Preparation of Diclofenac Diethylamine Nanoemulsions by Ultrasonication-Stability and Process Parameter Evaluation under Various Conditions

Praveen Kumar Gupta¹*, J.K. Pandit, P.J. Narain, R.N. Gupta and Sanjiv Kumar Gupta

¹Agra Public Institute of Technology and Computer Education, Agra

Department of Pharmaceutics, IIT-B.H.U., Varanasi.

Department of Pharmaceutical Sciences, B.I.T. Mesra

ABSTRACT:

In this study, oil-in-water nanoemulsions of Diclofenac Diethylamine were produced by Ultrasonication. The influence of emulsifying conditions including emulsifier type and concentration, homogenization pressure, temperature, cycle, on time, off time and total time on the properties and stability of the nanoemulsions were investigated using a Zetasizer. The mean diameters (z-average) of the dispersed particles containing Diclofenac Diethylamine ranged from 50.57 to 154.9 nm and the polydispersity index ranged from 0.318 to 0.719 and the zeta potential ranged from19.2 to35.3. The nanoemulsions produced with Tween-80 had the smallest particle sizes and narrowest size distribution. The particle sizes decreased with increases in homogenization pressure and cycle, and also with temperature up to 40ºC. The physical stability of the nanoemulsions increased with the elevation of temperature up to 40º C, with pressure up to 200 MPa and homogenization cycle (up to three cycles).

KEYWORDS: Nanoemulsion; Diclofenac Diethylamine, Ultrasonication; Particle size; Zetasizer;

INTRODUCTION:

In the last two decades, nanotechnology is rapidly emerging as one of the most promising and attractive research fields. The technology offers the potential to significantly improve the solubility and bioavailability of many functional ingredients including Diclofenac Diethylamine and numerous other compounds. Hitherto, however, researches into the application of this technology in the industry have been limited and there are only a few publications that explored the use of this technology for preparing nanoemulsions by ultrasonication.¹

Ultrasonic emulsification is believed to occur through two mechanisms. Firstly, the application of an acoustic field produces interfacial waves which become unstable, eventually resulting in the eruption of the oil phase into the water medium in the form of droplets ² Secondly, the application of low frequency ultrasound causes acoustic cavitations, that is, the formation and subsequent collapse of micro bubbles by the pressure fluctuations of a simple sound wave. Each bubble collapse (an implosion on a microscopic scale) event causes extreme levels of highly localized turbulence. The turbulent micro-implosions act as a very effective method of breaking up primary droplets of dispersed oil into droplets of sub-micron size³.

Studies to date comparing ultrasonic emulsification with rotor–stator dispersing have found ultrasound to be competitive or even superior in terms of droplet size and energy efficiency ⁴ It may also be more practicable with respect to production cost, equipment contamination and aseptic processing than a microfluidisation approach.⁵comparing mechanical agitation to ultrasound at low frequency,⁶ found that for a given desired diameter, the surfactant amount required was reduced, energy consumption (through heat loss) was lower and the ultrasonic emulsions were less polydisperse and more stable.

Diclofenac is a well-established Non steroidal anti-inflammatory agent, widely used in musculoskeletal disorders, arthritis, toothache, dysmenorrhea, symptomatic relief of pain and inflammation⁷. Diethylamine salt of diclofenac is reportedly used for topical application⁸Diclofenac Diethylamine possess the ideal characteristics for the preparation of novel drug delivery system, such as short biological half-life (2-3 hr), smaller dose (25-50mg) one of the approaches that can be used to improve the solubility and bioavailability of Diclofenac Diethylamine is to incorporate them in the fine particles of oil-in water (O/W) nanoemulsions.

In this article, Diclofenac Diethylamine nanoemulsion by ultrasonication were prepared and investigated the influence of phase ratio of emulsifier and their concentration, homogenization pressure, homogenization cycle, temperature ,on time , off time and total time exposure on particle size parameters, polydispersity and zeta potential of the nanoemulsions was systematically examined using a dynamic light scattering (DLS) technique.

MATERIALS AND METHODS:

Materials:

Diclofenac Diethylamine (Pulverised) was a gift sample from Pee-Medica(Agra, India). Isopropyl myristate (IPM), Tween-80 and Tween-20 were purchased from E-Merck (Mumbai, India). Diethylene glycol monoethyl ether (Transcutol ), were purchased from CDH. All other chemicals used in the study were of analytical reagent grade.

Preparation of Diclofenac Diethylamine nanoemulsions:

Oil-in-water (O/W) nanoemulsions were prepared using Isopropyl Myristate oil, Tween-80 and Tween-20 as the emulsifier, Transcutol as cosurfactant, Diclofenac Diethylamine as the drug, as dispersed phase and Milli-Q water as the continuous phase. The emulsifier was used Tween-20 and Tween-80 the concentration (in the final emulsion) were 5% and 10% at a fixed homogenization temperature, stage pressure and cycle (40ºC, 150 MPa, and three cycles), respectively. The emulsions were sampled and their particle size, size distribution and emulsion stability were measured.

Using the above procedure, several batches were prepared by varying the emulsifier and their concentration, and homogenization pressure, temperature and cycle to study their effects on the characteristics of the nanoemulsions.

Ultrasonication Process:

Ultrasonication process performed under optimized operating conditions of power intensity, homogenization cycle, temperature, total time and reaction time (On time and off time). 100ml dilution formulation mixes were taken into reaction vessel the horn probe of ultrasonicator was directly immersed into the reaction vessel at a depth of about 1cm for every running.

Figure 1: Probe Sonicator Used In Laboratory Scale for Preparation of Nanoemulsion

Analysis of particle size and distribution:

The average particle size and size distribution of the nanoemulsions were determined by dynamic light scattering using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). The measurement was carried out at a fixed angle of 90º with the samples diluted approximately 100 times with Milli-Q water. The particle size of the emulsions was described by the cumulants mean (z-average) diameter and the size distribution by the polydispersity index (PdI).

Evaluation of emulsion stability:

The stability of the nanoemulsions was evaluated by Zeta potential using Zetasizer.

Statistical analysis:

The whole experiment was conducted in duplicate and all analyses were done at least in triplicate. The data were analyzed by one way analysis of variance (ANOVA) using the SPSS 12.0 package. Significant differences of means were determined by the Duncan’s multiple range tests.

Table 1: Praticle size, Polydispersity index and Zeta Potential of Diclofenac Diethylamine nanoemulsions prepared with different emulsifiers concentration

(mean ± SD, n=3)^a

Emulsifiers	Property	Concentration of emulsifiers
Emulsifiers	Property	5%	10%
Tween-20	D(nm)^b PdI^c Zeta potential (mV)	152.6±4.28 0.537±0.050 -24.6±0.6	68.97±1.12 0.363±0.033 -27.2±0.4
Tween-80	D(nm) PdI Zeta potential (mV)	115.1±1.82 0.337±0.026 -23.9±0.4	57.50±1.62 0.318±0.024 -35.3±0.3

A–I For the PdI values, data followed significantly different (P < 0.05).

a. The emulsions were prepared at the homogenization temperature, pressure and cycle of 40ºC, 150 MPa and 3 cycles, respectively.

b. D (nm), cumulant mean (z-average) diameter of the nanoemulsion particles.

c. PdI, polydispersity index.

Figure 2: Particle Size and Zeta Potential of Diclofenac Diethylamine nanoemulsion different emulsifier and concentration

(i-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion Prepared with Tween-20 at a Concentration 5% w/w and a 150 MPa and 40ºC

(i-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion Prepared with Tween-20 at a Concentration 5% w/w

(ii-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion Prepared with Tween-80 at a Concentration 5% w/w and a 150 MPa and 40ºC

(iii-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion Prepared with Tween-20 at a Concentration 10% w/w and a 150 MPa and 40ºC

(iv-a)Particle Size distribution in the Diclofenac Diethylamine nanoemulsion Prepared with Tween-80 at a Concentration 10% w/w and a 150 MPa and 40ºC

(ii-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion Prepared with Tween-80 at a Concentration 5% w/w

(iii-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion Prepared with Tween-20 at a Concentration 10% w/w

(iv-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion Prepared with Tween-80 at a Concentration 10% w/w

Table 2: Praticle size, Polydispersity index and Zeta Potential of Diclofenac Diethylamine nanoemulsions prepared at different homogenization Cycles

(mean ± SD, n=3)^a

Homogenization Cycles	I	II	III
D (nm)	154.9 ± 1.6	112.4 ± 1.3	57.88 ± 0.9
PdI	0.532 ± 0.005	0.447 ± 0.003	0.386 ± 0.002
Zeta potential (mV)	-23.3 ± 0.7	-28.4 ± 0.4	-34.8 ± 0.7

A–I For the PdI values, data followed significantly different (P < 0.05).

Figure 3: Particle Size and Zeta Potential of Diclofenac Diethylamine nanoemulsions prepared at different homogenization cycles

(i-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion I^st Homogenization Cycle

(i-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion in

I^st Homogenization Cycle

(ii-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion II^nd Homogenization Cycle

(iii-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion in II^nd Homogenization Cycle

(iv-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion III^rd Homogenization Cycles

(iv-b)Zeta Potential of Diclofenac Diethylamine nanoemulsion in III^rd Homogenization Cycle

Table 3: Praticle size, Polydispersity index and Zeta Potential of Diclofenac Diethylamine nanoemulsions prepared at different homogenization temperatures (mean ± SD, n=3)^a

Temperature (ºC)	20	40	60
D (nm)	64.54±1.2	50.57±1.1	96.9±1.4
PdI	0.471±0.009	0.456±0.004	0.719±0.048
Zeta potential (mV)	-25.6±0.8	-33.9±0.7	-31.6±0.9

A–I For the PdI values, data followed significantly different (P < 0.05).

Figure 4: Particle Size and Zeta Potential of Diclofenac Diethylamine nanoemulsions prepared at different homogenization Temperature

(i-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 20⁰C Homogenization Temperature

(i-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion

at 20⁰C Homogenization Temperature

(ii-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 40⁰C Homogenization Temperature

(ii-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion

at 40⁰C Homogenization Temperature

(iii-a) Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 60⁰C Homogenization Temperature

(iii-b)Zeta Potential of Diclofenac Diethylamine nanoemulsion

at 60⁰C Homogenization Temperature

Figure 5: Particle Size and Zeta Potential of Diclofenac Diethylamine nanoemulsions prepared at different homogenization pressure

(i-a)Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 50 MPa Homogenization Pressure

(i-b)Zeta Potential of Diclofenac Diethylamine nanoemulsion

at 50 MPa Homogenization Pressure

(ii-a)Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 100 MPa Homogenization Pressure

(iii-a)Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 150 MPa Homogenization Pressure

(iv-a)Particle Size distribution in the Diclofenac Diethylamine nanoemulsion at 200 MPa Homogenization Pressure

(ii-b)Zeta Potential of Diclofenac Diethylamine nanoemulsion at 100 MPa Homogenization Pressure

(iii-b) Zeta Potential of Diclofenac Diethylamine nanoemulsion at 150 MPa Homogenization Pressure

(iv-b) Zeta Potential of Diclofenac Diethylamine nanoemulsionat 200 MPa H omogenization Pressure

RESULTS AND DISCUSSION:

Ultrasonication is one of the most frequently used techniques for preparing emulsions. The Diclofenac Diethylamine nanoemulsions prepared in this study were typical oil-in-water (O/W) emulsions. It is well known that emulsifying parameters such as the type and concentration of emulsifiers, homogenization temperature, pressure and cycle can affect the physicochemical properties and stability of the emulsions. Therefore, this study was conducted to investigate the effect of these parameters on the properties of the Diclofenac Diethylamine nanoemulsions with the aim of finding the optimal conditions.

Effect of the emulsifiers and their concentration change on the Praticle size and Polydispersity index:

Particle size parameters of Diclofenac Diethylamine nanoemulsions prepared with different emulsifiers Tween-80 and Tween-20 at various concentration 5% and 10%. The cumulates mean diameters (z-average) of the nanoemulsion particles of Tween-80 at 5% is 115.1 and at 10% is 57.5, with the polydispersity index (PdI) 0.337 and 0.318 respectively, while Tween-20 at 5% particle size is 152.6 and at 10% 68.97 with polydipersity index 0.537 and 0.363 respectively.

These results therefore proved that the nonionic emulsifiers, Tween-80 and Tween-20, can be used to produce Diclofenac Diethylamine emulsions with the dispersed droplets in the nanometer range. Of the two different emulsifiers used, Tween-80 produced nanoemulsions with the smallest particle size at 10% concentrations studied. These observations can probably be explained by the higher solubility of the drug in the emulsifier, emulsifiers with greater solubility could wrap and stabilize the particles in an O/W emulsion more efficiently, thus resulting in smaller particles.

As expected, increasing the emulsifier concentration from 5% to 10% generally resulted in a significant (P < 0.05) decrease in the particle size. This is because smaller particle sizes meant greater surface areas, which would require more emulsifiers to cover. However, the effect of emulsifier concentration on the particle size reached a plateau for Tween-80 at 10% as reported. This is most likely due to that at certain emulsifier concentrations, all the droplets in the emulsion were fully covered by the emulsifiers and excessive emulsifiers in the system would not be utilized unless the particle sizes could be reduced further.

The PdI value measures the spread of the particle size distribution and, thus, a small PdI value indicates a narrow particle size distribution.⁹In general, the Diclofenac Diethylamine nanoemulsions all exhibited a relatively narrow range of size distribution with the PdI values range 0.318-0.537 (0 being the smallest and 1 the largest possible values).

Nanoemulsions prepared with Tween-80 at 10% had the narrowest of size distribution with PdI 0.318.

Effect of homogenization cycle on the Praticle size and Polydispersity index:

The effect of homogenization cycle on the properties of Diclofenac Diethylamine nanoemulsions is as expected; increasing the homogenization cycle resulted in significant decreases (P < 0.05) in both the particle size and the range of particle distribution. However, after passing the emulsion through the ultrasonication for three times, subsequent passes had no further effect on the particle size. Furthermore, and in successive homogenization cycles, no further improvement on the size distribution was observed .¹⁵

Effect of homogenization temperature on the Praticle size and Polydispersity index:

Temperature can influence the particle size of the droplets in emulsions produced by ultrasonication. The influence may come from a number of ways including its effect on the viscosity, and the interfacial tension between, the oil and aqueous phases, both of which are temperature dependent ¹⁰, increasing the homogenization temperature from 20 to 40ºC resulted in significant (P < 0.05) decreases in the particle size. However, increasing the temperature 60ºC, the size actually increased significantly, at 60ºC and above it seems that Diclofenac Diethylamine was not stable.

This result is not unexpected because Tween-20 (the emulsifier used in this experiment) has a cloud point of about 76ºC ¹¹ and as the temperature approached this point, it could begin to lose the ability to prevent aggregation of emulsion droplets with consequent coalescence of some droplets¹². The effect of homogenization temperature on the size distribution, however, did not show a consistent pattern, although there were significant differences in the PdI values of emulsions prepared at different temperatures. The emulsion produced at the 60ºC had the highest PdI values, while that prepared at 40ºC had the lowest value.

Effect of homogenization pressure on the Praticle size and Polydispersity index:

Homogenization pressure can significantly influence the properties of emulsions as the shear forces and turbulence, both of which are pressure dependent, produced during homogenization can affect the particle size and size distribution^13,14. In this study, the effect of homogenization pressure on the properties of the Diclofenac Diethylamine nanoemulsions were studied by varying at 50 MPa, 100 MPa, 150 MPa and 200 MPa increasing the homogenization pressure resulted in significant (P < 0.05) decreases in the particle sizes over the entire pressure ranges studied, which agreed with the findings ¹⁵. However, at 200 MPa the slight increase in the particle size distribution.

Effect of emulsifiers and their concentration, homogenization cycle, temperature and pressure on Zeta potential:

The stability of the emulsion decreased with a rise in homogenization temperature over the 60ºC temperature range studied. The emulsion stability increased initially when the homogenization pressure was raised within the range of 50–200MPa. Similarly, when the homogenization cycle was increased to up to three cycles, the emulsion stability increased, but with a further increase in homogenization cycle, not a constant pattern in emulsion stability was observed. Emulsion stability is a complex issue and can be influenced by a number of factors including particle size, viscosity and environmental conditions such as temperature and shear force. In general, smaller particles have a lesser tendency to cream but a greater tendency to aggregate because they are more numerous at a given phase ratio and more susceptible to the influence of Brownian motion, both of which would lead to greater chance of collision .¹²It has been shown that when the particle sizes are smaller than 100 nm (many particles in the present study fell into this range), creaming would be greatly reduced and aggregation become a dominant mechanism for emulsion instability ¹². This may partly explain the results of the present study. As the homogenization temperature increased, the viscosity of the emulsions would decrease and the Brownian motion become more rapid, both of which would lead to more frequent particle collisions, with consequent aggregation and greater emulsion instability. The decreases in the stability of the nanoemulsions observed at high homogenization pressures (>200 MPa) and cycles (>3) could also be partly attributable to the increased particle collision and aggregation. This is because under these conditions, the collision frequency of the particles, which is recognized as one of the main contributing factors to aggregation in emulsions¹², would increase.

CONCLUSION:

This study confirmed that ultrasonication is a relatively simple and effective technique for producing Diclofenac Diethylamine oil-in-water nanoemulsions. The particle size and size distribution of the nanoemulsions were influenced by the emulsifiers and their concentrations, as well as homogenization temperature, pressure and cycle. The Diclofenac Diethylamine nanoemulsions had moderate physical stabilities.

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Received on 02.09.2011

Accepted on 11.09.2011

Research Journal of Pharmaceutical Dosage Forms and Technology. 3(6): Nov.- Dec., 2011, 285-293

Pressures (MPa)	50	100	150	200
D (nm)	150.9 ± 0.9	92.2 ± 0.7	59.64 ± 0.5	68.08 ± 0.6
PdI	0.511 ± 0.024	0.622 ± 0.036	0.324 ± 0.014	0.351 ± 0.019
Zeta potential (mV)	-19.2 ± 0.3	-25.8 ± 0.4	-32.5 ± 0.5	-34 ± 0.8