Formulation and evaluation of phenylephrine and amikacin nanoemulsion

The central perspective of this study is to formulate phenylephrine, and Amikacin loaded nanoemulsion by designing a pseudo ternary phase method. Selection of oils (castor oil, captex 200, ethyl oleate, triacetin) was made on the bases of the solubility studies. Captex 200 was chosen as the ideal oil phase for the formulation of emulsion due to its high solubility of both the drugs 1e., 106.54 (cid:6) 3.84 mg/ml and 116.32 (cid:6) 2.72mg/ml. The formulated Nanoemuslion was evaluated for thermodynamic stability, phase separation, transmission studies, percentage drug content, zeta potential(mV), Polydispersity index (PI) and particle size(nm). Among nine formulations of nanoemulsion, S6 formulation shows better and desired results like 95.27 (cid:6) 1.66% of drug content; 154.7nm of Particle size; 0.264 of PI and -44.5mV of zeta potential. Form the data. It was concluded that the reduction in particle size of S6 nanoemulsion might play a signi(cid:977)icant role in the enhancement of bioavailability of phenylephrine and Amikacin.


INTRODUCTION
Nanoemulsion is used as a carrier for many drugs to improve the delivery of the therapeutically active agent. Nanoemulsion is of submicron size. They consist of submicron size colloidal particles which are considered to be stable isotropic dispersion both in terms of thermodynamically and kinetically. It consists of two immiscible liquids, for example, oil and water due to which interfacial tension is developed between the two phases. To reduce the interfacial tension, suitable surfactants and co-surfactant are used to form a single stable phase. (Patel et al., 2013) A numerous variety of surfactant with different characteristic (.i.e. ionic or non-ionic) are used for the preparation of nanoemulsion (Gurpreet and Singh, 2018;Moreira et al., 2019;Savardekar and Bajaj, 2016).
The nanoemulsion is one of the most advanced drug delivery systems for targeting, controlled and sustained release of the pharmaceuticals. They are the stable isotropic system which consists of two immiscible liquid .i.e. water and oil, which is mixed together to form a single-phase by reducing the interfacial tension with the help of suitable surfactants. The globules size of nanoemulsion ranges from 10-100nm and also shows a narrow size distribution. In the upcoming years, the nanoemulsion will be having a wide range of scope in the ield of biotechnology, drug therapies, diagnostics and cosmetics (Marzuki et al., 2019;Rodrigues et al., 2018). This technique is mainly suitable for the drug delivery of a lipophilic bioactive compound, for example, beta-carotene which can be encapsulated, solubilized and transported with nanoemulsion directly to the epithelial cell where they are absorbed. The absorption of the ingested carotenoids in the cell mainly depends on lipid digestion. Therefore lipid digestion is considered as an important phase to ensure the absorption of carotenoids. However, the bioavailability of bioactive components will be increased due to encapsulation in nanoemulsion, which leads to a reduction in the droplet size (Suyal et al., 2018;Chandrasekaran et al., 2015).
The nanoemulsion is a multiphase colloidal dispersion which can be characterized based on their stability and clarity. To reduce the globule size in nanoemulsion is carried out with the help of ultrasonicator or micro luid, which produce high shear that will help to reduce the size of the globule (Pawan et al., 2015): low membrane permeation and pre systemic elimination. Any of the above approaches, which can alter the drug (Phenylepherine and Amikacin) permeability to enhance the solubility and bioavailability, should help in improving the bioavailability of the drugs (Aggarwal et al., 2014).
The central perspective of this present study was to evolve and to characterize the ideal thermostable nanoemulsion formulation of Amikacin and phenylephrine.

MATERIALS
Micro Labs Pvt. Ltd offers phenylephrine and Amikacin. Captex-200, Polaxomer, Tween 80 is purchased from Himedia Pvt. Ltd, Mumbai. All the other ingredients and solvents used in this research are analytical grade.

Optimization of formulation variables (Oil, Surfactants)
For the construction of each phase diagram, the oil and Smix ratio were mixed in distinct ratios starting from 1:9 to 9:1 in different test tubes. For this study maximum combination of oil and Smix ratios are prepared they are 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 ,9:1, 10:0 i.e. eleven combination are prepared. With the help of water titration, pseudo ternary phase diagrams were constructed to determine the existences zone of microemulsion and nanoemulsion. The initial step involved in the formation of the phase diagram, was to mix the oil phase (captex-200) in different ratios of co-surfactant and surfactant (Tween 80 and poloxamer respectively) later the resultant mixture was titrated against distilled water till the mixture turned turbid. The endpoint values were observed and noted down. With the help of the values obtained from the water titration, pseudo ternary phase diagram was constructed. For the given amount of oil concentration, the amount of water added ranges of 5-95% of total volume at 5% interval. On every 5% addition of the water to the oil and Smix mixture, the observation was made visually, and the results are cited in Table 1 and Figure 1. The ratio of surfactant and co-surfactant (Polaxomer and Tween 80) were used for the titration are, 1:1,1:2,1:3, 1:4, 2:1,3:1 and 4:1 respectively (Bajerski et al., 2016;Baboota et al., 2007;Sukanya et al., 2013).

High-Pressure Homogenization
This technique is used to reduce the globule and particle size (less than 100nm) in nanoemulsion with the help of an apparatus known as a homogenizer. The drug was dispersed selected oil phase with required quantity, as shown in Table 1. The required quantities of surfactant are dissolved in the aqueous phase (5 ml). Keep the aqueous phase in a homogenizer and add the oil phase with the drug in aqueous phase with constant rpm for 10 min, to get a P/W type of translucent nanoemulsion. In many cases, an emulsion is usually prepared in such a manner in which the volume of dispersing will be in a high fraction which can be diluted afterwards. For the smaller droplet size, if conceivable the surfac-      (Sukanya et al., 2013;Marzuki et al., 2019;Majeed et al., 2014).

Solubility study
Solubility studies are performed for various oils (castor oil, captex 200, ethyl oleate, triacetin) was done based on solubility studies. From the above study, Captex 200 was found to be an ideal oil phase for the formulation of emulsion due to its high solubility of both the drugs, i.e., 106.54±3.84 mg/ml and 116.32±2.72mg/ml (Marzuki et al., 2019;Majeed et al., 2014).

Phase separation study
Accurately about 1 ml of Amikacin and Phenylephrine Nanoemulsion was added to100 ml of distilled water in a beaker at 37 0 C and vortexed for 2 min. Later the mixture is left undisturbed for 2hours at the room temperature (37ºc) and observed visually for any phase separation (Gupta et al., 2016;Zeng et al., 2019).

Visual assessment
Approximately about 100 µl Amikacin and Phenylephrine nanoemulsion was diluted with puri ied water (250 ml) and gently stirred with a magnetic stirrer. The temperature should be 37 0 C. Formulation S4 and S5 were slight white milk-like emulsion and S6 was less clear emulsion, which has a bluishwhite appearance. Formulations S7, S8, and S9 were clear, slightly bluish appearance with good stability (Zeng et al., 2019;Tripathi et al., 2014).

Transmission test
The transmittance of light from selected Nanoemulsion formulations as well as it's 50 times, 100 times and UV-Spectrophotometer checked 200 times dilution with water at 665 nm by using water as a blank. The formulations S4 and S5 were less clear and turbid. Formulations S6, S7, S8, and S9 are clear and transparent (Sukanya et al., 2013;Marzuki et al., 2019;Majeed et al., 2014).

Determination of % drug content
The lipid-based dispersed system of Amikacin and Phenylephrine were dissolved in dichloromethane analyzed spectrophotometrically for the drug content at wavelength 665 nm with a proper dilution of formulations taking dichloromethane as a blank. Percentage of drug content was calculated from the standard and test sample absorbance.

Determination of DLS characteristics
Horiba Nanoparticle size analyzer equipped with the Horiba software was used for nanoparticles size characterization. The parameters like toxicity, biological fat, in-vivo distribution and targeting ability of the nanosystem can be determined by measuring the particle size, size distribution of the particles in the nanoemulsion and the data obtained from the above studies were also used to relate other parameters like drug release, stability and drug loading of the nanoparticle. The obtained result is doublechecked by Scanning Electron Microscopy (SEM). Zeta potential is de ined as the potential difference between the surface of a solid particle that is present in the bulk of the liquid. It predicts the electrical potential of particles and is dependent on the composition of the nanoformulation and the p H of the medium (Reddy and Sundari, 2019). Nanosuspension with zeta potential above +30mV or less than -30mV are said to be stable, as the surface charge prevents particles aggregation. Surface morphology of the prepared silver nanoparticles was analyzed using Horiba nanoparticle analyzer. About 30 microns of silver nanoparticles solution was initially dried, then mounted on the sample holder. This was followed by coating with gold using a sputter coater (Zeng et al., 2019;Tripathi et al., 2014;Reddy and Sundari, 2019) and further scanned for nanoparticles size and shape characterization. The measure of non-uniformity of the size distribution of particles is known as polydispersity index, which is dimensionless, and it indicates the degree of homogeneity of the medium. Nanoparticles with PIs less than 0.5 (Reddy and Sundari, 2019) are considered to be monodisperse and exhibit fewer particles aggregation (Zeng et al., 2019;Tripathi et al., 2014;Reddy and Sundari, 2019).

In-vitro release Study:
It was performed by using a dialysis membrane model which was performed at a rotation speed of 50rpm. The prepared nanoemulsion was packed in a dialysis bag, and tie is at both the ends. Make sure there is no leakage of emulsion on the tied end. This dialysis bag was taken in the basket. Then it was immersed in the vessel which containing 500ml of phosphate buffer solution of pH 7.4, which was maintained at a temperature of 37±0.20 • C. Later at speci ic time interval 5ml of the sample was collected from the dissolution medium, and the same volume of fresh medium was replaced in the lask to maintain the constant volume. The samples collected from the medium were analyzed by using UV spectrophotometer at 337nm and 520nm to determine the absorbance and calculate the percentage of drug release for 12 hrs (Zeng et al., 2019;Tripathi et al., 2014;Reddy and Sundari, 2019).

RESULTS AND DISCUSSION
Thermodynamic stability study and Phase separation studies are performed for all formulations from S1-S9 in different stress conditions like Heating Cooling Cycles (4 0 C to 45 0 C for 72 hrs); Freeze-Thaw Cycle between -21 0 C to -25 0 C; Centrifugation (3500 rpm 48 hrs). The results are tabulated in Table 2, which shown that the formulation S1 to S3 shows more phase separation while undergoing Freeze-Thaw Cycle and Centrifugation. But from S4 to S9, there is no phase separation takes place in all the stress condition. They are thermodynamically stable. On comparing to all the formulation, S6 formulation shows no phase separation, less clear emulsion, which has a bluish-white appearance. The transmission test results are shown in Table 3, from the thermodynamic stability data, formulation S1 to S3 was ignored for transmission studies.  -44.5mV. From the data, it shows that S6 formulation shows the least particle size with enhanced surface charge potential, so that it will not leads to phase separation. From the data shown in Table 4 and Figure 2, the S6 formulation was found to be the best nanoemulsion and selected for in-vitro drug release studies. The in-vitro drug release studies for S6 formulation shows the maximum amount of drug release at 12 hrs time, i.e. 94.56% of Amikacin and 98.9% of phenylephrine, this may lead to enhanced amount of bioavailable dose in the systemic circulation, which leads to more bioavailability.

CONCLUSION
From the data, it was concluded that S6 formulation was found to be better nanoemulsion in all the characteristics like phase separation studies, thermodynamic stability studies, particle characteristics studies, in-vitro drug release studies. So S6 formulation is concluded as a suitable nanoemulsion to enhance the solubility, dissolution and bioavailability of Phenylephrine and Amikacin drugs.