INTRODUCTION:

“There is plenty of room at the bottom” was one told by Richard Feynman, a novel laureate who in 1959 first proposed the concept of nano scale structure for use. This was the phrase which give birth to the nanotechnology. Nanotechnology scales up to one billionth of a meter. Generally, they are considered to be in range of 100nm to 1000nm. Various effect Such as surface area and area to volume ratio and many other physical properties get magnified when reduced to nanoscale. Most of the current research work in almost all technical and biomedical field is based on nanosize. Nanoemulsions are thermodynamically stable transparent (translucent) dispersion of oil and water stabilized by an interfacial film of surfactant and co-surfactant molecules having a droplet size of less than 1000 nm. Nanoemulsion, which is categorized as multiphase colloidal dispersion, is generally characterized by stabilized and clarity.

There is an application of high shear generally obtained by micro fluid or ultrasonic approach generally used to reduce the droplet size to nanoscale. Various works are done to correlate the different transdermal drug release from various delivery system. Studies suggested that the drug release from various formulation such as solid lipid nanoparticals, nanoemulsion and polymeric nanoemulsion, nanoemulsion proved to be efficient transdermal delivery vehicle.

Nanoemulsion are thermodynamically stable dispersions of two immiscible liquid (oil and water) which are stabilized using a surfactant co-surfactant molecules. They may be either transparent or translucent and have a droplet size of 5- 200nm. They are well tolerated orally on the skin and mucus membrane when used to deliver topically active drug. Nowadays increasing drug loading enhancing drug solubility, and bioavailability are there most important advantages encouraging the uses of Nanoemulsion as drug delivery carriers’ nanoemulsion is form of delivery for a drug that is difficult to dissolve and has side effects when administered orally by increasing the penetration of drug through the skin. Nanoemulsion comprise safe surfactant with or without other emulsifier to improve stability, oil (natural/ synthetic/semi-synthetic) and cosurfactant.

ADVANTAGES OF NANOEMULSION

· Increase the rate of absorption.

· Eliminate variability in absorption

· Provides aqueous dosage form for water insoluble drug.

· Increase the bioavailability.

· Various routes like topical, oral and intravenous can be used to deliver the product.

· Help in taste masking.

· Less amount of energy required.

· Provides protection from hydrolysis and oxidation as drug in oil phase in o/w emulsion.

· Liquid dosage form increase patient compliance.

DISADVANTAGES OF NANOEMULSION:

· Used of large concentration of surfactant and co surfactant necessary for stabilizing the nano droplet.

· Limited solubilizing capacity for high melting substance.

· The surfactant must be nontoxic for pharmaceutical application.

· Nanoemulsion stability in influenced by environmental parameters such as temperature and pH. These parameters change upon nanoemusion delivery to patients

Table No-1 Various example of oils used in Nanoemulsion

Sr. No	Name	Chemical name
1	Isopropyl myristate	Myristic acid isopropyl ester
2	Myritol 318	C8/C10triglycerides
3	Cocunt oil	Various
4	Seasem oil	Various
5	Olic acid	Various
6	Witepsol	90:10%w/wc12glycerides tri: diesters
7	Castor oil	Various

Table No-2 Various example of surfactant used in Nanoemulsion

Sr. No	Name	Chemical Name
1	Tween 20	Polyoxyethylene 20 sorbitanmonolaurate
2	Tween 40	Polyoxyethelene20sorbitan monopalmitae
3	Tween 80	Polyoxyethylene 20 sorbitanmonooleate
4	SPAN80	Sorbitanmonooleate
5	Cremphor EL	Polyoxyl 35 castor oil
6	Labrasol	PEG-8 Caprylic/Capric glycerides

Table No-3 Various example of co surfactant used in Nanoemulsion

Sr. No	Name	Chemical Name
1	Transcutol P	Diethylene glycol monoethyl ether
2	Ethylene glycol	Ethene 1,2 diol
3	Propylene glycol	1,2 propanediol

OBJECTIVE:

· To carry out the preformulation studies of the drug sample.

· To carry out the solubility of the ketoconazole being BCS class II drug

· To study the optimization of formulated Nanoemulsion.

· To carry out the in vitro drug content study of prepared Nanoemulsion.

· To carry out stability studies.

METHOD OF PREPARATION:

Several method have been suggested for the preparation of nanoemulsion. The basic objective of the nanoemulsion preparation to achieve the droplet size range of 100-600 nm and another is to provide the stability condition. formation of nanoemulsion system required a high amount of energy. This energy can be provided either by mechanical equipment or the chemical potential inherent within the component.

a. High Pressure Homogenization:

This technique makes used of high pressure homogenizer/piston homogenizer to produce nanoemulsion of extremely low particle size (up to 1 nm). during this process, several force, such as hydraulic shear. Intense turbulence and cavitation, act together to yield nanoemulsion with extremely small droplet size. The resultant product can be subjected to high pressure homogenization until nanoemulsion with desired droplet size and polydispersity index is obtained. The production of small droplet (submicron) requires application of high energy several procedures may applied to enhance the efficiency of emulsification when producing nanoemulsion. The emulsion is preferably prepared at high volume faction of the disperse phase and diluted afterwards. however, very high phase volume ratio may result in coalescence during emulsification, but more surfactant could be added to create a smaller reduction in effective surface tension and possibly coalescences. Surfactant mixture that show more reduction in surface is dissolved in the disperse phase rather than the continuous phase; this often leads to smaller droplets. It may be useful to emulsify in steps of increasing intensity, particularly with emulsion s having highly viscous disperse phase.

Effect of homogenization pressure

It should be form100 to 150 bars. The higher the pressure the lower is the particle size obtained.

No. of Homogenization cycles:

The higher the homogenization cycles the smaller is the particle size obtained. The cycles are carried out in 3,4 or 10 cycles. The number of cycles is analysed by polydispersity index of drug after each cycles.

Fig No: 1 High Pressure Homogenizer

b. Phase Titration Method:

Nanoemulsion are prepared by the spontaneous emulsification method (phase titration method) and can be depicted with the help of phase diagram. Construction of phase diagram is useful approach to study the complex serious of interaction that can occur when different component are mixed. Nanoemulsion are formed along with various association structure (including emulsion, micelles, lamellar hexagonal, cubic, and various gel and oily dispersion depending on the chemical composition and concentration of each component. The understanding of their phase equilibrium and demarcation of the phase boundaries are essential aspect of the study. as quaternary phase diagram (four component system) is time consuming and difficult to interpret, pseudo ternary phase diagram is often constructed to find the different zone including nanoemulsion zone in which each corner of the diagram represents 100% of the particular component. The region can be separated into w/o or o/w nanoemulsion by simply considering the composition that is whether it is oil rich or water rich. Observation should be made carefully so that the metastable system is not included.

c. Sonication method

Sonication method is another best way to prepare nanoemulsion. In this method the droplet size conventional emulsion or even micro emulsion are reduced with the help of sonication mechanism. This method is not suitable for large batches of nanoemulsion can be prepared by this method.

d. Phase inversion method:

In this method fine dispersion is obtained by chemical energy resulting of phase transitions taking place through emulsification path. The adequate phase transition are produced by varying the composition at constant temperature at constant composition, phase inversion temperature (PIT) Method was introduced by shinoda et al. based on the changes of solubility of polyoxyethylene - type surfactant with temperature. This surfactant becomes lipophilic with increase in temperature due to dehydration of polymer chain. but at low temperature, The surfactant monolayer has a large positive spontaneous curvature forming oil-swolllen micelle solution phase.

MATERIAL AND METHODOLOGY:

Ketoconazole was gifted by Swapnrup Drug Agency, Aurangabad, seaseam oil – Research Lab Fine Chem. Industries-Mumbai, tween 80 - Research Lab Fine Chem. Industries Mumbai, ethanol- Research Lab Fine Chem. Industries-Mumbai

Screening of oil, surfactants and cosurfactant for nanoemulsion formation:

The most important criteria for the screening of oil for nanoemulsion is the maximum solubility and compatibility with drug. Basing on the biocompatibility profile of oil from literature review, drug solubility was determined. Excess amount of drug (100mg) in 3ml selected oil (olive oil, castor oil, seaseam oil, coconuts oil) was taken in stopper vials and was then mixed by vortex mixer. The mixture vials were then kept at 37-2.0c in an isothermal orbital shaker for 72hr. To reach equilibrium. The equilibrated sample was removed from shaker at centrifuged at 5000rpm for 15 min. The solubility profile of drug in oil was determined from the supernatant using UV-VIS spectrophotometer at 257nm. insoluble drug from the settled material was determined and mass balance was then found it.

FORMULATION OF NANOEMULSON:

Nanoemulsion formulation were prepared using selected oil, surfactant, cosurfactant along with aqueous phase by water titration method. Required quantity of drug was added to adequate amount of oil in a cleaned and dry beaker having a small magnetic bead and was mixsed completely to magnetic stirrer. Then to the mixture, surfactant and cosurfactant at definite ratio (Smix) were added and was mixed by gentle stirring. The above mixture was finally allowed to titrate by distilled water under constant stirring on a magnetic stirrer. Store prepared nanoemulsion in sealed container at room temperature till further use.

Construction of Pseudo–Ternary Phase Diagram:

The phase diagram with different ratio of surfactant: co surfactant (1:2, 1:2, 2:1% w/w) with selected oil were constructed to explore the nanoemulsion region. The area of the monophasic region was used as a tool for the selection of suitable surfactant and cosurfactant mixture. Aliquot’s of each surfactant and cosurfactant mixture (Smix) were mixed with oil at ambient temperature. For each phase diagram the ratio of oil to the Smix was varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 (%w/w).water was added drop wise to each oil Smix mixture under constant stirring.After equilibrium the sample were visually checked and determine as being clear nanoemulsion. Phase diagram were constructed using chemix softwere. (chemix schhol, version 7.00)

Evaluation of Nanoemulsion:

The nanoemulsion was evaluated for the following characteristics:

a. Optical transparency:

The formulation was determined by inspecting the sample in clear and transparent container under the presence of light against reflection into the eyes.

b. Viscosity Measurement:

The viscosities of nanoemulsion were measured using a Brookfield rotational viscometer at 24.9 at 10rpm.

c. Phase Separation:

Nanoemulsion system subjected to centrifugation at 3000rpm for a period of 2 hour and examined for any evidence of phase separation.

d. Determination of Ph:

A 10% dispersion of formulation was prepared in distilled water and pH was determined by pH meter which was prior standardized with standard buffer of pH 4 and pH 7.

e. Measurement of Globule Size:

The average globule size of the nanoemulsion was determined by Zetasizer Nano-ZS (Malvern Instrument, UK). Measurement were carried at angle of 90 at 25c Nanoemulsion was diluted with double distilled water to ensure that the light scattering intensity was within the instruments sensitivity range. All the measurement was carried out at 25c The polydispersity index of the formulation was determined by the same instrument. The width of the size distribution was indicated by the polydispersity index (P.I).

f. Measurement of Zeta Potential:

The zeta potential was determined to verify stability of nanoemulsion due to charge interaction. Zeta potential was measured by using Zetasizer Nano-ZS (Malvern instrument, UK). The measurement was preformed at 25c.

g. Drug Content

A define volume of formulation was taken in a 10ml volumetric flask and diluted with ethanol. Absorbance of the resultant solution was sonicated for 3 min at ambient temperatures and the absorbance of the resultant solution was measured at λ maxof 240nm against blank.

h. In vitro diffusion study:

In vitro diffusion was carried out by modified franz diffusion cell. A glass cylinder with both ends open 10 cm height 3.7cm outer diameter and 3.1cm inner diameter was used as diifusion cell.a shep mucosa was fixed to one end of the cylinder with the aid of an to result as a diffusion cell.1 ml of nanoemulsion was taken in the cell and cell was immersed in a beaker containing 100ml of pH 6.8 phosphate buffer as receptor compartment. The entire surface of the cell was in contact with the receptor compartment which was againsed using magnetic stirred and a temperature of 37 c was maintained. Samples 10ml of the receptor compartment were taken and with same amount replaced to maintain sink condition. The sample was analyzed for ketoconazole at 240nm against blank using UV Spectrophotometer. Amount of ketoconazole released at various time intervals was calculated with the help of calibration curve with phosphate buffer and plotted against time.

i) Stability Studies:

The nanoemulsion were subjected to stability study at 37◦C for 1 month respectively. The sample were evaluated for transparency. drug content, pH and in vitro drug realest every one month for three month period.

RESULT AND DISCUSSION:

Solubility study: Result of solubility study of drug in oil, surfactant, co surfactant are shown in table

Ketoconazole solubility study data:

Sr. No	Oil	Solubility(mg/ml)
1	Arachise oil	8.8
2	Seaseam oil	8.9
3	Oleic acid	49.23
4	Castor oil	9.2
5	Soybean oil	7.2

Sr. No.	Surfactant	Solubility(mg/ml)
1	Tween 20	32
2	Span 20	38
3	Tween 80	28
4	Campul PG8	18.32

Sr. No.	Co-surfactant	Solubility(mg/ml)
1	PEG400n	44
2	Ethanol	28
3	Isopropylene Glycol	32
4	PEG20	30

Construction of Pseudo-Ternary Hase Diagram:

Surfactant was blended with co surfactant in fixed weight ratio (1:1, 1:2, 2:1) Aliquots of each surfactant mixture (Smix) were then mixed with oil at room temperature 25C. For each phase diagram, the ratio of oil to the Smix was varied at 9:1, 8:2, 7:3, 6:4, 5:5,4:6 3:7, 2:8, 1:9. (w/w). Water was added drop wise to each oil. Mix mixture under the vigorous stirring. After equilibrium the sample were visually checked and determined to clear nanoemulsion. For the determination of existence zone of Nanoemulsion, pseudoternary phase diagrams were constructed using water titration method. To constructed pseudoternary phase diagrams, the oil phase was mixed with different ratio of surfactant and co-surfactant and mixtures was titrated with distilled water until turned turbid. Examine each and every point I detailed and note it down. Pseudo ternary phase diagrams were drawn by using data obtain in aquase titration method as shown in figure. The amount of water added to give water the water to the oil and Smix mixture, visual observation were made as shown in figure. The ratio of surfactant and co surfactant were used for the titration.

COMPOSITION OF FINAL NANOEMULSION:

Sr No	Ingredient	% w/w
1	Seaseam oil	10ml
2	Tween 80	35ml
3	Ethanol	35ml
4	Water	20ml

Sr. No	Test	Nanoemulsion
1.	pH	5.10+-0.46
2.	Viscosity	248.3+-0.67
3.	Globule size	122.0
4.	Zeta potential (	-32.9mv
5.	Optical transparency	Transparent
6.	Drug content	88.85+-0.75
7.	Phase separation	No phase separation

CHARACTERIZATION OF NONAEMULSION:

THE RELEASE PROFILE OF KETOCONAZOLE:

In vitro drug release study:

Fig No:2 In Vitro Drug Release

STABILITY STUDY:

The nanoemulsion were subjected to stability study at 37◦C for 1 month respectively. The sample were evaluated for transparency. drug content, pH, and in vitro drug release every month for three months period shown in table.

Table No: 1.25 Stability Studies

Sr. No	Observation	Before accelerated Stability Study	After Accelerated Stability Testing
Sr. No	Observation	Before accelerated Stability Study	30days
1	Visual appearance (Transparency)	Transparent	Transparent
2	Drug Content (%)	88.85+0.75	88.58+-0.64
3	pH (±)	5.10+-0.46	5.18+-0.50
4	In Vitro drug releasee (±)	83.09+-0.54	83.07+-0.51

Value expressed as mean SD

CONCLUSION:

Many of strategies are described to show enhancement in the penetration of the low molecular weight drug and several of which have been successfully employed in commercial system.in this study, nanoemulsion drug delivery system a mixture containing surfactant, co-surfactant and oil, were prepared nanoemulsion solubility studied was determined in various oils for selection of oil phase, surfactant ,co-surfactant with distilled water being the aqueous phase. Various oil in water nanoemulsion prepared by the water titration method. This property was dependent on the composition of the excipients as well as their individual concentration in te mixture. The transparent emulsification are was identified by constructing pseudo phase ternary phase diagram. Excipients evaluated in nanoemulsion drug delivery system. Tween 80 as surfactant, ethanol as co surfactant and seaseam oil as oil. All the excipients showed a tendency to form a nanoemulsion with varying degree of efficiency. A particular nanoemulsion mixture comprising of tween80, ethanol, and seaseam oil was selected and optimized for the purpose for delivering a model drug, ketoconazole. nanoemulsion drug delivery system is known to improve dissolution characteristics of a poorly water soluble drug since they maintain the drug in a dissolution characteristics of a poorly water soluble drug since they maintain the drug in solubilized state. Using the optimized nanoemulsion of ketoconazole loaded were prepared. The prepared liquid nanoemulsion were subjected to thermodynamic stability testing and zeta potential due to their characteristics size and properties, which included kinetic stability; they are effective in solubilizing the drug and transferring them to words the target areas. It is characterized for droplet size, zeta potential, viscosity, in vitro drug release study were performed.

The following conclusion can be drown form the present study:

a) Solubility analysis aided the selection of suitable seaseam oil as a oil phase, tween 80 as a surfactant, ethanol as co surfactant.

b) From the pseudo ternary phase diagram construction, it aided to find the proper construction of seaseam oil as oil, tween 80 as a surfactant, ethanol as a co-surfactant and aqueous phase.

c) Globule size of optimized nanoemulsion (NE3) was found to be 122nm.

d) Drug content of optimized nanoemulsion (NE3) was found to be 88.85%.

e) Drug release study showed that, developed namoemulsion formulation (NE3) give higher percentages of drug release 83.09% in 60 min.

f) Stability studies indicated that prepared nanoemulsion formulation did not show any significant change in physical appearance, drug content, turbidity or phase separation incase of formulation NE3 up to 3 month.

Finally, it can be summarized and concluded that the nanoemulsion of ketoconazole can be one of promising tool in improve bioavailability, increases the rate of absorption due to the small globule size and by avoiding first pass metabolism and direct transport into systemic circulation.

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Received on 25.11.2019 Modified on 10.12.2019

Res. J. Pharma. Dosage Forms and Tech.2019; 11(4):269-274.

DOI: 10.5958/0975-4377.2019.00045.4