) to reduce particle size and achieve homogeneity.

2.2.2. Experimental Design

The formulation of BRT-loaded ethosomes was optimized using response surface methodology. Guided by preliminary experiments and literature evidence, the concentrations of SPC and EtOH were selected key formulation variables due to their substantial influence on vesicle characteristics [15,16]. A central composite design (CCD) with five coded levels (−α, −1, 0, +1, +α) was employed to investigate the effects of these independent variables. An α value of 1.44 was adopted to ensure rotatability and maintain model robustness. In total, 13 experimental runs were generated, comprising four factorial points, four axial points, and five replicates at the center point to estimate the experimental error and verify reproducibility (details are provided in

Table 1. Chosen parameters in CCD.

Variables		Level of Variables
		−1.41	−1	0	1	1.41
A	SPC (% w/v)	1.59	2	3	4	4.41
B	EtOH (% w/v)	7.93	10	15	20	22.07

). Encapsulation efficiency and particle size were designated as the primary response variables.

Design optimization and statistical analyses were performed using Design-Expert software (version 13.0.5.0). The experimental data was modeled using a nonlinear quadratic regression equation of the form:

Y: β₀ + β₁A + β₂B + β₁₂AB + β₁₁A² + β₂₂B²

where Y is the predicted response, A and B represent the independent variables, AB denotes their interaction term, and A² and B² account for the quadratic contributions. Here, β₀ is the intercept, while β₁ and β₂ are linear coefficients, β₁₂ is the interaction coefficient, β₁₁ and β₂₂ are the quadratic coefficients. The significance of the model and individual terms was assessed using analysis of variance (ANOVA), adopting a statistical significance level of p < 0.05. The adequacy of the model was evaluated by examining the adjusted and predicted coefficients of determination. Three-dimensional response surface plots were generated to provide a graphical interpretation of the influence and interactions of the independent variables on critical quality attributes.

An optimized ethosomal formulation was identified by targeting the highest encapsulation efficiency alongside the lowest particle size. The predicted optimal conditions were subsequently verified through triplicate validation experiments. The experimental results were then compared with the predicted values to confirm the accuracy of the optimization model.

2.2.3. Determination of the Particle Size, Polydispersity, and Zeta Potential

The particle size and polydispersity index (PDI) of the BRT-loaded ethosomal formulations were analyzed using a Zetasizer Nano ZSP (Malvern Instruments Ltd., Malvern, UK) equipped with dynamic light scattering technology, operated at 25 °C and a fixed scattering angle of 173°. Zeta potential measurements were conducted on the same instrument by the electrophoretic light scattering technique to evaluate the surface charge and electrokinetic stability of the vesicles. Prior to analyses, the samples were diluted 1:100 with ultrapure water to minimize multiple scattering and maintain appropriate count rates. All measurements were performed in triplicate, and the results are presented as mean ± standard deviation. This characterization strategy enabled a comprehensive assessment of vesicle size distribution, dispersion uniformity, and colloidal stability, which are critical attributes for ophthalmic delivery systems.

2.2.4. Morphological Characterization (FE-SEM/STEM Analysis)

Surface morphology and structural characterization of the optimized ethosomal formulation was examined using a field-emission scanning electron microscope (FEI Quattro S SEM, Thermo Fisher Scientific, Waltham, MA, USA) operated in FE-SEM/STEM mode. Imaging conditions were selected to allow visualization of nanoscale vesicular structures while minimizing drying-related artifacts, in line with WetSTEM-based imaging principles for soft and suspension-based samples.

2.2.5. Raman Spectroscopy Analysis

Raman spectroscopic measurements were performed to evaluate the molecular integrity of BRT and its encapsulation within ethosomal formulations. Spectra were obtained using a Cora 5001 Raman spectrometer (Anton Paar GmbH, Graz, Austria) equipped with a 785 nm laser source. Both spectra were collected in the range of 200–2200 cm⁻¹. Data processing included automatic background subtraction and baseline correction using the instrument’s software.

2.2.6. Analytical Quantification of BRT

Reverse Phase High performance liquid chromatography (RP-HPLC) (Agilent 1260, Santa Clara, CA, USA) was employed for the quantitative determination of BRT. An InertSustain C18 column (5 μm, 4.6 mm I.D. × 150 mm; GL Sciences, Tokyo, Japan) served as the stationary phase, with isocratic elution using methanol and 0.01 M ammonium acetate buffer (20:80, v/v) as the mobile phase. Detection was carried out at 260 nm using a UV—visible detector (Agilent 1260, Infinity II Diode Array Detector WR, Santa Clara, CA, USA). The flow rate was maintained at 1.3 mL/min, and the injection volume was set to 10 μL. The calibration curve, constructed from standard solutions ranging between 0.1 and 100 μg/mL, yielded the equation y = 16,645x + 5044, with a coefficient of determination (R²) of 0.9996. Method validation for the assay was performed in accordance with the ICH Q2 (R2) guideline [17].

2.2.7. Determination of the Encapsulation Efficiency

The encapsulation efficiency of ethosomal formulations was determined using a modified centrifugation technique, adapted for ethosomal vesicles [18]. Briefly, the freshly prepared ethosomal dispersion was subjected to centrifugation at 15,000 rpm for 30 min to effectively separate the free (unentrapped) drug from the encapsulated drug within the ethosomes. Following centrifugation, the supernatant, containing the free drug and ethanol-rich continuous phase, was carefully withdrawn, diluted appropriately with acetonitrile, and subsequently analyzed using a previously validated RP-HPLC method.

The resulting pellet, consisting of BRT-loaded ethosomal vesicles, was gently resuspended in phosphate-buffered saline (PBS, pH 7.4) for subsequent physicochemical characterization.

Encapsulation efficiency (EE%) was calculated according to the following equation:

$$\mathrm{E}\mathrm{E}\mathrm{\%}={\displaystyle \frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}-\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{u}\mathrm{n}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}}\times 100$$

(1)

2.2.8. In Vitro Release Study

In vitro release experiments were conducted using freshly prepared optimized ethosomal dispersions after separation and removal of non-encapsulated (free) BRT. Accordingly, the release medium and experimental conditions were selected to maintain sink conditions with respect to the encapsulated drug fraction throughout the release study. The in vitro release profile of BRT was evaluated using the dialysis bag diffusion method in phosphate-buffered saline (PBS, pH 7.4) containing 5% Tween 80. Prior to the experiment, cellulose dialysis membranes with a molecular weight cut-off of 12,000–14,000 Da were soaked in the receptor medium for 12 h to ensure full hydration. A 1 mL of the formulation suspension was carefully loaded into each membrane and securely sealed with weighted dialysis clips. The dialysis bags were immersed in 50 mL of pre-equilibrated receptor medium maintained at 35.5 °C in 100 mL beakers, and the beakers were placed in a shaking incubator set at 100 rpm. At specified time intervals (0, 0.25, 0.5, 1, 2, 3, 4, 8 and 12 h), 0.5 mL samples were withdrawn from the receptor compartment and immediately replaced with an equal volume of fresh medium at the same temperature to preserve sink conditions. Each collected sample was immediately mixed with 0.5 mL of methanol:0.01 M ammonium acetate:acetonitrile (1:4, v/v) to perform a dilution step. The resulting solutions were filtered through a 0.45 µm membrane filter and transferred to HPLC vials for analysis. Quantification of BRT was performed using a previously validated RP-HPLC method. All experiments were conducted in triplicate (n = 3) to ensure reproducibility and statistical validity of the release data.

To elucidate the mechanism governing BRT release from the optimized ethosomal formulation, the in vitro release data were fitted to various kinetic models. The cumulative percentage of drug released at predetermined time intervals was calculated and evaluated using mathematical models, including zero-order, first-order, Higuchi, and Korsmeyer–Peppas models. The corresponding linear equations applied were as follows [19,20,21,22]:

Zero-order: C = k₀t + C₀

(2)

First-order: In C = In C₀ +k₁t

(3)

Higuchi model: C = k₂t½

(4)

Hixson–Crowell: W₀^1/3 − W_t^1/3 = k_H

(5)

In these models, C represents the cumulative amount of drug released at time t, while C₀ denotes the initial drug concentration. The constants k₀, k₁, and k₂ correspond to the release rate constants of the zero-order, first-order, and Higuchi models, respectively. Additionally, W₀ refers to the initial amount of drug present in the formulation, and Wₜ is the amount of drug remaining within the vesicles at time t. The term k_H incorporates factors related to the surface area and volume of the diffusion matrix.

The most appropriate release kinetic model was determined based on the highest correlation coefficient, ensuring accurate interpretation of the drug release mechanism from the formulations.

2.2.9. Evaluation of Ocular Compatibility (HET-CAM Study)

Hen egg test—chorioallantoic membrane (HET-CAM) test was performed to evaluate the irritation potential of ophthalmic formulations. The complete optimized ethosomal formulation was evaluated without separation of the unentrapped fraction for the HET-CAM assay. This approach was intentionally selected to assess the overall irritation potential and biocompatibility of the final formulation as intended for ocular administration, independent of the physicochemical characterization and in vitro release studies.

Fertilized hen eggs were incubated in an upside position for 9 days. The temperature and humidity were adjusted to 38.3 ± 0.2 °C and 58 ± 2% RH, respectively. After incubation (10th day), light control was performed to detect the presence of embryo in embryonated eggs, and a 1–2 cm diameter window was opened in the shell with the help of a circular saw (Dremel^® Flex Shaft, Racine, WI, USA). Embryo eggs were divided into groups as negative control (0.9% NaCl), positive control (0.1 N NaOH) and BRT-ET formulations and applied as 0.3 mL. Vascularization was evaluated for 5 min to determine the effect of the formulations (n = 3). The effect of the substance was determined and scoring was done. Irritation scores (IS) were determined by the following formula according to the time (in seconds) based on points of hemorrhage (H), lysis (L) or coagulation (C) onset (Equation (6)):

$$\mathrm{IS} = \left[\left({\displaystyle \frac{301-H}{300}}\right)\times 5+\left({\displaystyle \frac{301-L}{300}}\right)\times 7+\left({\displaystyle \frac{301-C}{300}}\right)\times 9\right]$$

(6)

Regarding IS values, formulations can be classified as nonirritating (0–0.9), weakly irritating (1–4.9), moderately irritating (5–8.9) or severely irritating (9–21).

2.2.10. In Vivo Studies

The in vivo experiments were performed on male Wistar albino rats (weighing approximately 290 ± 10 g) obtained from the Experimental Animal Research Center (DEHAM) of Acibadem University. The animals were housed under controlled environmental conditions with free access to standard food and water. All experimental procedures were conducted in compliance with the principles of the Declaration of Helsinki and were approved by the Animal Experiments Ethics Committee of Acibadem Mehmet Ali Aydınlar University (Approval No. 2024/22, dated 3 July 2024).

In compliance with the 3R principle (Replacement, Reduction, and Refinement), a power analysis was conducted to determine the minimum required sample size. Assuming a one-way ANOVA design with 80% statistical power, a significance level of 0.05, and an anticipated size of 0.80, a total of 20 rats were required, with five animals assigned to each of the four groups:

Group 1 (C, n = 5): Control group; no intervention.

Group 2 (G, n = 5): Glaucoma model; no treatment.

Group 3 (G + TI, n = 5): Glaucoma model treated with standard BRT (0.2%) ophthalmic drops.

Group 4 (G + DL, n = 5): Glaucoma model treated with ethosomal formulation containing 0.2% BRT.

This grouping design enables direct comparison between conventional BRT treatment and the ethosomal delivery system, providing insights into their relative efficacy in glaucoma management.

The experimental glaucoma model was established using the episcleral vein cauterization (EVC) technique, as previously described in the literature [10]. Before the surgical intervention, rats were anesthetized via intramuscular injection of ketamine (30 mg/kg) and xylazine (20 mg/kg). After proper ocular sterilization, a 4-0 silk suture was applied to retract the eyelid, followed by a conjunctival peritomy to expose the episcleral veins. Two of the four episcleral veins in each eye were carefully cauterized under a surgical microscope using a pencil-tip cautery device. The conjunctiva was then closed with 8-0 vicryl sutures, and topical antibiotic and anti-inflammatory eye drops (moxifloxacin 0.5% and dexamethasone sodium phosphate 0.1%) were administered to prevent postoperative infection and inflammation. When necessary, topical anesthesia (Alcaine^®, proparacaine hydrochloride 0.5%) was applied to eliminate corneal reflexes during manipulation. Elevation of IOP to approximately 25–30 mmHg following episcleral vein cauterization verified the successful development of the experimental glaucoma model.

A commercially available ophthalmic solution containing BRT 0.2% (w/v) was administered topically three times daily (TID) in Group 3 (G + TI), following the dosing frequency commonly adopted in clinical practice. This regimen aligns with published clinical studies demonstrating the suitability of the TID schedule for maintaining consistent IOP reduction throughout the day [23,24,25,26,27]. Animals in Group 4 were treated with the optimized ethosomal formulation containing 0.2% (2 mg/mL) BRT, administered as the complete final formulation without separation of the unentrapped fraction, to ensure direct dose equivalence with the marketed BRT ophthalmic solution. The ethosomal formulation was administered topically as a single drop once daily in Group 4 (G + DL). This dosing schedule was designed to evaluate whether the administration frequency could be reduced without compromising (IOP) control, owing to the sustained-release and enhanced ocular retention characteristics of the ethosomal delivery system. Previous experimental evidence has indicated that such nanovesicular formulations can maintain IOP reduction comparable to conventional ophthalmic solutions applied three times daily, supporting the feasibility of a once-daily therapeutic regimen [10].

Topical administration was performed unilaterally to the same eye (right eye) in all treated animals, while the contralateral eye was left untreated to serve as an internal control. This experimental design is widely adopted in vivo glaucoma studies to minimize inter-animal variability and potential contralateral or systemic effects following topical ocular administration [28,29]

Group 3 (G + TI) received a commercially available BRT 0.2% (w/v) ophthalmic solution, administered topically three times daily (TID) at 08:00, 16:00, and 24:00 (±15 min), whereas Group 4 (G + DL) was treated with the ethosomal BRT 0.2% formulation, administered once daily (QD) at 08:00 (±15 min). A single drop (~50 µL) was instilled per dose. IOP was measured twice daily at 10:00 AM and 6:00 PM for 30 consecutive days using a digital handheld tonometer (Diaton^®, Bicom Inc., Long Beach, NY, USA), which was calibrated prior to each measurement session. IOP was measured twice daily to account for potential diurnal variations in IOP. Preliminary analysis indicated no significant differences between the two measurement time points; therefore, the mean of the two daily measurements was used for data analysis and presentation in the Results section. Before measurement, one drop of 0.5% (w/v) benoxinate hydrochloride ophthalmic solution (Alcaine^®, Alcon, Fort Worth, TX, USA) was applied to eliminate the corneal reflex. For each measurement, three consecutive readings with a standard error below 10% were obtained, and their mean value was recorded as the representative IOP. The percentage reduction in %IOP was calculated at each time point using the following equation:

$$\mathrm{\%}IOP reduction={\displaystyle \frac{(IO{P}_{baseline}-IO{P}_t)}{IO{P}_{baseline}}}\times 100$$

(7)

where $IO{P}_{baseline}$ represents the mean IOP recorded immediately before treatment initiation, and $IO{P}_t$ corresponds to the IOP value at each measurement time. The percentage decrease was used to compare the efficacy of the ethosomal formulation (QD) with the conventional market product (TID) across the 30-day experimental period, as previously described in the literature [30]. All measurements were performed by a single examiner blinded to group allocation. Following the final measurements, animals were euthanized under deep isoflurane anesthesia according to the approved ethical protocol.

2.2.11. Stability Studies

The physical stability of the optimized BRT loaded ethosomal formulation was evaluated during storage. Aliquots of the freshly prepared dispersion were transferred into amber glass vials, sealed, and stored at 4 °C and 25 °C. Samples were analyzed for particle size, polydispersity index, zeta potential and encapsulation efficiency to assess possible changes in vesicle characteristics. All measurements were performed in triplicate, and the results were expressed as mean ± standard deviation.

2.2.12. Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation. Normality of the datasets was evaluated using the Shapiro–Wilk test. When comparisons involved the four independent experimental groups, one-way ANOVA was applied if parametric assumptions were satisfied, followed by Tukey’s multiple comparisons test. When either normality or variance assumptions were not met, the Brown–Forsythe and Welch ANOVA tests were used, followed by the Games–Howell multiple comparisons test. Pairwise comparison between the two treatment groups in the in vivo IOP-lowering study (BRT-OD vs. BRT-ET) was performed using an unpaired two-tailed t-test when normality was confirmed. In cases where normality was not satisfied, the Mann–Whitney U test was employed. Statistical significance was defined as p < 0.05.

3. Results and Discussion

3.1. Experimental Design of Ethosomes

Although the formulation design involved two primary variables, a central composite design (CCD) was deliberately selected to enable a more comprehensive and mechanistic evaluation of the formulation space. CCD allows not only the assessment of linear effects but also the identification of quadratic effects and potential curvature in the response surface, which are particularly relevant for complex vesicular systems such as ethosomes. In lipid-based nanocarrier formulations, formulation responses often exhibit non-linear behavior due to coupled physicochemical interactions between formulation components.

Unlike a simple two-level factorial design, which is primarily suited for preliminary factor screening, CCD facilitates true formulation optimization by providing sufficient information to model response surface topology and to identify optimal operating regions within the design space. This approach enables a more robust and predictive formulation strategy, ensuring that the selected formulation represents a genuine optimum rather than a local or comparative outcome.

3.1.1. Impact of Independent Variables on Encapsulation Efficiency

The encapsulation efficiency of the BRT-loaded ethosomal dispersions ranged from 75.6% to 91.32%, as presented in

Table 2. CCD matrix and obtained responses for ethosome.

	Variables		Responses
Run	A: SPC (% w/v)	B: EtOH (% w/v)	Particle Size (nm)	EE (%)
1	1.59	15	152.5	85.6
2	3	15	195.4	88.02
3	3	15	169.4	89.77
4	2	20	121.8	86.14
5	3	15	141.2	91.27
6	4	10	163.8	84.45
7	4	20	120.7	75.6
8	2	10	179.6	90.74
9	4.41	15	176.2	79.8
10	3	22.07	88.6	81.15
11	3	15	162.2	90.2
12	3	7.93	168.4	91.32
13	3	15	185.4	90.8

. Achieving high encapsulation efficiency is essential for ophthalmic delivery systems, as limited drug loading may compromise therapeutic efficacy due to the restricted instillation volumes permitted for ocular administration. To mathematically interpret these outcomes, the experimental data were fitted to a quadratic regression model described as follows:

R1: 90.01 − 3.13A − 3.48B − 1.6AB − 3.71A² − 1.95B²

where R1 represents the encapsulation efficiency, and A and B denote the coded levels of SPC and EtOH concentrations, respectively.

The ANOVA results presented in

Table 3. ANOVA results for Encapsulation Efficiency.

	Sum of		Mean	F	p-Value
Source	Squares	df	Square	Value	Prob > F
Model	290.81	5	58.16	25.79	0.0002	significant
A-SPC (%)	78.33	1	78.33	34.73	0.0006
B-EtOH (%)	96.83	1	96.83	42.93	0.0003
AB	4.52	1	4.52	2.00	0.2000
A2	96.00	1	96.00	42.56	0.0003
B2	26.38	1	26.38	11.69	0.0111
Residual	15.79	7	2.26
Lack of Fit	9.52	3	3.17	2.03	0.2527	not significant
Pure Error	6.27	4	1.57
Cor Total	306.60	12

confirmed that the fitted quadratic model was statistically significant (F = 25.79, p = 0.0002), indicating its strong predictive capability for encapsulation efficiency within the investigated formulation domain. Both SPC concentration (p = 0.0006) and EtOH concentration (p = 0.0003) exerted highly significant influences on encapsulation efficiency, while their interaction term was not significant (p = 0.2000). The lack-of-fit test (p = 0.2527) was not significant, demonstrating adequate model fit.

The three-dimensional response surface plot in Figure 1A provides a clear visualization of the effects of formulation variables on encapsulation efficiency. As SPC concentration increased, the encapsulation efficiency initially showed a slight improvement, which could be attributed to the formation of a more cohesive and stable phospholipid bilayer, facilitating better accommodation of the lipophilic BRT molecules within the ethosomal vesicles. However, at higher SPC levels, encapsulation efficiency declined. This reduction could be associated with increased viscosity and rigidity of the vesicle system, potentially hindering vesicle formation and promoting drug leakage due to impaired bilayer flexibility [31].

Similarly, EtOH concentration exhibited a negative impact on encapsulation efficiency within the tested range. Although ethosomal systems typically rely on relatively high EtOH content (20–45% w/v) to enhance membrane fluidity and promote improved ocular permeability, excessive ethanol can disrupt phospholipid packing and destabilize vesicular structures [32]. At such high EtOH levels, the phospholipid packing may become excessively loose, reducing the vesicle’s capacity to retain the drug effectively and promoting drug leakage through the highly fluidized bilayer structure [33]. Therefore, while the presence of EtOH is essential for ethosome formation and their characteristic deformability, surpassing the optimal threshold compromises vesicle integrity and adversely affects encapsulation efficiency.

3.1.2. Impact of Independent Variables on Particle Size

The particle size of the BRT-loaded ethosomal formulations ranged from 88.6 to 195.4 nm, as shown in Table 2. This particle size distribution is considered suitable for ophthalmic drug delivery systems, as nanosized carriers can enhance precorneal retention, improve corneal permeability, and reduce irritation compared to larger particles. To describe these outcomes, a quadratic polynomial model was established as follows:

R2: 170.72 + 2.08 A − 26.72 B + 3.68AB − 3.17A² − 21.10B²

where R2 represents the particle size, and A and B denote the coded levels of SPC and EtOH concentrations, respectively. ANOVA results (

Table 4. ANOVA results for Particle Size.

	Sum of		Mean	F	p-Value
Source	Squares	df	Square	Value	Prob > F
Model	8897.53	5	1779.51	5.91	0.0187	significant
A-SPC (%)	34.52	1	34.52	0.1147	0.7448
B-EtOH (%)	5711.36	1	5711.36	18.98	0.0033
AB	54.02	1	54.02	0.1796	0.6845
A2	70.02	1	70.02	0.2327	0.6442
B2	3096.38	1	3096.38	10.29	0.0149
Residual	2105.97	7	300.85
Lack of Fit	335.60	3	111.87	0.2528	0.8562	not significant
Pure Error	1770.37	4	442.59
Cor Total	11,003.50	12

) demonstrated that the model was statistically significant (F = 5.91, p = 0.0187), confirming its suitability for predicting particle size within the studied design space. Among the factors studied, EtOH concentration showed a highly significant effect on particle size (p = 0.0033), while SPC concentration (p = 0.7448) was not significant. The lack-of-fit test was not significant (p = 0.8562), suggesting a good fit of the model to the data.

To further elucidate the influence of formulation variables on particle size, a three-dimensional response surface plot was constructed (Figure 1B). As illustrated, increasing EtOH concentration led to a marked reduction in vesicle size at the lower end of the experimental range. This behavior can be attributed to the ability of EtOH to lower interfacial tension and enhance phospholipid bilayer fluidity, thereby facilitating the formation of smaller and more deformable vesicles [34].

3.1.3. Optimization Study

Optimization of the BRT-loaded ethosomal formulation was conducted using Design-Expert software (v13.0.5.0), based on the experimental outcomes obtained from the central composite design. The optimization criteria were established by prioritizing maximum encapsulation efficiency while simultaneously achieving minimum particle size, which is of particular relevance for ophthalmic administration to enhance both drug retention and corneal penetration. The optimization algorithm identified the optimal formulation conditions as 2.45% SPC and 20% EtOH, representing the most favorable balance between vesicle stability and nano-scale size distribution for ophthalmic delivery.

The optimized formulation was subsequently prepared to validate the predictive performance of the optimization model under controlled laboratory conditions. As presented in

Table 5. Predicted and experimental responses of the optimized ethosome.

Responses	Predicted Value	Experimental Value	Prediction Error (%)
EE (%)	85.77	87.33 ± 0.04	1.82
Particle Size (nm)	120.55	122.6 ± 0.7	1.70

, the experimentally obtained encapsulation efficiency was 87.33 ± 0.04%, while the measured particle size was 122.6 ± 0.7 nm, both of which were in close agreement with the model-predicted values. This strong correlation confirms the reliability and robustness of the optimization approach and supports its applicability for rational ethosomal formulation development.

Further characterization of the optimized ethosomal dispersion was conducted to assess its physicochemical properties. The polydispersity index (PDI) of the optimized formulation was determined to be 0.15, indicating a narrow and homogeneous particle size distribution. A narrow distribution reduces the tendency for particle interactions, thereby minimizing the risk of aggregation during storage and ocular application.

Corneal epithelium, due to its tight junctions, restricts the passage of nanocarriers larger than a few nanometers [35]. Considering the particle size of optimized ethosomes, it is clear that they cannot pass through these tight junctions, which are characterized by narrow paracellular pathways and highly organized lipid domains. However, it is important to note that ethosomes are not intended to reach posterior tissues by passing through ocular pores like other transport systems. In ethosomal systems, the lipid bilayer is expected to penetrate throughout the entire thickness of the corneal epithelium, rather than passing through pores [36].

The zeta potential of the optimized formulation was −1.8 ± 0.9 mV. Although the absolute surface charge is relatively low, the combination of phospholipid-based steric stabilization and the narrow polydispersity index support acceptable colloidal stability of the vesicles for ophthalmic delivery. This low negative charge is attributed to the nonionic nature of SPC, which stabilizes vesicles predominantly through steric rather than electrostatic mechanisms.

In ethosomal systems, membrane fluidity is primarily governed by the high ethanol content, which induces lipid bilayer disordering and enhances vesicle deformability. A comprehensive review has reported that increasing cholesterol content may lead to higher bilayer order and reduced membrane flexibility, thereby partially counteracting the fluidizing effect of ethanol [37]. Accordingly, in the present formulation, cholesterol was maintained at a minimal level (0.1%) to preserve the characteristic soft and highly fluid membrane structure of ethosomes while still supporting membrane integrity. Low cholesterol levels (≤1%) have been described for ethosomal and related phospholipid-based vesicular systems aiming to balance membrane stability and fluidity [38].

When producing vesicular carrier systems, stabilization of the vesicular system occurs through different mechanisms such as electrostatic interactions or steric mechanisms. SPC is a zwitterionic phospholipid that contributes to vesicle stabilization through steric mechanisms. The phosphocholine head groups in its structure have a high hydration capacity. Thus, it enables the formation of a hydration layer around the vesicles with the aqueous phase. This hydrated interface layer creates short-range repulsive forces, preventing vesicle–vesicle convergence and aggregation [39,40,41].

Examples of stabilization occurring through hydration-based steric mechanisms for liposomal and ethosomal carrier systems produced using phosphatidylcholine are frequently encountered in the literature. In these examples, it is accepted that the stability of the vesicular system can be achieved even without loaded lipids or polymeric stabilizers [39,40,41]. In the current formulation, the ultimate stability of the vesicular system is attributed to the combined effects of hydrated SPC head groups, optimized bilayer packing, and balanced membrane fluidity, rather than electrostatic repulsion or polymer-derived steric barriers.

The micrographs shown in Figure 2 demonstrate discrete nanosized vesicular structures with relatively uniform morphology, confirming the formation of intact vesicles. Imaging was performed using an FEI Quattro S SEM operated in FE-SEM/STEM mode under conditions selected to minimize drying-related artifacts and preserve vesicular integrity. Although dynamic light scattering (DLS) was used as the primary technique for particle size analysis, the vesicular morphology of the optimized ethosomal formulation was further evaluated by FE-SEM/STEM imaging. The micrographs obtained revealed discrete, nanosized vesicular structures with a relatively uniform morphology, providing visual confirmation of intact vesicle formation. This morphological evidence supports the DLS findings and reduces ambiguity related to potential aggregation effects that may arise in ethanol-rich vesicular systems. The combined use of DLS and STEM-based imaging therefore provides a more reliable assessment of particle size and structural integrity in the present ethosomal formulation.

3.2. In Vitro Release Study

In the present study, in vitro release experiments were conducted using freshly prepared optimized ethosomal dispersions after separation and removal of non-encapsulated (free) BRT. Accordingly, the reported release profiles represent the release of the initially encapsulated drug fraction. Release was evaluated using the dialysis membrane diffusion method, where released BRT diffuses across the membrane while vesicular structures are retained. Entrapment efficiency was determined in parallel using the same freshly prepared dispersions, and the release data was interpreted based on the encapsulated fraction. Multiple washing cycles or lyophilization were not applied prior to release testing to avoid potential alterations in vesicle integrity. After separation of the free fraction, the vesicular pellet was gently resuspended and used directly for the release experiment to best reflect the native formulation characteristics.

The cumulative release profile presented in Figure 3 exhibited a biphasic pattern consisting of an initial burst phase and a subsequent sustained release phase. During the first 120 min, approximately 70.99 ± 3.07% of BRT was released, reflecting the rapid diffusion of drug molecules positioned on the vesicle surface or loosely associated with the outer bilayer. In a study reported in the literature, nanovesicular BRT formulations similarly demonstrated an early burst effect that was attributed to superficial drug deposition and increased flexibility of surfactant-based vesicles [28]. Another investigation involving proniosomal gel derived niosomes showed a comparable early release pattern with high Q2h values, further confirming the contribution of surface located drug molecules to the initial phase of release [42].

Following this rapid phase, the ethosomal system transitioned into a more sustained release behavior, reaching 88.55 ± 9.19% cumulative release at 24 h. Considering that the release study was performed with the optimized formulation after removal of non-encapsulated drug, this value corresponds to the fraction of initially encapsulated BRT released and is equivalent to an absolute amount of 1.55 ± 0.16 mg/mL BRT at 24 h.

This pattern indicates diffusion-controlled release of drug entrapped within the inner bilayers, where ethanol induced membrane fluidity facilitates gradual diffusion. A separate study examining cubosomal BRT formulations also demonstrated an extended-release period of 24 h, which was explained by the structural properties of cubic liquid crystalline phases that slow down drug mobility through lipidic regions [30]. In another recent investigation, hyaluronic acid coated cubosomes incorporated into an in situ gel system exhibited prolonged release with a diffusion dependent mechanism, providing additional support for the role of vesicular nanostructures in sustaining drug delivery [43]. Furthermore, niosomes integrated into contact lenses were shown to maintain drug release for up to 20 h, demonstrating that multilayered vesicular assemblies can effectively prolong ocular drug availability [44].

Because the cumulative release profile reached a plateau after approximately 3 h, release kinetics were evaluated using data from the initial 3 h period. The corresponding kinetic parameters are presented in

Table 6. Regression values of kinetic release models of the optimized formulations.

Kinetic Models	Ethosome Formulation
Zero-order (r2)	0.5313
First-order (r2)	0.5239
Hixson–Crowell (r2)	0.5299
Higuchi (r2)	0.7982

. Among the tested kinetic models, the Higuchi model showed the best correlation with the experimental data. The Higuchi model describes drug release as a diffusion-controlled process that is proportional to the square root of time, which is typical for matrix-based delivery systems. This indicates that BRT release from the ethosomal formulation occurs mainly through diffusion from the vesicular structure rather than erosion or dissolution of the carrier. Similar diffusion dominated release behavior has been reported for other vesicular formulations containing BRT [28].

Overall, the in vitro release profile of ethosomal formulation aligned closely with findings from other vesicular drug delivery systems described in the literature. The observed biphasic pattern, characterized by an initial burst followed by sustained Fickian diffusion, is a common feature of vesicular carriers and is supported by multiple independent studies. This extended-release behavior is particularly advantageous for ophthalmic delivery because it may help maintain therapeutic concentrations for longer periods while reducing dosing frequency and improving patient adherence.

3.3. Assessment of Interactions (Raman Evaluation)

The Raman spectral analysis provided critical insight into the molecular fingerprint of BRT and its transformation upon encapsulation into ethosomal vesicles. The Raman spectrum revealed that the vibrational spectrum of pure BRT (Figure 4A) did not show any distinct or isolated absorption bands in the 1600–1800 cm⁻¹ region. This finding is consistent with data in the literature [45]. It is known that dominant peaks in this region may originate from amide I and carbonyl groups [46,47]. The absence of these structures in the structure of pure BRT explains the absence of peaks or bands in the 1600–1800 cm⁻¹ region.

In contrast, the spectrum of the ethosomal formulation (Figure 4B) shows a broadened band centered around 1650 cm⁻¹. However, the assumption that this band is a vibrational mode related to the newly formed drug is not an accurate approach to explaining the situation. It is thought that phospholipid-related vibrational contributions, including amide I type vibrations and ester carbonyl (C=O) stretching modes originating from the phospholipid bilayer of the ethosomal system, are the main factors in the formation of this band. Such spectral properties are well-documented for lipid-based vesicular carriers and are known to be sensitive to hydration status and microenvironmental effects [46].

It is known that the characteristic peaks in the spectrum of pure API in drug-loaded liposomal and ethosomal formulations are either not seen or partially attenuated in drug-loaded carrier systems. There are several reasons for this. These include dilution of the drug within the vesicular matrix, molecular shielding resulting from the embedding of the API in the lipid bilayer and overlapping spectral contributions from excipients and carrier components. In such cases, it has been shown that the chemical structure of the encapsulated API is not degraded or lost [47]. A similar situation applies to the spectra in our study. A relative attenuation of the characteristic BRT peaks was observed in the ethosomal spectrum.

In conclusion, it is seen that the intrinsic vibrational signatures of BRT are not completely eliminated in the spectrum data, and changes in the local microenvironment surrounding the drug can be observed. The spectral changes observed upon loading BRT into the ethosomal carrier system demonstrate that BRT is successfully encapsulated.

3.4. Evaluation of Ocular Compatibility (HET-CAM Study)

The ocular irritation potential of the BRT loaded ethosomal formulation was evaluated using the HET-CAM method, which is an established in ovo alternative to the classical Draize rabbit eye test. All experimental procedures were conducted in accordance with the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) guidelines to ensure ethical compliance and reproducibility. The HET-CAM model enables direct observation of vascular reactions such as hemorrhage, lysis, and coagulation within a 300 s period after topical application. This model provides a reliable indication of ocular tolerance while eliminating the ethical and procedural limitations associated with animal-based tests.

Representative images from the HET-CAM assay illustrating vascular reactions after the application of the test samples are shown in Figure 5. The optimized BRT-loaded ethosomal formulation and its placebo produced mild irritation (score = 1) in the HET-CAM assay, whereas the aqueous drug solution was classified as non-irritant (score = 0) as summarized in

Table 7. Cumulative scores and results of the HET-CAM test.

Formulation	Cumulative Score	Irritation Assessment
BRT ethosomal formulation	1	Slight
BRT solution	0	Practically none
Placebo	1	Slight

Lysis was observed at 300 s in both the BRT formulation and the placebo, and therefore a score of ‘1’ was recorded.

. In contrast, the NaOH-treated positive control exhibited rapid and pronounced vascular damage within the initial seconds of exposure, confirming the discriminative capacity and validity of the HET-CAM assay. The mild and delayed lysis observed in the ethosomal and placebo samples was mainly attributed to the excipients rather than the active drug. Ethanol, which serves as a penetration enhancer in ethosomal systems, has been reported to be non-irritant even at concentrations exceeding 40%, as demonstrated by Ahmed et al. in ketoconazole-loaded trans-ethosomes [11]. Similarly, Üner et al. confirmed the ocular safety of timolol-loaded ethosomes using the HET-CAM model [10]. In the present formulation, which contained 20% ethanol, only mild vascular changes were detected, remaining within the non-to-mild irritation range. These findings indicate that ethanol concentrations typically employed in ethosomal systems are well tolerated and that the developed BRT-loaded ethosomal formulation possesses an acceptable safety margin for ophthalmic application.

The phospholipid matrix appeared to attenuate the transient irritant potential of ethanol by stabilizing the ocular surface, improving hydration, and enhancing overall biocompatibility. These results indicate that formulation possesses a favorable safety profile for ophthalmic administration. Consistent with the 3R principles of reduction and replacement, the HET-CAM method was adopted as a validated in ovo alternative to the traditional Draize eye test, as also implemented by Üner et al., in order to minimize animal use while ensuring reliable ocular tolerance assessment [10].

Previous investigations have demonstrated that BRT can be incorporated into various advanced ocular delivery systems, including cubosomes [30], nanoemulsions [48], and in situ gels [49], all of which exhibited minimal or no ocular irritation. Collectively, these studies indicate that lipid-based nanocarriers can be formulated safely when the compositions of the excipients (ethanol content and surfactant composition) are properly optimized. The results of the present work are consistent with this evidence, confirming that the BRT-loaded ethosomal formulation ensures ocular compatibility while providing improved drug penetration and prolonged residence time, thereby offering a promising alternative for sustained glaucoma therapy.

While MTT assays are often used to complement HET-CAM findings, the latter was selected in this study as a reliable and ethically favorable method for primary ocular irritancy screening. The HET-CAM model allows direct observation of vascular responses that closely mimic ocular surface reactions and has been shown to correlate well with cell-based cytotoxicity assays such as MTT [50]. The method provides high reproducibility, rapid evaluation, and compliance with the 3R principles, making it particularly suitable for preliminary safety assessments of ophthalmic formulations. Although the absence of an MTT assay may be considered a limitation, the application of the validated HET-CAM system provided robust and sufficient evidence of ocular tolerability for the developed ethosomal formulation.

Long-term local ocular toxicity parameters, such as histological evaluation of corneal epithelium, stroma, and endothelium, were not investigated in this study. However, in vivo studies observed rats for ocular discomfort, inflammation, or behavioral abnormalities following 30 days of drug administration. These observations indicate an acceptable pre-ocular tolerance profile. On the other hand, a retrospective study comparing the results of the HET-CAM and Draize tests found that the test results were consistent in 80–90% of all pathological cases [51].

In conclusion, the BRT-loaded ethosomal formulation demonstrated a high level of ocular safety in the HET-CAM model. The mild and transient vascular reactions observed were comparable to those of the placebo and remained within acceptable tolerability limits. These outcomes confirm that the optimized formulation achieves a balance between enhanced ocular penetration and minimal irritation. Overall, the developed ethosomal system can be considered a promising and biocompatible alternative to conventional BRT eye drops for sustained IOP control in glaucoma therapy.

3.5. In Vivo Assessment

The percentage change values obtained from the BRT ophthalmic drops (BRT-OD) (%) and BRT Ethosome (BRT-ET) (%) formulations over a 30-day period were statistically analyzed. Normality testing using the Shapiro–Wilk test indicated that the BRT-OD data were normally distributed (p = 0.154), whereas the BRT-ET data deviated from normality (p = 0.003). Consequently, a non-parametric Mann–Whitney U test was applied to compare the two groups.

The analysis revealed a statistically significant difference between the BRT-OD and BRT-ET formulations (U = 227.0, p = 0.001). The BRT-ET group exhibited higher percent change values (mean ± SD: 18.09 ± 6.56%) compared to the BRT-OD group (12.69 ± 5.16%). As illustrated in Figure 6, the BRT-ET formulation produced a more pronounced and sustained IOP-lowering effect throughout the 30-day evaluation period.

Throughout the study period, IOP values in Groups 1 and 2 showed only minor, non-directional fluctuations below 1 mmHg. Importantly, IOP levels in Group 2 consistently remained above the predefined glaucoma threshold of 25 mmHg, indicating stability of the disease model without spontaneous regression. In Groups 3 and 4, IOP outcomes were therefore expressed as percentage reduction from baseline to allow meaningful evaluation of treatment-related effects. In these groups, the contralateral untreated eye was monitored as a within-animal reference to minimize inter-animal variability rather than as an independent comparator. No consistent IOP changes exceeding minor physiological fluctuations were observed in these untreated eyes, suggesting the absence of a measurable cross-talk or systemic effect following topical administration.

The optimized BRT-loaded ethosomal formulation is designed for application as an ophthalmic solution. Under normal conditions, the concept of “ophthalmic solutions” might be perceived as describing a conventional dosage form. However, since the ethosomes produced in this study are lipid-based nanosystems, they can exhibit greater permeation to ocular surfaces compared to conventional ophthalmic solutions. Thus, drug loss in the precorneal region, especially due to nasolacrimal drainage, is reduced [9,52].

Although ocular permeation studies were not performed ex vivo with tissues in this study, the retention potential of the formulation was indirectly estimated based on its physicochemical and functional properties. It is expected that the optimized ethosomes, thanks to their ethanol-reinforced flexible lipid bilayer, will be able to interact readily with the corneal and conjunctival epithelium [52]. On the other hand, in vivo pharmacodynamic results showed a longer-lasting and more consistent reduction in intraocular pressure in BRT-loaded ethosomal formulations with lower drug application frequency compared to conventional eye drops. This result provides functional evidence supporting longer ocular retention and prolonged drug presence at the site of action. Taken together, these findings suggest that ethosomal formulation has the potential to improve ocular retention and therapeutic performance, even without direct permeation and penetration studies.

The results obtained in this study are consistent with the in vivo results of carrier systems for the ocular use of BRT previously. In one study, it was found that BRT-loaded Eudragit RL/RS-based nanoparticles, despite their high particle size (between 220 and 410 nm), showed a sustained IOP-lowering effect lasting up to 72 h in rabbits with α-chymotrypsin-induced glaucoma [53]. In another study, nanoparticles produced with chitosan showed a statistically significant and long-lasting reduction in IOP compared to a standard BRT solution, again despite their high particle size (between 270 and 370 nm) [54]. In yet another nanoparticle system produced with chitosan, a 3-fold extended drug release and an IOP-lowering effect approaching 25 h were detected compared to the commercial product Alphagan^® P (Allergan, Irvine, CA, USA) [55]. Fedorchak et al. applied the BRT-loaded PLGA microspheres they developed to rabbits once a day and obtained clinical findings similar to the effect of conventional eye drops used twice a day on IOP [56]. In formulations of BRT prepared with lipid carriers (NLCs), the reduction in IOP was found to be significantly greater and longer lasting than both solid lipid nanoparticles and commercially available BRT formulations [57]. Similar results have been reported for BRT microemulsions [58], nanoparticles produced with different polymers [59,60], and nano-resin/drug complex suspensions [61]. In studies on vesicular carrier systems, it has been reported that the ability of BRT-loaded niosomes to reduce IOP is clinically more than 30% higher than conventional eye drops [42,44,62]. In the study conducted by Maiti et al., it was observed that the BRT nanovesicular system showed a hypotensive effect twice as long as the commercial product [28]. In a study in which BRT-loaded liposomes were developed, it was reported that the onset of clinical effect was shorter than Alphagan^®, but the IOP reduction effect lasted longer [29,63]. In cubosome studies, it was observed that BRT showed a significantly higher and longer-lasting effect (approximately 24 h) compared to the commercial 0.2% BRT solution [30,43].

BRT’s short ocular half-life typically necessitates dosing two to three times per day to sustain IOP reduction. This frequent dosing regimen is associated with challenges in chronic therapy, including reduced patient adherence and an increased risk of cumulative side effects. A simplified once-daily dosing schedule can offer significant benefits by improving patient compliance and maintaining more consistent IOP control. Enhanced adherence to therapy is particularly critical in glaucoma management, as poor compliance with multi-dose regimens has been linked to suboptimal long-term outcomes. The sustained-release profile of the ethosomal formulation addresses this issue by reducing the administration frequency without compromising efficacy. By delivering BRT in a controlled manner, the ethosomal carrier helps avoid the troughs in drug concentration that occur with conventional eye drops, thereby providing stable IOP-lowering activity throughout the day.

These results highlight the potential of ocular nanocarriers to maintain therapeutic efficacy while reducing the dosing frequency. Other advanced ophthalmic formulations, such as in situ gelling systems for BRT, have similarly been designed to prolong drug action and decrease the need for multiple daily instillations. The present study’s positive outcomes with ethosomal formulation align with this trend and demonstrates a viable strategy to improve glaucoma therapy. Reducing the overall drug burden on the eye may also mitigate dose-dependent adverse effects over long-term use. The ethosomal BRT system offers a promising approach to achieve effective and sustained IOP reduction with a more convenient dosing regimen. This nanocarrier-based therapy could thus enhance patient acceptability and therapeutic outcomes in glaucoma by combining improved drug delivery with improved adherence.

3.6. Stability Assessment

Over a 3-month storage period, the BRT-loaded ethosomal formulation exhibited moderate yet statistically significant changes in certain physicochemical parameters, particularly in entrapment efficiency (EE) (Figure 7). The initial EE of 87.33% declined to 71.97% and 71.05% at 4 °C and 25 °C, respectively, representing a reduction of approximately 16–18%. This decrease was found to be statistically significant (p = 0.0185), suggesting partial drug leakage from the vesicle bilayers during storage. In contrast, particle size and polydispersity index (PDI) remained largely stable, with mean sizes ranging from 121.3 nm at day 0 to 124.3 nm (4 °C) and 123.5 nm (25 °C) at day 90, and PDI values consistently below 0.3, indicating maintenance of a narrow and homogeneous size distribution. These findings suggest the absence of vesicle aggregation or fusion during storage.

Interestingly, a notable shift in zeta potential was observed under refrigerated conditions, changing from −1.82 mV at baseline to +0.83 mV after 90 days at 4 °C, whereas the value remained negative at 25 °C (−1.37 mV). This inversion may be attributed to desorption of the drug from the vesicle core and subsequent adsorption onto the vesicle surface, altering its interfacial charge. Alternatively, cold-induced membrane restructuring may have affected the distribution of surface-active components.

Published research on vesicular drug carriers demonstrates that non-ionic surfactant vesicles (niosomes) and soft lipid-based vesicles (liposomes) may undergo gradual drug leakage or entrapment efficiency (EE) decline over storage, depending on bilayer composition, surfactant/lipid ratio and environmental conditions. Comprehensive studies on niosomes notes that despite their relative stability compared to traditional liposomes, issues such as bilayer destabilization, progressive drug leakage and payload loss remain key limitations affecting long-term shelf-life [64,65].

Indeed, in a formulation of piroxicam-loaded niosomes stored for three months at 5 °C or room temperature, drug leakage in the range of 1.6–6.6% was observed, even though the vesicle morphology and dispersion remained acceptable [66]. Another study optimizing ocular niosomal delivery of pilocarpine hydrochloride reported that although sonication and proper surfactant choice improved EE initially, long-term chemical stability and maintenance of EE remain challenging for hydrophilic drugs in niosomes [67].

The decrease in EE observed in this study may be mechanistically explained by increased bilayer permeability over time, a known limitation of ethanol-containing vesicular systems. The fluidizing effect of ethanol facilitates high initial drug loading but may also predispose vesicles to slow drug diffusion out of the bilayer matrix. Moreover, temperature-dependent membrane dynamics could accelerate such leakage at higher storage temperatures, although a similar decline at 4 °C implies that cold-induced bilayer stress or phase transitions may also play a role. The stability of particle size and PDI throughout the study indicates that the formulation remained physically intact, and the modest changes in zeta potential, while noteworthy, are not expected to compromise colloidal stability over the time frame tested.

Overall, the formulation demonstrated good physical stability over 90 days, with a modest but quantifiable decline in drug retention. These findings align with existing literature on ophthalmic vesicular systems and emphasize the need for further optimization of bilayer composition or use of stabilizers to mitigate long-term drug leakage.

4. Conclusions

This study successfully demonstrates that BRT can be efficiently integrated into an ethosomal nanocarrier system that combines positive qualitative and quantitative characteristics with improved therapeutic performance. The optimized ethosomal formulation exhibited homogeneous vesicular properties, maintained its structural integrity throughout the evaluation period, and demonstrated favorable physicochemical characteristics, including effective drug encapsulation and controlled release behavior. The diffusion-controlled drug release profile of the resulting carrier system demonstrates the ability of the lipid bilayer in the ethosomal structure to modulate drug transport. Thus, long-term permeation and penetration into ocular tissues are ensured, supporting the potential for therapeutic effect.

In vivo findings confirmed that the optimized ethosomal system tends to reduce intraocular pressure more consistently and for a longer period than conventional eye drops, even when applied less frequently. This improvement in the pharmacodynamic performance of BRT, combined with the tolerability of the optimized formulation by ocular tissues, clearly highlights the importance of the generated ethosomal carrier systems. Overall, the integrated qualitative and quantitative results of this study demonstrate that ethosomal nanocarriers are a promising and effective strategy for enhancing ocular delivery and therapeutic efficacy of BRT in glaucoma treatment.