INTRODUCTION:

Nanoemulsions are colloidal dispersions composed of an oil phase, aqueous phase, surfactant and cosurfactant at appropriate ratios. Unlike coarse emulsions micronized with external energy, nanoemulsions are based on low interfacial tension. This is achieved by adding a co-surfactant, which leads to spontaneous formation of a thermodynamically stable nanoemulsion. The droplet size in the dispersed phase is very small, usually below 140 nm in diameter, which makes the nanoemulsions transparent liquids.

In principle, nanoemulsions can be used to deliver drugs to the patients via several routes, but the topical application of nanoemulsions has gained increasing interest. The three main factors determining the transdermal permeation of drugs are the mobility of drug in the vehicle, release of drug from the vehicle, and permeation of drug into the skin. Nanoemulsions improve the transdermal delivery of several drugs over the conventional topical preparations such as emulsionsand gels.Mobility of drugs in nanoemulsions is more facileas compared to the nanoemulsion with gel former which will increase its viscosity and further decrease the permeation in the skin. The superior transdermal flux from nanoemulsions has been shown to be mainly due to their high solubilization potential for lipophilic and hydrophilic drugs. This generates an increased thermodynamic activity towards the skin ¹.The non-steroidal anti-inflammatory drugs (NSAIDs) are having the limitation of poor aqueous solubility and bioavailability. In addition to, shorter half-life and prominent GI side effects like ulceration and high first pass metabolism are the prime obstacles that led to commercial withdrawal of certain drugs like valdecoxib. Researchers have been trying to overcome GI side effects by the topical delivery of NSAIDs². Therefore, application of valdecoxib topically has another important role in inhibiting Ultraviolet radiation-B mediated inflammation. Duration of action of valdecoxib can be increased by improving its release patterns from formulation and systemic anti-inflammatory effects without major GI side effects ^3,4. It was found that Nanoemulsions could be a very good carrier for topical delivery of highly lipophilic drugs. Valdecoxib, a new non-steroidal anti-inflammatory drug (NSAID) is widerly used in rheumatoid arthritis, osteoarthritis and joint disorders. Valdecoxib having short half-life, low aqueous solubility and high lipophilicity.Valdecoxib belongs to class II category under the biopharmaceutical classification system (BCS), i.e. it is inherently highly permeable through biological membranes, but exhibits low aqueous solubility.Therefore, the present work was developed and evaluated a nanoemulsion based gel for transdermal delivery of valdecoxib to increase the solubility and to increase the permeability of valdecoxib.Physical properties, dissolution properties and permeability of nanoemulsion gel were evaluated in comparision to normal valdecoxib gel.

MATERIAL AND METHODS:

Materials:

Valdecoxib was a gift sample from Hetero Pharma Ltd,Jeedemetla, Hyderabad, India.Oleic acid, Triacetin, Isopropyl myristate, Ethyl oleate and carbopol 934P were purchased from CDH, New Delhi, India. Tween 80, Cremophore, Gelucire, Propylene Glycol, Triethanolamine, Polyethylene glycol 400 and Isopropyl alcohol were purchased from SD Fine Chemicals Ltd.Mumbai. All other chemicals and solvents used were of analytical grade.

Determination of drug solubility in various vehicles.:

Screening of Oil:

Since the aim of this study is to develop a transdermal formulation, therefore, solubility of drug in oils is one of the most important criteria. Correlating the solubility of drug with loading of the drug in oil phase can affect the ability of the nanoemulsion to maintain the drug in a solubilized form. The solubility of valdecoxib was determined in various oils by adding an excess amount of valdecoxib in 5 mL of selected oils (Labrafac, Triacetin, IPM, Ethyl oleate and Oleic acid), in 15mL capacity glass tubes.The drug suspension was equilibrated at 25°C in a thermostatically controlled bath for 72 h. After equilibration, the tubes were centrifuged at 12,000 rpm for 20 min and clear supernatants were analyzed for valdecoxib with a UV spectrophotometer at 245 nm⁵.

Screening of Surfactants:

Emulsions are the formulations where two immiscible liquid phases are dispersed in one another by using a mechanical shear ⁶. It is an inherent tendency of dispersed liquid and continuous phase to attract the molecules of each other to form a successive phase. An interfacial-tension(σ) exists between the two liquids, which can be reduced by incorporating a suitable surfactant into the system.The ability of the emulsion formation was monitored by noting the number of volumetric. Hence, to select the best surfactant from a pool, the emulsification ability of the surfactants was screened. 300 mg of surfactant and oily phase were added to each other, and the mixture was gently heated at 45-60°C for homogenizing the components. Then 50 mg of the homogenized mixture was diluted with distilled water to 50 ml to yield fine emulsion.The ability of the emulsion formation was monitored by noting the number of volumetric flask inversions required to give an uniform emulsion.Flask inversions required to give a uniform emulsion.The resulting emulsions were observed visually for the relative turbidity. The emulsions were allowed to stand for 2 hrs to check any change in turbidity, and their transmittance was assessed at 245 nm by UV- Visible spectroscopy r using distilled water as blank⁶.

Screening of Co-surfactants:

Co surfactants are the excipients added to the emulsion for further stabilizing the interfacial film and prevent coalescence of the droplets.100 mg of co-surfactant was added to 200 mg of the surfactant selected previously and the surfactant mixture (Smix) was then added to the selected oily phase. The effect of co-surfactant on stability was checked in the identical way as screening of surfactant. As the ratio of co-surfactants to surfactants is same, the turbidity of resulting Nanoemulsions will help in assessing the relative efficacy of the co-surfactants to improve the nanoemulsification ability of surfactants⁷.

Construction of Pseudo-ternary phase diagram:

On the basis of the solubility studies, a combination of Elainic acid was selected as the oil phase.Tween-80 and Ethanol were selected as surfactant and co-surfactant, respectively. Distilled water was used as an aqueous phase. Surfactant and co-surfactant (Smix) were mixed at different mass ratios (1:1, 1:2, 2:1, 3:1).These ratios were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co-surfactant with respect to surfactant for a detailed study of the phase diagrams. For each phase diagram, oil and Smix at a specific ratio was mixed thoroughly at different mass ratios from 1:9 to 9:1 in different glass vials.Sixteen different combinations of oil and Smix were made so that maximum ratios were covered for the study to delineate the boundaries of phases precisely formed in the phase diagrams. Pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method. Slow titration with aqueous phase was performed for each mass ratio of oil and Smix and visual observations were made for transparent and easily flowable o/w Nanoemulsions.The physical state of the Nanoemulsion was marked on a pseudo-three-component phase diagram with one axis representing the aqueous phase, the second one representing oil and the third representing a mixture of surfactant and co-surfactant at a fixed mass ratio and plotted on triangular coordinates to construct the pseudo ternary phase diagrams using CHEMIX ternary diagram software⁹.

Preparation of Nanoemulsion from Phase Diagrams:

Formulations were selected from the Nanoemulsion region of the constructed phase diagram to incorporate drug into the oil phase. The formulation was chosen with the criteria of maximum oil being emulsified with the minimum amount of Smix. Constant amount of Valdecoxib that is 1% w/w of Valdecoxib, was selected for the formulations, was dissolved in the oil phase of the emulsion formulation.Selected formulations were subjected to different thermodynamic stability tests⁷. The Nanoemulsion formulations were prepared by spontaneous emulsification method as follow. Appropriate quantities of oil Elainic acid, surfactant tween 80 and co-surfactant ethanol were weighed and mixed well. The drug was accurately weighted to represent 1% of the total weight of the formulation and added to the previous mixture and stirred with a magnetic bar on magnetic stirrer, at room temperature until the drug completely dissolved. The weighed amount of water then added drop wise with continuous mixing. Then droplet size is further reduced by using sonication method⁸.

Characterization and Evaluation of Nanoemulsion:

Different characterization parameters for Nanoemulsion include thermodynamic stability studies, Scanning electron microscopy, Droplet size analysis, viscosity, refractive index, in-vitro skin permeation studies, skin irritation test and In-vivo efficacy study.

Thermodynamic Stability Studies¹⁰:

To overcome the problem regarding the thermodynamic stability, stability study were performed, which are as follows:

Heating Cooling Cycle:

Heating and cooling cycle was done in refrigerator ranging the temperature between 4°C and 45°C for 48 hours. The formulations which were stable at these temperatures were subjected to centrifugation test

Centrifugation:

Centrifugation study for the selected formulations was done at 3500 rpm for 30 min. Formulations that did not show any phase separation were taken for the freeze thaw stress test

Freeze Thaw Cycle:

Three freeze thaw cycles were carried out between a temperature - 21°C and +25°C where the formulation was stored for not less than 48 hours at each temperature. Those formulations, which passed these thermodynamic stress tests, were selected for further study

Nanoemulsion Droplet Size Analysis:

Droplet size distribution of the Nanoemulsion was determined by photon correlation spectroscopy, which analyzes the fluctuations in light scattering due to Brownian motion of the particles, using a Zetasizer 1000 HS (Malvern Instruments, UK). Light scattering was monitored at 25 °C at a 90° angle.Droplet size distribution studies were performed at a fixed refractive index of the respective formulation ¹¹.

Polydispersity Index:

The average diameters and polydispersity index of samples were measured by photon correlation spectroscopy. The measurements were performed at 25⁰ C using He-Ne laser.

Viscosity:

Brookfield DVE viscometer (Brookfield Engineering Laboratories, Inc, Middleboro, MA) was used for the determination of viscosity of the formulations. About 0.5 g of sample was taken for analysis without dilution the sample by using spindle no. 63 at different rpm at 25±0.5°C¹².

Refractive Index:

The refractive index of placebo formulation and drug loaded formulations was determined using an Abbe-type refractometer (Macro Scientific Works, Delhi, India)¹³.

pH:

The apparent pH of the formulation was measured by using digital pH meter which is standardized previously¹⁴.

Scanning Electron Microscopy (SEM):

Morphology and structure of the Nanoemulsion were studied using Scanning electron microscopy. It was used to reveal the form and size of Nanoemulsion droplets. Observations was performed as, a drop of the Nanoemulsion was directly deposited on the holey film grid and observed after drying.

Zeta Potential:

Zeta potential for microemulsion was determined using Zetasizer HSA 3000 (Malvern Instrument Ltd., UK). Samples were placed in clear disposable zeta cells and results were recorded. Before putting the fresh sample, cuvettes were washed with the methanol and rinsed using the sample to be measured before each experiment¹¹.

In-vitro skin permeation studies

In-vitro skin permeation studies were performed on a Franz diffusion cell with an effective diffusion area of 3.14 cm² and 25 mL of receiver chamber capacity, using rat abdominal skin. The full thickness of rat skin was excised from the abdominal region and hairs were removed with an electric clipper. The subcutaneous tissue was removed surgically and the dermis side was wiped with isopropyl alcohol to remove adhering fat. The cleaned skin was washed with distilled water and stored at –21 °C until further use. The skin was brought to room temperature and mounted between the donor and receiver compartments of the Franz diffusion cell where the stratum corneum side was facing the donor compartment and the dermal side was facing the receiver compartment. Initially, the donor compartment was empty and the receiver chamber was filled with phosphate buffer saline (PBS) pH 7.4. The receiver fluid was stirred with a magnetic rotor at a speed of 100 rpm. After complete stabilization of the skin, 5 mL nanoemulsion formulation (20 mg mL–1 Valdecoxib or 1 g of Valdecoxib gel (20 mg g^-1) was placed into the donor compartment and sealed with paraffin film to provide occlusive conditions. Samples were withdrawn at regular intervals (0.5,1, 2, 3, 4, 5, 6, 8, 10, 12, 14 and 24 h) filtered through 0.45mm membrane filter and analyzed for drug content by using UV- Visible spectroscopy at 245nm¹⁵.

Permeation Data Analysis:

The permeation profiles were constructed by plotting the cumulative amount of Valdecoxib permeated per unit dialysis membrane area (g/cm²) versus time. Linear regression analysis was used to calculate the steady state flux (Jss, g/cm²/hr) (20) of Valdecoxib by using the slope of the plot. The following equation was used to determine the permeability co-efficient (Kp) of the drug through the stratum corneum¹⁶.

Kp = Jss/c

Where,

C is the initial concentration of the drug in the donor compartment.

The penetration enhancing effect was calculated in terms of enhancement ratio (ER) by using the following equation¹².

Er = Jss of formulation/Jss of control

Skin irritancy test:

Skin irritancy test was done on male Swiss albino mice, weighing 25–30 g. The animals were kept under standard laboratory conditions, temperature (25±1°C) and relative humidity (55 ± 5%). The animals were housed in polypropylene cages, six per cage, with free access to standard laboratory diet (Lipton Feed, India) and water ad libitum. A single dose of 10 μL of the Nanoemulsion was applied to the left ear of the mice, with the right ear as a control. The development of erythema was monitored for 6 days¹⁷.

Preparation of Valdecoxib gel:

Valdecoxib gel was prepared by dispersing 1 g of Carbopol-940 in a sufficient quantity of distilled water. After complete dispersion, the solution was kept in dark for 24 h for complete swelling of Carbopol-940.Then 1 g of Valdecoxib was dissolved in a specified quantity of polyethylene glycol-400 (PEG-400). This drug solution was added slowly into the aqueous dispersion of Carbopol-940.Then other ingredients such as isopropyl alcohol (IPA), propylene glycol (PG) and triethanolamine (TEA) were added to get homogeneous dispersion of the gel¹⁸.

Table 01- Formula for Valdecoxib gel preparation.

Ingredient	Mass (g)
Valdecoxib	1
Carbopol-940	1
Isopropyl alcohol	10
Polyethylene glycol-400	10
Propylene glycol	10
Triethanolamine	0.5
Distilled water	q.s

In-vivo studies:

Approval to carry out in vivo studies was obtained from the Institutional Animal Ethics Committee, bearing approval no. MRCP/CPCSEA/IAEC/2016-17/Pecu/02 and their guidelines were followed throughout the studies.The anti-inflammatory and sustaining actions of the optimized formulations were evaluated by the carrageenean-induced hind paw edema method developed by Winter et al. ¹⁹ in Wistar rats. Young male Wistar rats, weighing 180–220 g, were randomly divided into 3 groups: control, NG₂ and Valdecoxib gel, each containing 6 rats. The animals were kept under standard laboratory conditions, temperature at 25±1°C and relative humidity (55 ± 5%). The animals were housed in polypropylene cages, six per cage, with free access to standard laboratory diet (Lipton Feed) and water ad libitum. Doses for the rats were calculated based on the mass of the animals according to the surface area ratio¹⁶. The abdominal region of the rats was shaved 12h before starting the experiments, except in the control group. Nanoemulsion gel and normal Valdecoxib gel formulations were applied on the shaved abdominal region of all animals (except in the control group) half an hour before subplanter injection of carrageenean into right paws. Paw edema was induced by injecting 0.1 mL of 1% (m/m) homogeneous suspension of carrageenean in distilled water. The paw volume was measured at 1, 2, 3, 6, 12 and 24 h after injection using a digital plethysmometer²⁰. The amount of paw swelling was determined from time to time and expressed as percent edema relative to the initial hind paw volume. Percent inhibition of edema produced by each formulation--treated group was calculated against the respective control group. Results of anti-inflammatory activity were compared using the following formulae ^21,22.

Edema Rate (E%) = Vt – Vo/Vo *100 (3)

Ihibition Rate (I%) = Ec – Et/Ec *100 (4)

Where, Vo is the mean paw volume before CFA injection (ml), Vt is the mean paw volume after CFA injection (ml), Ec is the edema rate of control group, and Et is the edema rate of the treated group.

RESULTS AND DISCUSSION:

Excipients Selection:

The physicochemical properties of Valdecoxib suggest that it has good potential for topical drug delivery.The important criteria for selection of excipients for Nanoemulsion formulation and development was that components were to be pharmaceutically acceptable, nonirritating, and nonsensitizing to the skin and to fall into the GRAS (generally regarded as safe) category. Higher solubility of the drug in the oil phase was another important criterion, as it would help the Nanoemulsion to maintain the drug in solubilized form. Safety was a major determining factor in choosing a surfactant, as a large amount of surfactants may cause skin irritation. Non-ionic surfactants are less toxic than ionic surfactants. An important criterion for selection of the surfactants was that the required hydrophilic lipophilic balance (HLB) value to form the o/w Nanoemulsion be greater than 10. The right blend of low and high HLB surfactants leads to the formation of a stable Nanoemulsion formulation.In this study, we selected Tween 80 as a surfactant with an HLB value of 15.Transient negative interfacial tension and fluid interfacial film are rarely achieved by the use of single surfactant; usually, addition of a co-surfactant is necessary. The presence of co-surfactant decreases the bending stress of interface and allows the interfacial film sufficient flexibility to take up different curvatures required to form Nanoemulsions over a wide range of composition. Thus, the co-surfactant selected for the study was Ethanol, which has an HLB value of 4.2. Valdecoxib is a highly lipophilic drug, and its physicochemical properties suggest that it has good potential for transdermal drug delivery.Therefore, in the present study different Nanoemulsions were prepared for transdermal delivery of Valdecoxib.

Solubility of Valdecoxib.:

The maximum solubility of Valdecoxib was found in Elainic acid (1.22±0.145mg/ml) as compared to other oils. High drug solubility was found in Tween 80 (5.49 ± 0.120 mg/ml) and Ethanol was selected as co-surfactant as it forms stable Nanoemulsion, also acts as permeation enhancer. Therefore, Tween 80 and Ethanol were selected as surfactant and cosurfactant, respectively, for the phase study.

Table 02: Solubility of Valdecoxib in oils.

Oil	Concentration of drug (mg/ml)
Triacetin	0.521 ± 0.010
Isopropyl myristate	0.358 ± 0.014
Ethyl oleate	0.136 ± 0.020
Elainic acid(oleic acid)	1.22 ± 0.145

Mean ± SD, n=3

Fig 01: Solubility of Valdecoxib drug in oils.

Table 03: Solubility of Valdecoxib drug in surfactants.

Surfactant	Concentration of drug (mg/ml)
Gelucire	4.16 ± 0.157
Cremophore	3.82 ± 0.132
Tween 80	5.49 ± 0.120

Mean ± SD, n=3

Fig 02: Solubility of Valdecoxib drug in surfactants.

Pseudo-ternary phase diagram:

Care was taken to ensure that observations were not made on metastable systems; although the free energy required to form an emulsion is very low, the formation is thermodynamically spontaneous.The relationship between the phase behaviour of a mixture and its composition can be captured with the aid of a phase diagram. Pseudoternary phase diagrams were constructed separately for each Smix ratio, so that o/w Nanoemulsion regions could be identified and Nanoemulsion formulations could be optimized.Pseudoternary phase diagrams were constructed separately for each Smix ratio as shown in the figure 03, 04, 05 and 06,which represents Smix 1:1, 1:2, 2:1 and 3:1 respectively. It was observed in 1:2 Smix (Fig. 04) that when co-surfactant was added along with surfactant, the interfacial film became more fluid and no liquid crystalline area was found in the phase diagram.A large o/w Nanoemulsion area was observed. The maximum amount of oil that could be solubilized was 23% (m/m) with around 35% (m/m) of Smix. As the surfactant concentration was increased in Smix (ratio1:1, Fig. 03), a higher Nanoemulsion region was observed. It may be due to further reduction of the interfacial tension, increasing the fluidity of the interface, thereby increasing the entropy of the system. There may be greater penetration of the oil phase in the hydrophobic region of the surfactant monomers. As we further increased surfactant concentration in Smix to 2:1 (Fig.05), Nanoemulsion region decreased as compared to 1:2, the maximum concentration of oil that could be solubilized by this ratio was 24% (m/m) utilizing 36% (m/m) of Smix. When the Smix ratio of 3:1 was studied (Fig.06), the small area of Nanoemulsion further decreased and the liquid crystalline area started to appear in the phase diagram, which may be due to increased surfactant concentration. The maximum concentration of oil that could be solubilized with 53% of Smix was 23%. When cosurfactant concentration was increased from 1:1 to 1:2 compared to surfactant, the Nanoemulsion area decreased.It is well known that large amounts of surfactants cause skin irritation, it is therefore important to determine the surfactant concentration properly and use the optimum concentration of surfactant in the formulation. From pseudoternary phase diagrams, the formulations in which the amount of oil phase completely solubilized the drug and which could accommodate the optimum quantity of Smix and distilled water were selected for the study.

Figure 03: Pseudo-ternary phase diagram sowing the o/w Nanoemulsion (shaded area) regions of Elainic acid (oil), tween80 (surfactant), Ethanol (cosurfactant) at S_mix ratio 1:1.

Figure 04: Pseudo-ternary phase diagram showing the o/w Nanoemulsion (shaded area) regions of Elainic acid (oil), tween80 (surfactant), Ethanol (cosurfactant) at S_mixratio 1:2.

Figure 05: Pseudo-ternary phase diagram showing the o/w Nanoemulsion (shaded area) regions of Elainic acid (oil), tween80 (surfactant), Ethanol (cosurfactant) at S_mix ratio 2:1.

Figure 06: Pseudo-ternary phase diagram showing the o/w Nanoemulsion (shaded area) regions of of Elainic acid (oil), tween80 (surfactant), Ethanol (cosurfactant) at S_mixratio 3:1.

Table 04: Composition of selected Nanoemulsion formulations

			Component %(w/w)
Code	Oil/S_mix ratio	Oil/S_mix	Oil	S_mix	Water
NE₁	1:1	1:4	10	40	50
NE₂	1:1	1:5	10	50	40
NE₃	1:2	1:2	15	35	50
NE₄	1:2	1:3	15	45	40
NE₅	2:1	1:4	10	40	50
NE₆	2:1	1:5	10	50	40
NE₇	2:1	1:2	15	35	50
NE₈	2:1	1:3	15	45	40
NE₉	3:1	1:4	10	40	50
NE₁₀	3:1	1:5	10	50	40
NE₁₁	3:1	1:2	15	35	50

A total of 11 formulations were selected based on their ability to form oil in water Nanoemulsions which are selected from pesudoternary phase diagram of each Smix as shown in the table no.4.

Dispersion stability studies:

Nanoemulsions are thermodynamically and physically stable systems and are formed at a particular concentration of oil, surfactant and water, making them stable to phase separation, creaming or cracking. It is the thermo stability that differentiates Nanoemulsion from emulsions with kinetic stability and eventually phase separation. Thus, the formulations were tested for their physical (dispersion) stability by using centrifugation, heating-cooling cycle and freeze-thaw cycle. Only those formulations which survived dispersion stability tests were selected for further study. The compositions of selected formulations are given in table 4. Except NE8and NE₁₁ remaining all the formulation were passed the stability tests.

Table 05: Stability studies of formulations.

Code	Heating and cooling	Centrifugation	Freeze-thaw cycle	Inferences
NE1	Pass	Pass	Pass	Pass
NE2	Pass	Pass	Pass	Pass
NE3	Pass	Pass	Pass	Pass
NE4	Pass	Pass	Pass	Pass
NE5	Pass	Pass	Pass	Pass
NE6	Pass	Pass	Pass	Pass
NE7	Pass	Pass	Pass	Pass
NE8	Pass	Fail	Fail	Fail
NE9	Pass	Pass	Pass	Pass
NE10	Pass	Pass	Pass	Pass
NE11	Pass	Pass	Fail	Fail

Droplet size measurements:

The mean droplet size and polydispersity index were calculated from intensity, volume and bimodal distribution assuming spherical particles. All the Nanoemulsion had small average droplet diameter between 10 to 100 nm. A small droplet sizes are very much prerequisite for drug delivery as the oil droplets tend to fuse with the skin thus providing a channel for drug delivery. Polydispersity index (PI) is a measure of particle homogenicity and it varies from 0.0 to 1.0. The closer to zero the polydispersity value the more homogenous are the particles. Formulations showed their PI in between 0.314 to 0.91 that indicates acceptable homogenicity. Zeta potential of all Nanoemulsion formulation was found between -9.22 to -0.044 mV in the 100 times diluted (Table 06). Nanoemulsion formulation consists of non-ionic components which show relatively neutral charge, it means it will not affected by body membrane charge during absorption.

Fig 07: Graph indicating the mean particle size of formulation NE₁

Fig 08: Graph indicating the mean particle size of formulation NE₂

Fig 09: Graph indicating the Zeta potential of the formulation NE₁

Fig 10: Graph indicating the Zeta potential of the formulation NE₂

Table 06: Droplet size analysis and zeta potential

Code	Mean Particle size (nm)	Polydispersity index	Zeta potential
NE₁	42.91 ± 7.16	0.366	-6.8
NE₂	28.6 ± 9.0	0.314	-9.22
NE₃	50.67 ± 6.03	0.44	-4.71
NE₄	65.83 ± 11.25	0.621	-4.31
NE₅	80.61 ± 13.45	0.81	-5.21
NE₆	73.62 ± 11.17	0.67	-2.34
NE₇	83.33 ± 9.73	0.56	-3.95
NE₈	86.81 ± 13.45	0.59	-2.55
NE₉	81.27 ± 13.01	0.83	-1.25
NE₁₀	86.67 ± 5.51	0.68	-2.42
NE₁₁	89.33 ± 4.04	0.91	-0.044

Fig 11: An image showing SEM report.

Scanning electron microscopy:

The formulation with least mean particle size among the eleven formulations was analyzed for scanning electron microscopy to confirm the particle size. From the results of particle size analysis NE2 was selected and analyzed for SEM the results of the analysis also confirms that the formulation shows least particle size that is less than 200nm which was within the required limit (5nm-200nm) as shown in figure 11.

Viscosity:

Formulation NE₂ had the least viscosity (27.33 ± 1.15cps) compared to other formulation. This may be due to the lower oil content. The difference in viscosity between formulations NE₁ and NE₂ was not significant. Viscosity of all Nanoemulsion formulations was very low as expected. As shown in the Table 07.

p^Hof Nanoemulsion formulations:

The pH value of all developed Nanoemulsion formulations was in the range of 6.7-7.21, which is well within the limits of skin pH i.e. 5.6-7.5. Hence, it was concluded that all the formulations could not produce any local irritation to the skin. The results are shown in table 07.

Table 07: viscosity and pH of Nanoemulsion formulation

SAMPLE CODE	VISCOSITY (cP)	P^H
NE₁	31.66 ± 1.52	7.16 ± 0.11
NE₂	27.33 ± 1.15	6.93 ± 0.05
NE₃	40 ± 2	6.86 ± 0.05
NE₄	50.66 ± 3.05	6.7 ± 0.10
NE₅	34 ± 2	7.06 ± 0.11
NE₆	30 ± 2	6.9 ± 0.10
NE₇	45.33 ± 3.05	6.83 ± 0.05
NE₈	54.66 ± 1.15	6.73 ± 0.05
NE₉	40 ± 2	7.1 ± 0.1
NE₁₀	37.33 ± 1.15	6.82 ± 0.11
NE₁₁	54 ± 2	6.73 ± 0.05
NG₂	1980	7.21 ± 0.11

Mean ± SD, n=3

Refractive index:

Te refractive index of placebo formulations and drug loaded formulations was determined using an Abbes refractometer.The values of the refractive index of drug loaded formulations and placebo formulations are given Table 8. When the refractive index values for formulations were compared with those of the placebo, it was found that there were no significant differences (p< 0.05) between the values. Therefore it can be concluded that the Nanoemulsion formulations were not only thermodynamically stable but also chemically stable and remained isotropic. Thus there were no interactions between Nanoemulsion excipients and drug.

Table 08: Refractive index of Nanoemulsion formulations

SAMPLE CODE	Refractive Index of Formulation	Refractive Index of Placebo formulation
NE₁	1.324 ± 0.021	1.326 ± 0.001
NE₂	1.313 ± 0.001	1.314 ± 0.001
NE₃	1.328 ± 0.02	1.329 ± 0.01
NE₄	1.333 ± 0.002	1.335 ± 0.001
NE₅	1.371 ± 0.001	1.372 ± 0.002
NE₆	1.374 ± 0.02	1.372 ± 0.04
NE₇	1.379 ± 0.001	1.375 ± 0.001
NE₈	1.382 ± 0.02	1.381 ± 0.002
NE₉	1.413 ± 0.04	1.425 ± 0.02
NE₁₀	1.401 ± 0.02	1.403 ± 0.01
NE₁₁	1.414 ± 0.01	1.416 ± 0.02

Mean ± SD, n=3

In-Vitro Diffusion Studies of Nanoemulsions:

In-vitro skin permeation studies were performed to compare the drug release from eleven different Nanoemulsion formulations (NE₁ to NE₁₁), Nanoemulsion gel (NG₂) and normal Valdecoxib gel, all having the same quantity (1%, m/m) of Valdecoxib. The study was carried out using pH 7.4 buffer in receiver compartment. In-vitro skin permeation was the highest in formulations NE₂ and NE₆ and the lowest for normal Valdecoxib gel. Formulation NG₂ showed an intermediate skin permeation profile. The skin permeation profiles of NE₂ and NE₆ were not significantly different but they were significantly different from simple gel and NG₂ (p < 0.05). The significant difference in Valdecoxib permeation between Nanoemulsion formulations, NG₂ and Valdecoxib gel was probably due to the mean size of internal phase droplets, which were significantly smaller in Nanoemulsions. The maximum release in NE₂ and NE₆ could be due to the smallest droplet size and lowest viscosity compared to other Nanoemulsions. To explain the probable mechanism by which Nanoemulsions enhance the skin permeation of drugs, the histological and histochemical structure of stratum corneum must be taken into consideration.Drugs permeate stratum corneum through two micro pathways, i.e., intercellular and transcellular pathways. of these, the intercellular pathway plays a major role in percutaneous uptake of drugs. It is well known that a complex mixture of essentially neutral lipids, which are arranged as a bilayer with their hydrophobic chains facing each others, forms a lipophilic bimolecular leaflet. Most of the lipophilic drugs pass through this region, and it is called a lipid pathway. The polar head group of lipids faces an aqueous region, forming a polar route that hydrophilic drugs generally prefer. A dermally applied Nanoemulsion is expected to penetrate the stratum corneum and to exist intact in the whole horney layer, alter both lipid and polar pathways. The drug dissolved in the lipid domain of the Nanoemulsions can directly penetrate the lipid of the stratum corneum, thereby destabilizing its bilayer structure. These interactions will increase the lipid pathway permeability to drugs. On the other hand, the hydrophilic domain of Nanoemulsions can hydrate the stratum corneum to a greater extent and play an important role in percutaneous uptake of drugs.

Figure 12: percentage drug release of Nanoemulsion formulations.

Comparative drug release study between Nanoemulsion, Nanogel and Valdecoxib gel.:

Nanoemulsion shows more drug release when compared to the Nanogel and normal Valdecoxib gel. Nanogel drug release was 76%, and Nanoemulsion shown 87.63% where as normal gel of Valdecoxib shows 23.34% at the end 24 hrs. This indicates that drug release was increased greatly when compared to the normal Valdecoxib gel. The drug release was found in the following order like Nanoemulsion ˃ Nanogel ˃ Normal Valdecoxib gel.

Figure 13: Comparative drug release profile.:

Permeation data analysis:

Permeability parameters like steady-state flux (J_ss), permeability coefficient (K_p), and enhancement ratio (E_r) were significantly increased in Nanoemulsions and the Nanogel (NG₂) formulation as compared with conventional gel. This is because Nanoemulsions and Nanogel decreased drug loaded globule size. The permeability parameters of different formulations are given in the following table no.09. Nano gel shows the steady state flux (511.13(μg/cm2/hr), permeability coefficient (0.255 cm/hr) and enhancement ratio was 3.30 when compared Valdecoxib gel.

Table 09: Permeability parameters of different Nanoemulsions and Nanogel.

Sample code	Jss± SD (μg/cm2/hr)	*Kp± SD 10^-2 (cm/hr)**	Enhancement Ratio (Er)
Valdecoxibgel	154.45	0.077	--
NE₁	553.78	0.276	3.58
NE₂	618.39	0.309	4.04
NE₃	542.32	0.271	3.51
NE₄	535.64	0.267	3.46
NE₅	554.74	0.277	3.59
NE₆	554.74	0.277	3.59
NE₇	570.01	0.285	3.69
NE₈	553.78	0.276	3.58
NE₉	551.55	0.275	3.57
NE₁₀	573.1	0.286	3.71
NE₁₁	416.16	0.208	2.69
NG₂	511.13	0.255	3.30

Solubility of optimized formulation:

Te solubility of Valdecoxib in optimized formulation NE₂ was determined and compared with aqueous solubility of Valdecoxib. Significant increase in solubility of Valdecoxib in Nanoemulsion NE₂ (9.89 mg/ml ) was found as compared to water(0.065μg/ml), which could be due to presence of surfactant (Tween 80) and co-surfactant (ethanol). Thus it was predicted the enhancement in the permeability of Valdecoxib through skin.

Skin irritation test:

Based on in-vitro diffusion study formulation NE₂ containing drug was optimized. Further, Skin irritation test was performed with optimized formulation NE₂by applying it to left ear of mice to study irritancy by comparing with its right ear to which formulation was not applied. The study was carried out for six days and it was found that the Nanogel of NE₂ causes no irritation or erythema.

First day Nanogel application At sixth day of study

Fig 14: Results of skin irritancy studies

In vivo studies:

Based on higher drug release, optimum droplet size, lower viscosity and lowest polydispersibility index, formulations NG₂ was selected for the in vivo anti-inflammatory effects. The percent inhibition value after 24 h administration was found to be high for NG₂, i.e., 72% as compared to normal Valdecoxib gel (38%). The difference was significant (p < 0.05) when compared formulation NG₂. The enhanced anti-inflammatory effects of formulation NG₂ could be due to the enhanced permeation of Valdecoxib through the skin. Based on higher drug permeation, smallest droplet size, minimum polydispersity, lower viscosity, optimum surfactant and cosurfactant concentration and higher solubility, formulation NG₂ was optimized as the Nanoemulsion formulation of gel for Valdecoxib¹⁶.

Table 10: Anti-inflammatory study of Group-1

Time	Edema (mean ± SD, %)
1	32.0 ± 1.2
2	46.0 ± 1.3
3	81.0 ± 1.2
6	51.0 ± 1.5
12	43.0 ± 1.5
24	15.0 ± 1.3

Table 11: Anti-inflammatory study of Group-2

Time	Edema (mean ± SD, %)	Inhibition (%)
1	19	32
2	27.2	38
3	43.4	44
6	25.6	58
12	16.4	66
24	9.4	72

Table 12: Anti-inflammatory study of Group-3

Time	Edema (mean ± SD, %)	Inhibition (%)
1	25.2	10
2	39.4	13
3	56.2	27
6	35.9	35
12	26.4	36
24	9.7	38

Fig 15: Percentage edema inhibition effect.

CONCLUSION:

The optimized formulation contained 10 % of oil phase (Oleic acid), 50 % of surfactant mixture (Tween 80 as surfactant and Ethanol as co-surfactant) and 40 % of distilled water. From the studies, it is observed that the formulated Nanoemulsions and Nanoemulsion gel released up to 87.63 and 76 % of the drug, respectively. The formulation was nonsensitizing and safe for use prepared with non-irritating, pharmaceutically acceptable ingredients. No additional permeation enhancers were needed to be added since the excipients themselves acted as permeation enhancers. A high percent inhibition of edema was observed with the Nanogel (72 %) as compared with the normal Valdecoxib gel (38 %). Thus, it can be concluded that the developed Nanoemulsion-based gel have a greater potential for topical drug delivery as compared to conventional formulations.

CONFLICT OF INTEREST:

No.

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Received on 19.10.2018 Modified on 14.11.2018

Research J. Pharm. and Tech 2019; 12(2):600-610.

DOI: 10.5958/0974-360X.2019.00107.0