INTRODUCTION:

One of the most frequently prescribed nonsteroidal anti-inflammatory drugs (NSAIDs) worldwide is diclofenac sodium. Joint diseases, such as osteoarthritis, are often treated with this drug¹. Diclofenac sodium frequently causes adverse effects in the gastrointestinal tract, including bleeding and gastric ulcers, due to its mechanism of action, which involves the inhibition of COX synthesis in both COX-1 and COX-2^2,3.

It is classified as BCS class II and possesses a low solubility^4,5. Diclofenac sodium is possible to formulate a topical preparation to enhance the efficacy of it and mitigate potential adverse effects. In contrast to oral formulations, topical preparation offers several benefits: circumvention of first-pass metabolism, prevention of gastrointestinal irritation, non-invasive drug delivery to the intended site of action, patient improvement, and convenience in medication administration and discontinuation^6–8.

Through the epidermis, topical preparations must be applied. The epidermis is the most exposed and expansive organ within the human body. Nevertheless, due to the stratum corneum's function as a protective barrier against external influences, not all active pharmaceutical ingredients are readily absorbed via the epidermis^9,10. As a result, pharmaceutical substances intended for topical administration via the skin should possess equivalent balance values of hydrophilicity and lipophilicity to those of the epidermis (log P 1-3)¹¹.

One method to increase the solubility and drug absorption through the skin is to design the drug as a nanoemulsion system. Nanoemulsion is a preparation consisting of two immiscible ingredients (water and oil), which are stabilized by surfactants and cosurfactants¹². The small particle size of nanoemulsion (50-500 nm) can increase the level of drug penetration through the skin, assisted by the mechanism of oil as a permeation enhancer^13,14. Due to its small size, nanoemulsion exhibits enhanced long-term stability by preventing particle aggregation, thereby preventing sedimentation, creaming, flocculation, and coalescence^15,16. Nanoemulsions possess several advantages over alternative novel carrier systems, including micelles and liposomes. These include superior drug loading and solubilizing capacities, ease of manufacturability, high stability, and controlled release patterns^7,15,17. In the context of topical delivery, such as in the management of joint diseases, nanoemulsion can be modified into a gel formulation by incorporating an appropriate gelling agent (nanoemulgel). Research has demonstrated that nanoemulgel achieves more effective drug release and penetration than conventional gel, which does not contain nanoemulsion^8,14,18.

The optimal proportions of constituent elements of nanoemulsion—water phase, oil phase, and emulsifier—can significantly influence the attributes of high-quality nanoemulsions, including small particle size, transparency, pH stability, and storage resistance. One potential solution to reduce the time and effort required to ensure optimal ratios is to use experimental design techniques, such as the simplex lattice design method. The utilization of this method allows for the determination of the optimal relative proportion of constituents in a formulation, with respect to predetermined variables^19,20. One hundred percent of the components that make up the preparation are in SLD. In addition, SLD can ensure a causal relationship between independent and dependent variables by managing the acceptability criteria of the dependent variable as desired^21–24.

MATERIALS AND METHODS:

Materials:

Diclofenac sodium, which was given by PT Dexa Medica (Indonesia); oleic acid, Span 80, Tween 80, which were purchased from PT Brataco (Indonesia); absolute ethanol, which was made by Merck (Germany); and distilled water. All excipients were pharmaceutical grade.

Methods:

Preparation of pseudo-ternary phase diagrams:

Using the aqueous titration method, a pseudo-ternary phase diagram of three components (water phase, oil phase, and emulsifier phase) was produced^12,21,25. The emulsifier phase, which consisted of a weight ratio of 2:1; 3:1; and 4:1 for Tween 80–Span 80 (HLB 14) as the surfactant (S) and ethanol as the cosurfactant (CoS), was dissolved in oleic acid (the oil phase) (Table 1). For five minutes, this mixture was stirred at 200 rpm. A titration was performed on each ratio of the S/CoS mixture and oil phase using aqueous phase drop by drop (100 µL), while the solution was stirred gently and continuously at 400 rpm. The quantity of water required to furnish the solution just prior to turbidity was recorded²⁵. The percentage of each component to form a transparent nanoemulsion system was calculated. Phase diagrams were plotted using CHEMIX School software version 12.

Table 1. Ratio of oil and S/CoS in aqueous titration method of nanoemulsions

S/CoS : Oil	Tween 80 (g)	Span 80 (g)	Ethanol (g)	Oleic Acid (g)
Ratio of surfactant and cosurfactant 2:1
1: 9	0.604	0.062	0.333	9.000
2: 8	1.209	0.125	0.667	8.000
3: 7	1.813	0.187	1.000	7.000
4: 6	2.417	0.249	1.333	6.000
5: 5	3.022	0.312	1.667	5.000
6: 4	3.626	0.374	2.000	4.000
7 : 3	4.231	0.436	2.333	3.000
8: 2	4.835	0.498	2.667	2.000
9: 1	5.439	0.561	3.000	1.000
Ratio of surfactant and cosurfactant 3:1
1: 9	0.680	0.070	0.250	9.000
2: 8	1.360	0.140	0.500	8.000
3: 7	2.040	0.210	0.750	7.000
4: 6	2.720	0.280	1.000	6.000
5: 5	3.400	0.350	1.250	5.000
6: 4	4.079	0.421	1.500	4.000
7: 3	4.759	0.491	1.750	3.000
8: 2	5.439	0.561	2.000	2.000
9: 1	6.119	0.631	2.250	1.000
Ratio of surfactant and cosurfactant 4:1
1: 9	0.725	0.075	0.200	9.000
2: 8	1.450	0.150	0.400	8.000
3: 7	2.176	0.224	0.600	7.000
4: 6	2.901	0.299	0.800	6.000
5: 5	3.626	0.374	1.000	5.000
6: 4	4.351	0.449	1.200	4.000
7: 3	5.077	0.523	1.400	3.000
8: 2	5.802	0.598	1.600	2.000
9: 1	6.527	0.673	1.800	1.000

Experimental design by simplex lattice design:

The nanoemulsion system was optimized utilizing the simplex lattice design implemented in Design-Expert software version 13. As causal factors, three components of the nanoemulsion were chosen: water phase (X₁), oil phase (X₂), and S/CoS (X₃). The levels of each component have been defined by the upper and lower limits of the pseudo-ternary diagram.

Preparation of nanoemulsion systems:

The nanoemulsion systems were produced utilizing the low energy emulsification method in accordance with the ratio acquired from SLD. S/CoS and the oil phase were mixed at 200 rpm for 5 minutes. Each formulation underwent a dropwise addition of water while being continuously stirred at 400 rpm for 30 minutes or until a transparent nanoemulsion was achieved.

Characterization of nanoemulsion systems:

a) Particle size and PDI:

The particle size and PDI were examined using a particle analyzer (Delsa^TM Nano C, US). The nanoemulsion sample was put into a cuvette and observed at an angle of 165^o and a temperature of 25^oC. The data (output) was the droplet size value calculated from the average fluctuation of light scattering intensity and PDI, which describes the particle size distribution.

b) Percent Transmittance:

The percent transmittance (%T) of the nanoemulsions was measured using a UV-Vis spectrophotometer (Hitachi UH5300, Japan) at 570 nm against distilled water as a blank.

c) pH value:

A calibrated pH meter (Eutech pH 700, US) was utilized to determine the nanoemulsion's pH. The electrode of the pH meter was submerged in the sample by dipping it. The pH result was denoted by the value displayed on the instrument.

Predicted optimal nanoemulsion:

By employing a simplex lattice design, the optimal nanoemulsion formulation was ascertained. The response variables were the properties of the nanoemulsion, including particle size (Y₁), PDI (Y₂), percent transmittance (Y₃), and pH (Y₄). The concurrent optimal nanoemulsion solution was hypothesized to possess the following characteristics: minimum particle size, minimum PDI, maximum percent transmittance, and pH within the range of 5.0 to 6.5. The observed and predicted values were compared in order to ascertain that the response surface was reliable.

RESULT:

Pseudo-ternary phase diagram:

There were three pseudo-ternary diagrams in Figure 1, each with a distinct S/CoS ratio. The region susceptible to nanoemulsion formation was marked by the grey-shaded areas. On the basis of these results, a S/CoS ratio of 2:1 was selected as it generated the largest nanoemulsion area in comparison to the other options (Table 2).

Figure 1. Pseudo-ternary phase diagrams containing the following components: water, oleic acid as oil, Tween 80 – Span 80 as surfactant, and ethanol as cosurfactant. Grey shaded area shows nanoemulsion region in different ratio of S/CoS in (A) 2:1 (B) 3:1 and (C) 4:1.

Table 2. Percentage of nanoemulsion region obtained for different ratios of S/CoS

Ratios of surfactant: cosurfactant	Nanoemulsion region (grey shaded area) (%)
2: 1	18.4
3: 1	16.8
4: 1	13.9

Experimental design by SLD for nanoemulsion formulation:

In accordance with Figure 1A, the upper and lower limits of each nanoemulsion component were determined by calculating the area under the nanoemulsion region that can accommodate the largest equilateral triangle (Figure 2). The total concentration of the three components was adjusted to 100%, so the following upper and lower limits were determined by equality:

5 ≤ X₁ ≤ 30 (%) (1)

10 ≤ X₂ ≤ 35 (%) (2)

60 ≤ X₃ ≤ 85 (%) (3)

X₁ + X₂ + X₃ = 100 (%) (4)

Figure 2. Simplex lattice design for the three factors (blue shaded area) under the nanoemulsion region.

The software received the upper and lower limit values for each nanoemulsion component. In response, SLD generated 14 formulations, each with a different component ratio, as represented in Table 3.

Table 3. Nanoemulsion formulation composition by SLD method

Formula	Component 1 A:Water %	Component 2 B:Oil %	Component 3 C:S/CoS %
F1	30.000	10.000	60.000
F2	17.500	22.500	60.000
F3	30.000	10.000	60.000
F4	9.167	14.167	76.667
F5	5.000	22.500	72.500
F6	9.167	26.667	64.167
F7	17.500	22.500	60.000
F8	21.667	14.167	64.167
F9	17.500	10.000	72.500
F10	13.333	18.333	68.333
F11	5.000	10.000	85.000
F12	5.000	10.000	85.000
F13	5.000	35.000	60.000
F14	5.000	35.000	60.000

Formulation of nanoemulsion systems:

The 14 nanoemulsions were produced, as in Figure 3. From a visual perspective, the nanoemulsion system exhibited a transparent appearance, no phase separation, which were typical nanoemulsion¹⁶, with a light yellow color.

Figure 3. Appearance of the nanoemulsion system that has been produced based on SLD method formulation (A) formula 1-7 & (B) formula 8-14

Characterization of nanoemulsion systems

a) Particle size and PDI:

The range of particle sizes observed in the formed nanoemulsion is 0-221 nm. At the nanoemulsion's particle size of zero, no droplets are detected. The particle size of the nanoemulsion could be augmented by the water phase, as indicated by the regression equation. Therefore, extremely minute quantities of water may result in the absence of droplet formation. The range of PDI observed in the formed nanoemulsion is 0-0.7 nm. The influence of the number of water phases on the PDI value is evident. As the water phase increases, so does the PDI value.

b) Percent transmittance:

The percent transmission value obtained falls within the range of 95.7 to 100. On the contrary, the R² value obtained from the response analysis is relatively low, suggesting that the factor variables do not exhibit a significant correlation with the resulting transmission value. Significant discrepancies may arise between the predicted values and the observed values as well (Adjusted R² and Predicted R² values are low).

c) pH:

The nanoemulsion exhibits pH characteristics, with a range of 5.104 to 6.046. According to the regression equation, the pH value is impacted by variable factor C, specifically surfactant and cosurfactant.

The optimal nanoemulsion:

SLD predicted that the optimal nanoemulsion formula includes 15.28% water, 21.25% oil, and 63.47% S/CoS which is in accordance with the desired response variable criteria (minimum particle size, minimum PDI, maximum percent transmittance, and pH within the range of 5.0 to 6.5). The mean predicted values determined by SLD were particle size of 83.816 nm, PDI of 0.225, transmittance of 100%, and pH of 5.203. To confirm thus predicted values, the nanoemulsion was produced by utilizing the ratio of this formula, and its properties were assessed through three replications. The nanoemulsion exhibited the following characteristics: particle size of 108.8 ± 7.03 nm, PDI of 0.443 ± 0.12, percent transmittance of 99.8 ± 0.15%, and pH value of 5.276 ± 0.04. There was no significant difference observed between the predicted and observed values (p > 0.05).

DISCUSSION:

The selection of oleic acid as the oil phase was based on the claim that diclofenac sodium dissolves more readily in oleic acid than in alternative oils, including ethyl oleate, myritol, or MCT (medium chain triglyceride) oil²⁶. Furthermore, by increasing the fluidity of stratum corneum lipids via the formation of water channels, oleic acid can facilitate the permeation of drugs into the epidermis¹¹. The primary purpose of surfactants in nanoemulsions is to inhibit droplet aggregation and decrease interfacial tension through adsorbing a monolayer of molecules or ions at the interface¹³. The surfactants utilized in this research were Tween 80 and Span 80, which were selected because they were non-toxic^16,25, had similar molecular structure to oleic acid, and their liquid state, which prevented the need for heating during nanoemulsion preparation. In comparison to nanoemulsions composed of single surfactants, those comprising combination surfactants exhibit enhanced storage stability and more minuscule droplet dimensions^27,28. In order to aid surfactants in the reduction of interfacial tension, cosurfactants are utilized. Furthermore, compared to the use of surfactants alone, cosurfactant can enhance the solubility of the drug⁸ and oil phase²⁹. Ethanol is selected as a cosurfactant due to its brief chain of carbon atoms^13,16, which enables it to dissolve diclofenac sodium more effectively than propranolol, PG, PEG 400, or glycerin²⁶.

Reducing the cosurfactant ratio in the pseudo-ternary diagram led to an increase in the nanoemulsion region¹⁴. The pseudo-ternary diagram showed that the greatest quantity of cosurfactant (S/CoS at 2:1) resulted in the largest nanoemulsion area, compared with S/CoS at 3:1 or 4:1. As a cosurfactant, ethanol's short chain of carbon atoms facilitates its passage between the surfactants, thereby aiding in the reduction of the nanoemulsion's surface tension^21,30.

The nanoemulsion exhibited a transparent appearance due to the small size of its constituent droplets. Surfactants and cosurfactants are critical to the formation of nanoemulsion systems, with a significant proportion contributing to their success in contrast with conventional emulsions. The surfactant effectively mitigates the surface tension of the nanoemulsion, thereby preventing aggregation and phase separation. At the same time, the cosurfactant serves to enhance the surfactant's functionality¹³. The light yellow color of the nanoemulsion is a result of the natural color of Tween 80 and Span 80 that were employed.

A low proportion of water phase was required to produce a nanoemulsion with small particles²¹ but where the water phase is present in extremely small quantities, nanoemulsion not formed. Thus, the particle size is adjusted within the range of 50-500 nm in the intended variable response criteria¹³. The small particle size will help increase the stability and the drug-loaded nanoemulsion penetration rate to be absorbed into the skin^15,17.

Each collection of droplets is referred to as polydispersion. An optimal index value is between 0 and 0.5³¹. The nanoemulsion system's PDI characterizes the physical stability of a dispersion system and the particle size distribution. A low PDI value signifies that the formed dispersion system exhibits more excellent long-term stability³². A significant degree of PDI illustrates an uneven distribution of particulate sizes. This phenomenon occurs due to the aggregation of droplets, which results in their asymmetrical dispersion and subsequent polydispersity, thereby impeding the stability of the nanoemulsion³³.

A characteristic of nanoemulsion, percent transmittance, can be utilized to estimate the dimensions of the particles that are produced. A greater percentage of transmittance (approaches 100%) indicates an enhanced capability of the nanoemulsion to permit light to traverse the system, thus ensuring the preservation of clarity. The higher percentage of transmittance observed in nanoemulsion may be attributed to the reduced droplet size and more homogeneous distribution of droplet sizes³⁴. The percent transmittance was determined using UV Vis at a wavelength of 570 in this investigation. This is due to the light yellow color of the resulting nanoemulsion³⁵. A high percent transmittance (>95%) was achieved with nanoemulsion in this study due to the presence of an adequate amount of emulsifier to generate particles on the nanometer scale.

The absorption of active drug ingredients into the epidermis depends on the compatibility between the pH of the skin and the pH of the drug preparation and also to prevent irritation. The pH value for human epidermis ranges between 4 and 6.5³¹. The pH range of the nanoemulsion produced in this study was comparable to the pH of the epidermis. The response variable criteria determine a pH range of 5 to 6.5, which is not only consistent with the pH of the epidermis but also guarantees the stability of diclofenac sodium as an active ingredient, namely between pH 5 and 8³⁶. S/CoS concentrations can increase the pH value, particularly Tween 80, which has a pH range of 6 to 8³⁷.

CONCLUSION:

The ideal composition of the nanoemulsion, comprising 15.28% water, 21.25% oil, and 63.47% emulsifier has been determined by implementing the simplex lattice design method. The nanoemulsion exhibits good characteristic values, including a particle size of 108.8 (<500 nm), a PDI of 0.443 (<0.5), a percent transmittance of 99.8 (approaches 100%), and a pH value of 5.276 (in the range of 5 to 6.5). No significant difference between the predicted and observed values indicates that the simplex lattice design method is advantageous for pharmaceutical development, such as optimizing nanoemulsions.

CONFLICTS OF INTEREST:

The authors have not disclosed any financial conflict of interest.

ACKNOWLEDGMENTS:

The authors expressed their gratitude to the Project Management Unit (PMU) of Maulana Malik Ibrahim State Islamic University for funding this research, which allowed it to be completed successfully.

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Received on 24.02.2024 Revised on 14.08.2024

Accepted on 19.11.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2237-2243.

DOI: 10.52711/0974-360X.2025.00320

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Response	Model	Regression equation	R²	Adjusted R²	Predicted R²
Particle size	Special quartic	Y = 118.61 A + 0.2147 B + 0.2147 C + 606.66 AB + 294.98 AC + 2.58 BC + 53.27 A²BC – 6948.73 AB²C – 4143.59 ABC²	0.9943	0.9851	0.9318
PDI	Special quartic	Y = 0.5504 A + 0.0014 B + 0.0014 C + 0.5477 AB + 1.72 AC + 0.0171 BC	0.9657	0.9109	0.6840
Transmittance	Special qubic	Y = 98.88 A + 99.44 B + 97.92 C – 1.35 AB – 3.59 AC – 5.41 BC	0.4778	0.0301	-1.1347
pH	Quadratic	Y = 5.34 A + 5.32 B + 6.03 C – 0.9428 AB – 0.2586 AC – 0.5943 BC	0.9824	0.9714	0.9508