Optimization, Characterization and Ex-vivo permeation of Nanoemulsion containing Diclofenac sodium as the development of Novel Nano-drug Delivery System

 

Wildan Khairi Muhtadi¹*, Bambang Hernawan Nugroho², Oktavia Indrati²,

Ronny Martien³, Nofriyanti¹

¹Department of Pharmacy, Sekolah Tinggi Ilmu Farmasi (STIFAR) Riau, Pekanbaru, Indonesia.

²Department of Pharmacy, Faculty of Mathematic and Natural Science,

Universitas Islam Indonesia, Yogyakarta, Indonesia.

³Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia.

 

ABSTRACT:

This study aimed to optimize the diclofenac sodium (DS)-loaded nanoemulsion (DSNE)s components, to characterize the optimum formula of DSNEs, including determining its skin penetration ability. DSNEs were optimized by the aqueous titration method to decide the optimum ratio of each component. The yielded diagram of pseudo-ternary phase was used on the considerationof  the optimum formulas. The characterization of three optimum DSNEs was done by measuring the globule size, polydispersity index, zeta potential, pH, viscosity, kinetic stability, and ex-vivo permeation. One Way ANOVA (95% confidence interval) was used to analyze the cumulative DS penetrated. The optimum formulations were found with the oil:smix (surfactant:cosurfactant) ratio of 1:7(1:1), 1:7(2:1), and 1:7(3:1), which coded as DSNE1, DSNE2, and DSNE3, respectively. The three optimum formulations possessed the average droplet size of below 200nm, polydispersity index of lower than 0.7, and zeta potential of above -30 mV, respectively. There were no phase separations in the centrifugation test. DSNE3 possessed the highest DS penetrated and flux compared to other formulations. The optimization of DSNEs yielded three optimum formulations with good characteristics in accordance with the acceptance criteria of nanoemulsions. Furthermore, the smaller particle size and higher concentration of T80 enhanced the ability of DS penetration.

 

 

 

INTRODUCTION: 

Diclofenac sodium (DS) is one of the NSAIDs (Nonsteroidal anti-inflammatory drugs) that generally used in order to cure inflammation. DS shows the ability to block cyclooxygenase-2 that produces inflammatory mediators. Regrettably, DS also inhibits cyclooxygenase-1 which is known as the producer of gastric mucous that prevents gastric damage. In the oral administration, DS undergoes hepatic first-pass metabolism extensively that further leads to low drug bioavailability^1,2.

 

In order to overcome several disadvantages of orally administered DS described above, transdermal drug delivery of DS need to be developed.

 

Nanoemulsions (NEs) are considered as the promising dosage form in transdermal delivery of DS. These dosage forms are defined as the dispersions of one within another liquid immiscibly with the droplets sized smaller than 200 nm containing active compounds. Due to the immensely small particle size that is formed by the presence of surfactant and cosurfactant that stabilize the dispersions, NEs possess the transparent appearance and thermodynamic stability^3–5. The particle size has been proven as a factor that affects the penetration of NEs, in which the smaller particles own an ability to penetrate into the layers in the deeper skin^6,7. Thus, the preparation of diclofenac sodium-loaded nanoemulsions (DSNEs) can be the effective transdermal delivery of DS.

 

One of the methods that can be used in the preparation of DSNEs is the spontaneous emulsification method. This method is preferred to be implemented due to its simplicity, since the absence of the use of homogenization equipment that eventually leads to low cost of preparation. DSNEs are formed by a gentle stirring of oil, water, surfactant and cosurfactant together at room temperature. Nevertheless, the method requires surfactant in the relatively high concentration, lowering the safety of NEs^8–10. Therefore, an optimization in the process of spontaneous emulsification is needed in in the strive to determine the optimum DSNEs components, especially the minimum amount of surfactant that able to form DSNEs.

 

This study aimed to obtain the optimum concentration of DSNEs components and also to characterize the optimum formula of DSNEs, including to determine its skin penetration ability. The spontaneous emulsification method was conducted using aqueous titration technique to construct the phase diagram and to observe the phase behavior in the emulsification process. To the best of our knowledge, the method described above had not been reported to be carried out in the preparation of DSNEs.

 

MATERIALS AND METHODS:

Materials:

Diclofenac sodium (DS) was purchased from Pharmaceutical Chemistry Laboratory, Universitas Islam Indonesia (Yogyakarta, Indonesia). Virgin coconut oil (VCO) was purchased from Lansida Group (Yogyakarta, Indonesia). Polysorbate 80 (tween 80) was purchased from Bratachem (Yogyakarta, Indonesia). Glycerin was purchased from P&G Chemicals (Singapore). Propylene glycol was purchased from Dow Chemical Pacific (Singapore). Methanol (analytical grade), potassium dihydrogen phosphate, and sodium hydroxide were purchased from Merck (New Jersey, USA).

 

Solubility Study:

DS solubility in the oil phase of NEs was determined by adding the excess amount of DS into a test tube loaded with 5ml of VCO. The mixture was mixed by using a vortex mixter and the test tube was then kept in a shaker at toom temperature for 24hours. Subsequently, the equilibrated samples were centrifugated at 3000rpm for 15minutes. The supernatant was collected and membrane with the pore size of 0.45µm was used to filter the sample. Moreover, the filtered sample was diluted with methanol and the DS concentration was analyzed using UV-Vis Spectrophotometer (Shimadzu UV-1650 PC) at 282nm.

Formula Optimization and Construction of Phase Diagram:

Formula optimization was conducted by using the aqueous titration method. Tween 80(T80) and Glycerin (GC) as the surfactant and cosurfactant, respectively, were combined (Smix) in several ratios of volume (1:1, 2:1, 3:1, 4:1, 5:1, and 6:1). The oil phase (VCO) and specific Smix ratios as mentioned previously were mixed in various volume ratios that were 1:9, 1:8, 1:7, 1:6, and 1:5. The final volume ratios of VCO:Smix (T80:GC) were 1:9(1:1); 1:9(2:1); 1:9(3:1); 1:9(4:1); 1:9(5:1); 1:9(6:1); 1:8(1:1); 1:8(2:1); 1:8(3:1); 1:8(4:1); 1:8(5:1); 1:8(6:1); 1:7(1:1); 1:7(2:1); 1:7(3:1); 1:7(4:1); 1:7(5:1); 1:7(6:1); 1:6(1:1); 1:6(2:1); 1:6(3:1); 1:6(4:1); 1:6(5:1); and 1:6(6:1). Aqueous phase was titrated slowly into each volume ratios and the phase behavior was observed visually in the interval of 5% of aqueous phase addition (Table 1). DS was incorporated initially into VCO with the final concentration of 1% (b/v) at each volume ratios. The physical states at each observation points of each volume ratios were plotted on pseudo-ternary phase diagrams representing the percentage of three main components of NEs, which were oil, aqueous phase, and Smix¹¹. The phase diagrams were made using ProSim Ternary Diagram 1.0 free software.

 

Table (1): Calculation of percentage of DSNEs components for the production of pseudo-ternary phase diagrams (ratio of oil:Smix was 1:7).

Oil (ml)	Smix (ml)	Water (ml)	Water Added (ml)	Total (ml)	Oil (%)	Smix (%)	Water (%)
2	14	1.8	0	17.8	11.24	78.65	10.11
2	14	3	1.2	19	10.53	73.68	15.79
2	14	4	1	20	10.00	70.00	20.00
2	14	5.4	1.4	21.4	9.35	65.42	25.23
2	14	7	1.6	23	8.70	60.87	30.43
2	14	8.6	1.6	24.6	8.13	56.91	34.96
2	14	10.7	2.1	26.7	7.49	52.43	40.07
2	14	13.5	2.8	29.5	6.78	47.46	45.76
2	14	16	2.5	32	6.25	43.75	50.00
2	14	19.6	3.6	35.6	5.62	39.33	55.06
2	14	24	4.4	40	5.00	35.00	60.00
2	14	30	6	46	4.35	30.43	65.22
2	14	37.5	7.5	53.5	3.74	26.17	70.09
2	14	48.5	11	64.5	3.10	21.71	75.19
2	14	64.5	16	80.5	2.48	17.39	80.12
2	14	90.5	26	106.5	1.88	13.15	84.98
2	14	143.5	53	159.5	1.25	8.78	89.97
2	14	303.5	160	319.5	0.63	4.38	94.99

 

Formula Selection:

Formulas that would be employed in the characterization studies were selected by considering the formed phase diagrams. The selected formulas were those with the maximum NEs area in the phase diagrams, lowest amount of surfactant, and maximum concentration of aqueous phase¹².

 

The Measurement of Particle Size, Polydispersity Index, and Zeta Potential:

Particle size, polydispersity index, and zeta potential of DSNEs were analyzed by using the instrument of particle size analyzer (Horiba SZ-100). 1ml of sample was added with demineralized water to the volume of 10 ml. Subsequently, the diluted sample was put into the cuvette and analyzed in triplicate.

 

pH Measurement:

The pH of DSNEs were determined by using pH meter (Horiba ®LaquaAct) in triplicate at room temperature.

 

Viscosity Studies:

The viscosity of DSNEs was determined in triplicate by using the viscometer of Brookfield KU-2.

 

Centrifugation Studies:

Kinetic stability of DSNEs were determined by conducting centrifugation study. The centrifugation of samples was conducted using centrifugator (Thermo Scientific) at the rate of 4000rpm for 15 minutes. Finally, the formulations was considered kinetically stable when no phase separation observed.

 

Ex-Vivo Skin Permeation Studies:

The study protocols of the ex-vivo skin permeation test were reviewed and approved by the Ethics Committee of the Faculty of Medicine, Islamic University of Indonesia with the registration number of 50/Ka.Kom.Et/70/KE/III/2015. The Ex-vivo skin permeation of DSNEs was observed using Franz diffusion cell. Skins were obtained from the dorsal region of male Wistar mouse with weight ranged 200-300g. Skin’s hair in the dorsal region was carefully removed and the skin was subsequently excised with an area of 2.5 x 2.5 cm of each mouse. Subcutaneous fat existed on the excised skins were trimmed and the fat-free skins were soaked in phosphate buffer pH 6.8 for 2 hours.

 

Prior to the beginning of the study, the skins were placed between the receptor and donor compartments of Franz diffusion cell. Phosphate buffer pH 6.8 was added to the receptor compartment and stirred with a magnetic bar. The area of effective diffusion was 1.77 cm². Samples (1 g) was situated on the upper side of skins on the donor compartment. The test was maintained at the temperature and stirring speed of respectively 37 ± 0.5°C and 100rpm. Aliquots from the receptor compartment were collected (1 ml) at the interval of 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours. Furthermore, the addition of a same volume of buffer solution was immediately conducted to the receptor compartment to maintain sink condition. The concentration of DS in the withdrawn aliquots were measured using UV-Vis spectrophotometer (Shimadzu UV-1650 PC) at 276nm. The cumulative penetrated amount of DSNEs were plotted with time function. The rate of penetration at the steady-state (J_ss, µg/cm².h) was determined as the linear section’s slope of permeation profiles. The coefficient of permeability of DSNEs was calculated by dividing the permeation rate with the initial concentration of DS within the DSNEs^13,14.

 

Data Analysis:

The characterization data were provided as mean ± standard deviation with three replications. One Way Anova was utilized to define the statistical difference of each formula with a confidence interval of 95%.

 

Results and Discussion:

Solubility Study of DS:

Based on the study, DS solubility in VCO was 39.40± 0.19mg/ml, which indicated that 1gram of DS can be solubilized by using 25.39±0.13ml of VCO. On the other hand, 37.43±0.05ml of water was needed to solubilize 1gram of DS, since the solubility of DS in water was 26.72±0.04mg/ml.

 

Formula Optimization and Production of Pseudo Ternary Phase Diagram:

 

Figure (1): Nanoemulsion region indicated by pseudo ternary phase diagram with the Smix ratio of 3:1. The region was marked by red dotes within the thick black line.

 

The optimization study indicated that NEs were formed from the beginning to the last drop of the aqueous phase titration at various ratios of oil:Smix (T80:GC). The ratios exhibited transparent appearance were 1:9(4:1); 1:9(5:1); 1:9(6:1); 1:8(4:1); 1:7(1:1); 1:7(2:1); and 1:7(3:1) (Table 2). The largest area of formed NEs was illustrated by the pseudo ternary phase diagram with T80:GC ratio of 3:1 (Figure 1).

 

Table (2): Observed appearances at certain points of water addition for the construction of phase diagram using various ratios of oil and Smix.

Ratio of oil:Smix (T80:GC) (ml)

Visuality observed after addition of water

1 ml

1 ml

0.5 ml

1 ml

1 ml

1 ml

1 ml

1.5 ml

2 ml

2 ml

3 ml

3.5 ml

5 ml

6.5 ml

10 ml

15 ml

35 ml

110 ml

1:9 (1:1)

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

1:9 (2:1)

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

1:9 (3:1)

NE

NE

NG

NG

NG

NG

NG

EG

EG

EG

EG

EG

E

E

E

E

E

E

1:9 (4:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

1:9 (5:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

1:9 (6:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

2 ml

1.2 ml

1.3 ml

1.5 ml

1.8 ml

1.9 ml

2.3 ml

2.7 ml

3.3 ml

4 ml

5 ml

6.5 ml

8.5 ml

12 ml

18 ml

30 ml

60 ml

180 ml

1:8 (1:1)

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

1:8 (2:1)

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

E

1:8 (3:1)

NE

NE

NE

NE

NG

NG

NG

NG

NG

NG

NG

E

E

E

E

E

E

E

1:8 (4:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

1.8 ml

1.2 ml

1 ml

1.4 ml

1.6 ml

1.6 ml

2.1 ml

2.8 ml

2.5 ml

3.6 ml

4.4 ml

6 ml

7.5 ml

11 ml

16 ml

26 ml

53 ml

160 ml

1:7 (1:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

1:7 (2:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

1:7 (3:1)

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

NE

*E = emulsion; NE = nanoemulsion; NG = nanogel; EG = emulgel

 

Formula Selection:

Based on the optimization study, the consideration in the selection of formula was that the formula possessed the largest area of NEs in the phase diagram and the low concentration of surfactant. Three selected formulations were 1:7(1:1); 1:7(2:1); and 1:7(3:1), which were coded as DSNE1, DSNE2, and DSNE3, respectively. The constituents of each formulation were provided in Table 3. The appearances of the three formulations were shown in Figure 2.

 

 

Figure (2): The appearance of DSNEs with the formulation code of DSNE1 (a), DSNE2 (b), and DSNE3 (c).

 

Characterization of DSNEs:

Table (3): Characterization results of selected formulations (n = 3)

Code	Percentage of Components				Particle Size (nm)	Polydispersity Index	Zeta Potential (mV)	Viscosity (cP)	pH	Centrifugation
	Drug (% w/v)	Oil (% v/v)	Smix (T80:GC) (% v/v)	Water (% v/v)						Before	After
DSNE1	1	3	21.7 (10.85:10.85)	75.3	44.7 ± 17.8	0.389 ± 1.103	-46.9 ± 0.86	570.67 ± 7.09	7.98 ± 0.01	NS	NS
DSNE2	1	3	21.7 (14.50:7.20)	75.3	39.8 ± 18.5	0.348 ± 0.089	-46.8 ± 0.79	622 ± 5.00	7.90 ± 0.02	NS	NS
DSNE3	1	3	21.7 (16.30:5.40)	75.3	14.1 ± 1.3	0.512 ± 0.093	-42.0 ± 0.72	638 ± 7.21	7.80 ± 0.03	NS	NS

*NS = No phase separation

 

 

 

 

The characterization results of three selected DSNEs formulations were shown in Table 3. The results met the requirements of good NEs. The mean size of particles of the three formulations were smaller than 200 nm. Moreover, the  polydispersity index and zeta potential values of selected formulations were lower than 0.7 and more than -30 mV, respectively. The distributions of dispersed particle dimensions and zeta potential of the three formulations were shown in Figure 3 and Figure 4, respectively. The results of pH and viscosity of the three selected formulations indicated the suitability of formulations to be applied on the surface of skin. The results of the centrifugation test indicated no phase separations observed in all of the selected formulations.

 

 

(a)

 

 

(b)

 

 

(c)

Figure (3): Particle size distributions of DSNE1 (a), DSNE2 (b), and DSNE3 (c).

 

(a)

 

(b)

 

(c)

Figure (4): Zeta potential distributions of DSNE1 (a), DSNE2 (b), and DSNE3 (c).

 

Ex-vivo Skin Permeation:

The cumulative DS permeated resulted in 8 hours was shown in Figure 5, which DSNE3 possessed the highest cumulative DS permeated (323.49 ± 170.52 µg/cm²) among three formulas (P<0.05). The values of flux and permeability coefficient (K_p) of DSNE3 were 35.63 µg/cm²/h and 3.5 x 10^-3 cm/h, respectively (Figure 6).

 

 

Figure (5): Cumulative of DS permeated from several NE formulations (n = 3).

 

 

(a)

 

(b)

Figure (6): The steady-state flux (a) and permeability coefficient (b) values of DSNE formulations (n = 3).

 

DISCUSSION:

The results of the solubility study indicated that the solubility of DS in VCO was higher than in the aqueous phase. The solubility results described that the system of oil in water (o/w) nanoemulsions were formed, in which DS was entrapped within the dispersed oil particles^15,16. The phase diagram with the Smix ratio of 3:1 indicated that a higher number of Smix were needed to solubilize the higher number of oil. It theoretically takes place due to the increase of VCO penetration into the hydrophobic region of the surfactant monomers, which lead to more Smix required to decrease the interfacial tension in the formation of nanoemulsions^11,17. Three selected formulas were chosen to be used in further studies based on the concentration of surfactants. Since the large amounts of surfactant can cause skin irritation, the formulas with the ratio of oil and smix of 1:7 were selected instead of 1:8 and 1:9¹⁸.

 

The size of the dispersed phase particles of the nanoemulsions is highly influenced by the surfactant concentration. As the amounts of surfactant in the formula were increased, the particle size lowered, which was clearly described by the particle size of three selected formulas. DSNE1 which contained lowest amounts of surfactant showed the highest particle size compared to the two other formulas, while DSNE3 with the highest concentration of surfactant possessed the lowest dispersed particle size. This finding is in accordance with the theory that the increase of surfactant concentration yields a larger oil-water interface. Co-surfactant also plays a role in lowering the size of dispersed particles. GC as the co-surfactant in the present study aided the surfactant with the additional reduction of interfacial tension. At the optimum ratios of T80 and GC, the suitable size of the dispersed particles was achieved and the stable nanoemulsions were formed^19,20. In addition, GC also hindered the viscosity rising of the obtained system by increasing the fluidity of the interfacial film formed by T80²¹.

 

Polydispersity index (PDI) illustrates the uniformity of the particles inside the NE system. PDI values of less than 0.7 are considered as a monodisperse system indicating a narrow size distribution of dispersed particles, which enable the dynamic light scattering method to analyze the nanoemulsion sample²². In contrast, PDI values that close to 1.0 trigger the condition of Ostwald ripening to take place. Ostwald ripening is a phenomenon in which the smaller particles with higher Laplace pressure tend to merge with the larger particles with lower Laplace pressure, which eventually form the larger dispersed particle size²³. Zeta potential of the optimized formulas showed that the three nanoemulsion formulas were negatively charged. The formed negative charged was generated by the existence of the anionic groups of glycol and fatty acids owned by VCO and smix¹⁷. Zeta potential plays a significant role in maintaining a stable condition of nanoemulsions in long term storage. The zeta potential value of a nanoemulsion preparation of above -30mV avoids the occurrence of coalescence in the formulation due to strong repulsive force between the particles²⁴. The centrifugation test was conducted as the part of stability observation of newly prepared nanoformulations. Centrifugation is considered as the stress condition that could promotes a faster breakage of emulsion²⁵. The three nanoformulations tend to be stable in the storage of one year period²⁶.

The skin permeation test indicated that the penetration of DS through the skin membrane was affected by the particle size of the formulations. DSNE3, as the formulation with the lowest mean particle size showed the highest cumulative of DS penetrated through the skin membrane, followed by DSNE2 and DSNE1, respectively. Based on the study conducted by Yokota and Kyotani, nanoformulations with the dispersed particle size ranged 70-80nm owned the ability penetrate the horny layer, as the outermost skin layer. It was caused by the approximately 50-100nm dimension range of the intercellular spaces situated in the horny layer²⁷. The small particles possess a higher possibility to stick with membranes, as well as providing a wide surface area for DS penetration into the skin¹⁵. Sinergistically, penetration of DS through the skin membrane was affected by the concentration of T80, which had known to played the role as the penetration enhancer beside of its main role as the surfactant. The penetration enhancers significantly extract the lipids of stratum corneum that lead to a decrease of stratum corneum’s barrier properties^28,29. This theory was confirmed by the result of the permeation test in the present study. DSNE3, which was the formula with the highest percentage of T80, penetrated the most amount of DS compared to the other formulations (P<0.05).

 

Results of the other permeation parameters, which were steady-state flux and permeability coefficient indicated insignificant differences between each formulation (P>0.05). Normally, the flux values of the same compound would be similar at every condition of formulations, in the case of the components of the formula showed no interactions with the skin membrane. However, the results of the other study indicated there was a difference of flux values due to the variation of surfactant concentration. A rise in surfactant amount generated a higher rate of drug permeated and subsequently, further increase of flux was delayed when the certain surfactant concentration was reached^30–32. In the present study, further investigation is expected to observe the presence of interaction of the formula’s components with the skin membrane. Thus, the effect of surfactant concentrations on the flux and permeability coefficient value could be determined.

 

CONCLUSION:

The optimization of DS-loaded nanoemulsion was succesfully conducted with the three obtained optimum formulations with the oil:smix (surfactant:cosurfactant) ratio of 1:7(1:1), 1:7(2:1), and 1:7(3:1), respectively. The characterization studies indicated that the three optimum formulations possessed good characteristics in accordance with the acceptable criteria of nanoemulsions. DSNE3, which possessed the lowest particle size and the highest amount of T80 among the three optimum formulations indicated the best ability to penetrate DS through the skin membrane (P<0.05).

 

CONFLICT OF INTEREST:

Authors declare that there is no conflict of interest is related with this study. The authors are fully responsible for all of data included of this article.

 

ACKNOWLEDGMENTS:

The authors would like to thank Sekolah Tinggi Ilmu Farmasi Riau for funding the present study, as well as Nanopharmacy Research Center, Department of Pharmacy, Universitas Islam Indonesia, for providing the facilities needed in this study.

 

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Accepted on 03.11.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2257-2264.