DEVELOPMENT AND EVALUATION OF NANOEMULSION FORMULATIONS FOR IMPROVED ORAL DELIVERY OF CARVEDILOL

Objective: The aim of the present investigation was to develop, optimize and evaluate nanoemulsion system of carvedilol to improve its solubility, and oral bioavailability. Carvedilol is a non-selective beta blocker used in the treatment of mild to moderate congestive heart failure and mild to moderate essential hypertension. It has both poor water solubility (0.583 mg/L) and oral bioavailability (23%) because of significant first-pass hepatic metabolism. Methods: Based on solubility testing, clove oil was used as oil, tween 20 was used as surfactants and PEG 400 was used as cosurfactants in construction of phase diagrams. Carvedilol nanoemulsions were prepared by aqueous phase titration method. Out of twelve formulations, eight thermodynamically stable formulations were selected for preparation of carvedilol loaded nanoemulsions and these nanoemulsions were subjected for characterization i.e. particle size, viscosity, polydispersity, zeta potential. A 12 hrs in-vitro release release study was performed on selected nanoemulsion formulations of carvedilol. Results: The results of viscosity of carvedilol nanoemulsions were found to be in range (60.42 –134.63 m Pa.sec.). The results of pH measurement for formulations explain that the pH values of drug free nanoemulsions were slightly acidic. All formulations have PDI value less than (1.0). Conclusion: Study concludes, nanoemulsion formulation of batch NEC4 (Smix ratio 1:3) was found to be optimum formulation.


INTRODUCTION
The term 'nanoemulsions' is used to designate emulsions with the internal phase droplets having size ranging from 50 to 1000 nm 1 . Nanoemulsions are dispersed particles used for pharmaceutical and biomedical aids and vehicles. Size of the droplets is governed by the surfactant phase structure (bicontinuous microemulsion or lamellar) at the inversion point induced by either temperature or composition 2 . They are composed of an oil phase, aqueous phase, surfactant and co surfactant at appropriate ratios. The particles can exist as water inoil and oil-in-water forms, where the core of the particle is either water or oil, respectively 3 . Nanoemulsions are based on low interfacial tension, achieved by adding a co-surfactant, which leads to spontaneous formation of a thermodynamically stable nanoemulsion. Nanoemulsions have high kinetic stability, low viscosity, and transparency/ translucency, are very attractive for a range of industrial applications, including the pharmaceutical field where they have been explored as drug delivery systems. The nanoemulsions are also termed as mini emulsions, ultrafine emulsions and submicron emulsions 4 . Carvedilol is a non-selective beta blocker used in the treatment of mild to moderate congestive heart failure and mild to moderate essential hypertension 5 . It is a poor water-soluble and highly permeable drug. Its oral bioavailability is low (23%) because of significant first-pass hepatic metabolism by cytochrome P450 6,7 . However, some sources suggest that this low bioavailability is the result of poor aqueous solubility. It is practically insoluble in water (0.583 mg/L) and have pH-dependent solubility. Carvedilol also has a short plasma half-life of 7-10 hrs. Many parameters like low oral dose (6.25-25.0 mg), suitable log P (octanol/ water) of 4.19, low oral bioavailability and the condition of being a BCS class II drug make it  The aim of this study was to assess  the feasibility of preparing Carvedilol nanoemulsion by  aqueous  phase  titration  method,  and  the  physicochemical properties of obtained Carvedilol  loaded nanoemulsions, such as particle size, viscosity, polydispersity, zeta potential, in vitro drug release behavior.

Screening of oils, surfactants and co-surfactants for nanoemulsion Solubility studies
The solubility of Carvedilol in various oils (Capryol 90, Isopropyl myristate, Oleic acid, Olive oil, Sunflower oil and Linseed oil), surfactants (Tween 20 and Tween 80) and cosurfactants (Transcutol P, Propylene glycol, PEG 400 and Glycerol) was determined by adding an excess amount of drug in oils, surfactants and co surfactants separately in stopper vials, and mixed 9 . The mixture vials were then kept at 25±1.0°C in an Orbital shaker for 72 hrs to reach equilibrium. The samples were removed after achieving equilibrium and centrifuged at 3000 rpm for 15 min. The supernatant was taken and filtered through a 0.45-μm membrane filter. The filtrate was solubilized in suitable solvent, diluted with the pH 7.4 buffer and the concentration of Carvedilol was determined using UV-Visible spectrophotometer (Finlab Ltd, Nigeria) at 241 nm.

Selection of Surfactant
Surfactant selection was done on the basis of percentage of transparency (% transparency) and ease of emulsification. Briefly, 0.3 ml of each surfactant was added to the selected 0.3 ml of oil phase 10 . The mixture was gently heated at 50ºC for homogenization of the components. Each 0.05 ml mixture was then diluted with water in a stoppered conical flask. Ease of emulsification was judged by the number of flask inversion required to yield a homogenous emulsion. Emulsion is allowed to stand for 2 hours and their % transparency was evaluated by UV spectrophotometer using distilled water as a blank at 241 nm.

Selection of Co-surfactant
Screening of co-surfactant was conducted on the basis of % transparency and ease of emulsification. 0.1 ml of each cosurfactant mixed with 0.2 ml of selected surfactant and the 0.3 ml of selected oil phase was added and evaluated in a similar fashion as described in the above section of surfactant 11 . Construction of phase diagrams Pseudo ternary phase diagrams were constructed for 1:1, 1:2 and 1:3 surfactants to cosurfactant ratios (S mix ). So that nanoemulsion regions could be identified. In construction of phase diagrams clove oil was used as oil, tween 20 was used as surfactants and PEG 400 was used as cosurfactant 12 . Nanoemulsions were prepared by aqueous phase titration method. The composition of the nanoemulsions was chosen according to the pseudo ternary phase diagram. The drug was dissolved in the oil, surfactant and co-surfactant mixture was added in the chosen concentration, and water was added drop wise with continuous stirring until clear nanoemulsion was formed. Resulting carvedilol containing nanoemulsion was subjected to homogenization for 10 minutes using ultra turrex (Silverson, U.K) at 8000 rpm to get uniform and stable nanoemulsion 13 .

Characterization of nanoemulsions
Nanoemulsions are thermodynamically stable systems and are formed at a particular concentration of oil, surfactant and water, with no phase separation, creaming or cracking. It is the thermostability which differentiates nanoemulsions from emulsions that have kinetic stability and will eventually phase separate. Thus, the selected formulations were subjected to different thermodynamic stability by using heating cooling cycle, centrifugation and freeze thaw cycle stress tests. a). Centrifugation test: Nanoemulsions were centrifuged for 20 min at 3000 rpm and checked for phase separation, creaming or cracking 14 . b). Freezing-thawing test: The formulations were subjected to two different temperatures which are (21 o C) and (-21 o C) using refrigerator and the time for each temperature not less than 24 hours. This test used to indicate accelerated stability of formulations 15 . c). Heating-cooling test: This test was done by keeping the formulations at 40 o C and at 0 o C by refrigerator for 48 hours. This test used to indicate the racking effect on the formulations stability 15 . Optimized formulations were taken for viscosity, refractive index, transmittance and in vitro release studies.

Preparation of Carvedilol loaded nanoemulsion:
Carvedilol nanoemulsions were produced by dissolving the quantity of drug in specialized amount of oil. Then the determined quantity of S mix added for oil loaded drug, after that the whole mixture was blended together by vortex mixer (Mon Scientific, Nigeria), at the speed of 100 rpm. Then the aqueous phase (deionized water) titrated drop by drop to obtain transparent, clear (o/w) nanoemulsion. Particle size and zeta potential (ZP) measurement An amount of 0.1 ml of each tested formulation was dispersed in 50 ml of water in volumetric flask and then mixed by inverting the flask. Globule size and zeta potential of the nanoemulsion was determined by particle size analyzer (QL-1076, Nigeria) that analyzes the fluctuations in light scattering due to Brownian motion of the particles. Light scattering was monitored at 25°C at a 90° angle 16 .

Poly dispersity index (PDI) assay
This assay is used to measures the uniformity of globules size in nanoemulsion. It can be obtained by ABT-9000 nanolaser particle size analyzer. The higher the poly dispersity value refers to the lower uniformity of globules size of nanoemulsion 17 .

Determination of pH
The pH value plays important role in determination of the stability of the nanoemulsion. Change in pH means occurrence of chemical reactions that can impair the quality of the final product. The digital pH meter was used to determine the pH of the formulations.

Refractive Index
Refractive Index was determined using Abbe's refractometer at 25°C. Viscosity Viscosity of the samples was measured as such without dilution using Brookfield viscometer at 25°C. A sample volume of 10 ml was used. The nanoemulsion formulations were subjected to different rpm (5, 10, 20, 30, 50, 60 and 100) and the rheological behavior of the disperse system was examined by constructing rheograms of shear stress vs. shear rate 18 .

Drug content
Drug content of RF in nanoemulsion was measured by using UV visible spectrophotometer at 241 nm. About 0.1 mL of the formulation was suitably diluted with 5 mL in pH 7.4 phosphate buffer and analyzed for drug content 19 .

In vitro drug release studies
The in vitro drug release of Carvedilol from the nanoemulsion formulation was determined by dialysis bag method 20 . The dissolution study was performed for 12 hrs. In this method 0.1N HCl and pH 7.4 phosphate buffer maintained at 37°C and stirred with a magnetic stirrer (STUART, Finlab, Nigeria) was selected as in vitro release medium. About 1 mL of formulation was placed in the dialysis bag which was immersed in 50 mL of 0.1 N HCl. Samples (2 mL) were withdrawn at predetermined time intervals and replenished with equal volume of fresh medium. The samples were analyzed by the UV-Visible spectrophotometer at 241 nm to determine the Carvedilol content 21 .

Kinetics models and drug release mechanism
Determination of release kinetic of drug was done by using various kinetics models. The results of dissolution must be fitted for these kinetics models which are (zero order kinetic, first order kinetic, Higuchi model) 22 .

Statistical analysis
All the data generated were expressed as mean±standard deviation. Three group comparisons, one-way analysis of variance with duplication was applied. Statistical significance was determined using Student's t-test, with P<0.05 considered to be statistically significant.

RESULTS AND DISCUSSION
Drug solubility in oils plays an important role, as the ability of the nanoemulsion to maintain the drug in solubilized form depends on the solubility of the drug in the oil phase. In case of inadequate solubilization, there may be chances of precipitation, particularly in case of oral or parenteral nanoemulsion. After screening the oils for carvedilol solubility, it was found that carvedilol exhibited maximum solubility in clove oil (Table 1). Hence clove oil was chosen as the oil phase. Similarly based on solubility parameter tween 20 was used as surfactants and PEG 400 was used as cosurfactant. The components of pseudo-ternary phase plot ( Figure 1) are oil, water and S mix (surfactant /co surfactant) which considered as variable component due to that it presents in different ratio such as 1:1, 1:2, 1:3 as shown in Figure 1. The shaded area represents the area of nanoemulsion and the larger shaded area indicates a good nanoemulsifying activity. Based on the results of thermodynamic stability tests as shown in Table 3 Table 4, i.e. droplet size, PDI, pH and % transmittance etc. For carvedilol loaded nanoemulsions explains that the droplets size was decrease with increase in the S mix ratio. The results of PDI as shown in Table 4 indicate the uniformity of droplets distribution within the formulations. All formulations have PDI value less than (1.0). The lower value of PDI was (0.088) for NEC2, this explains that NEC2 has higher uniformity of droplets distribution within formulation. The results of viscosity of carvedilol nanoemulsions were found to be in range (60.42 -134.63 m Pa.sec.). Viscosity has an important aspect to ensure the smoothen formulations, packing 23 . The refractive index of the formulated nanoemulsion was similar to the refractive index of the water (1.333).  The results of % transmittance explain that the formulated nanoemulsions were clear and transparent, and the transparency of nanoemulsions indicates that the droplets size was in nano-scale.

Figure 2: In vitro dissolution profiles of Carvedilol nanoemulsion formulations of batch NEA1-NEB2
The higher value of % transmittance in carvedilol loaded nanoemulsions was (98.68 %) for NEC4. The results of pH measurement for formulations explain that the pH values of drug free nanoemulsions were slightly acidic. The higher pH value in the formulations of carvedilol nanoemulsions was (6.23) for NEA3 and this pH value is suitable for oral administration. Zeta potential of different formulations was calculated to explain the electro kinetic potential in colloidal dispersions. The results of zeta potential of carvedilol nanoemulsions were in range -18.34 mV to -26.47 mV). According to rule of thumb, the values of zeta potential which are: range -5 mV to +5 mV indicate fast aggregation, about -20 mV or +20 mV provide short term stability, above +30 mV or below -30 mV indicate a good stability and above +60 mV or below -60 mV offers excellent stability 24 . The results of drug content in nine formulations of carvedilol loaded nanoemulsions were in range (92.1-98.9%). The higher percent of drug content (99.04 %) was found in NEA4 that has S mix (1:1), and the lowest percent of drug content (96.48 %) was found in NEC1 that has S mix (1:3). A 12 hrs invitro release study was performed on selected nanoemulsion formulations of carvedilol ( Figure 2 and Figure 3). The highest in-vitro release is shown by formulation of batch NEC4 (84.57 %), while the lowest release is shown by NEA4 (42.786 %). Analysis of variance (ANOVA), study was performed by means of ezANOVA software. It reveals that there was a significant difference (P<0.05) between the release of carvedilol in all formulations. The release of drug from carvedilol nanoemulsions in dissolution media explains the influence of surfactant concentration on the release of carvedilol for each ratio of S mix . In each S mix ratio, as the concentration of tween 20 increase, the release of carvedilol decrease. This may be due to increasing the concentration of tween 20 results in increasing the diffusion of carvedilol molecules from dialysis bag for the dissolution medium 15 . The data of dissolution were fitted for various kinetic models (Table 5). It was found that the higher regression coefficient (R 2 ) values in the zero order kinetic. So, the kinetic of drug release in all Nanoemulsions was zero-order kinetic and the values of diffusion exponent (n) for all nanoemulsions of carvedilol was significantly lower than 0.41 (P<0.05), this explains that the mechanism of release of nimodipine from nanoemulsion formulations was Fikian release (diffusion).

CONCLUSION
Nanoemulsions are considered as an advance technique for improving the bioavailability of poorly watersoluble drugs by enhancing the solubility and minimizing the first pass metabolism. A correct combination of oil, surfactant, cosurfactant, and water is a major consideration factor in nanoemulsion preparation. Clove oil, tween 20, PEG 400 were selected for preparation of nanoemulsion by aqueous phase titration method. On the basis of different invitro release study, carvedilol nanoemulsion formulation of batch NEC4 (S mix Ratio 1:3) was found to be optimum formulation. The optimized formulation showed low particle size, low viscosity and high percentage transmittance. The present study was clearly indicated that the usefulness of nanoemulsion in the improvement of the solubility, dissolution rate and there by oral bioavailability of carvedilol.

AUTHOR'S CONTRIBUTION
The manuscript was carried out, written, and approved in collaboration with all authors.