International Journal of Innovative Pharmaceutical ...ijipsr.com/sites/default/files/articles/IJIPSRMNR-91.pdf · loaded nanoemulsion was prepared successfully by spontaneous self-emulsification

RESEARCH ARTICLE Kanu Saini et.al / IJIPSR / 2 (6), 2014, 1168-1191

Department of Pharmaceutics ISSN (online) 2347-2154

Available online: www.ijipsr.com June Issue 1168

e

DESIGN, DEVELOPMENT AND EVALUATION OF NANO-

EMULSION GEL OF SALICYLIC ACID

1Kanu Saini*,

2Punam Gaba,

3Jasvir Singh

1,2Amar Saheed Baba Ajit Singh Jujhar Singh Memorial College of Pharmacy, Bela-Ropar,

Punjab-140 001, INDIA 3School of Pharmaceutical Sciences, Shoolini University, Solan (HP), Oachghat -173 212,

INDIA

Corresponding Author:

Kanu Saini

M.Pharmacy Student

Amar Saheed Baba,

Ajit Singh Jujhar Singh Memorial College of Pharmacy,

Bela-Ropar, Punjab-140 001, INDIA

Email: [email protected]

Phone: +91-9569221501

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

The objective of this work was to increase the solubility, permeability and also stability of nanoemulsion

formulation of salicylic acid. Using pseudo-ternary phase diagram nanoemulsion area was selected and

according to that ratio of oil, surfactant and cosurfactant nanoemulsion was optimized. Optimized salicylic acid

loaded nanoemulsion was prepared successfully by spontaneous self-emulsification method and characterized

for viscosity, droplet size and transmission electron microscopy (TEM). Further the optimized nanoemulsion

was incorporated into 0.75% Carbopol 980 to get a gel for improving convenience in superficial application of

the drug. The particle size of the nanoemulsion prepared ranging from 100nm-500nm. In vitro drug release was

performed upto 12 hours. Further compatibility studies were performed to confirm any drug polymer interaction

and it was found that there were no special peaks indicating no drug polymer interaction that is significant. The

optimized formulation showed 52.19% release in about 12 hours but the marketed formulation showed 17.81%

release in about 12 hours. The release studies of optimized formulation was fitted in different kinetic models

and found that they are following Zero order release. This work showed for the first time that salicylic acid can

be formulated into nanoemulsions.

Key words: Salicylic acid, nanoemulsions, topical delivery.

mailto:[email protected]




INTRODUCTION

Salicylic Acid (SA) or 2-Hydroxybenzoic acid is commonly referred to as a beta hydroxyl acid

(BHA). Today, Salicylic Acid continues to play an important role as an active ingredient in over-

the counter (OTC) topical drug products. SA is in a class of medications called keratolytic agents

that induces corneocyte disaggregation in the upper stratum corneum, thereby enhancing the

shedding of scales and softening the stratum corneum. SA is readily absorbed from the skin and

may induce toxicity (Salicylism). A limitation of the currently available keratolytic topical

formulations is their potential to cause such unwanted reactions as stinging, burning, peeling,

dryness and erythema due to their rapid absorption [1-3]. These reasons lead to the study of

nano-emulsion based gel of salicylic acid. Nanoemulsions (NE) have received a growing

attention as colloidal drug carriers for pharmaceutical applications. Nanoemulsions are a class of

emulsions with very small and uniform droplet size, typically in the range of 20-500nm and are

thermodynamically stable [4], transparent (or translucent), dispersion of oil and water stabilized

by an interfacial film of surfactant molecules having the droplet size in nanometer range. These

system require relatively appropriate amount of surfactant is stabilize the large interfacial area

created by the nano-droplets often addition of cosurfactant such as alcohol is also required to

attain appropriate fluidity or viscosity of the interface. Nanoemulsion provides ultra low

interfacial tension and large o/w interfacial areas. Nanoemulsion have a higher solubilization

capacity than simple micellar solutions and their thermodynamic stability offers advantages over

unstable dispersions, such emulsions and suspensions, because they can be manufactured with

little energy input (heat or mixing) and have a long shelf life. However long term physical

stability of nanoemulsion (which no apparent flocculation or coalescence) make them unique and

they are sometime referred as approaching thermodynamic stability [5,6]. The use of

nanoemulsions could be a very good carrier for topical delivery of highly lipophilic drugs. It was

also found that the excipients which are used to developed nanoemulsion helps to augment the

solubilizing and permeation capacity. Because of the smaller droplets contained in nanoemulsion

there by facilitate close contact with the statum corneum. That is why the amount of

encapsulated agent penetrating into the viable skin facilitates the drug transport by changing the

vehicle/stratum corneum partition coefficient. Salicylic acid having shorter half-life (2-4 hours),

lower molecular weight (< 138.12g/mol), low aqueous solubility & high lipophilicity (Partition

co-efficient, 2.2) eventually becomes a suitable candidate for the topical delivery.




MATERIALS AND METHOD

Materials

Salicylic acid (SA) was kindly supplied from Central Drug House, New Delhi, India. Oleic acid

was purchased from Molychem, Mumbai, India. Tween-20 was obtained from Thomas baker,

chemicals, Mumbai, India. Polyethylene glycol 200 (PEG 200) was purchased from Finar

Chemicals Ltd., Ahmedabad, India. Carbopol 980 was obtained from Lubrizol Advanced

Materials, Europe. All chemicals and solvents used in this study were of analytical reagent grade.

Freshly distilled water was used throughout the work.

Methods

Solubility Studies of Salicylic Acid

To find out the appropriate oils, surfactants and co-surfactants as a composition of nanoemulsion

the solubility of salicylic acid in various oils (Castor oil, Sunflower oil, IPM, Myritol, MCT oil,

Oleic acid, Olive oil), surfactants (Cremophor RH 40, Kolliphor ELP, Tween 80, Tween 60,

Tween 20, Span 80, Span 20) and co-surfactants (Glycerin, Propylene Glycol, Ethanol, PEG 400,

PEG 200, Propanol) was determined by using shake flask method. Briefly, an excess amount of

salicylic acid (10mg) was added to each vial containing 5 mL of the selected vehicle, i.e. oil,

surfactant and co-surfactant. After sealing, the mixture was vortex for 5 min and sonicated by

using bath sonicator for 3 mins in order to facilitate proper mixing of salicylic acid with the

vehicles and reduce the particle size of the drug. Mixtures were shaken for 72 hrs in an

isothermal shaker (Remi, Mumbai, India), maintained at 37±1ºC, and afterwards, mixtures were

centrifuged at 1500 rpm for 10 min and then supernatant was filtered through membrane filter

(0.45µm) to remove the remaining salicylic acid. After the appropriate dilution with methanol

the concentration of SA in the filtrate was determined at 304 nm by UV spectrophotometer and

solubility of SA in different oils, surfactants, and co-surfactants was calculated with the help of

standard calibration curve [7].

Selection of Oil, Surfactant and Co-Surfactant

On the basis of solubility studies oil, surfactant and co-surfactant were selected for nano-

emulsion formulation.

Screening of Surfactants

Different surfactants were screened for emulsification ability. For this study, 150mg of surfactant

were added to 150mg of oily phase and then this mixture was heated at 50ºC for homogenization




of the components. Then from each mixture prepared 100 mg was withdrawn and diluted to

100ml in a volumetric flask. The ease of emulsification was judged by the number of flask

inversions required to yield homogeneous emulsion. The emulsions were allowed to stand for 24

hrs and then % transmittance was evaluated at 604nm by using UV spectrophotometer. They

were also observed for turbidity or phase separation visually [7].

Screening of Co-Surfactants

For screening of co-surfactants the best combinations of oil and surfactant as before were

selected depending on the stability and %transmittance. For which the oil: surfactant: co-

surfactant were taken as 600mg: 400mg: 200mg i.e. in the ratios of 3:2:1. Out of the total

1200mg of the mixture 100 mg was withdrawn and then added dropwise in a 100 ml volumetric

flask containing distilled water dropwise; then it was inverted 50-60 times and kept overnight.

After which the % transmittance was determined by scanning in the range from 800-200 nm

(wavelength 604 nm) using UV-visible spectrophotometer. After the completion of screening the

next step was to optimize the combination showing good % transmittance. For this purpose the

effect of oil, surfactant and co-surfactant on transmittance was studied using different mass

ratios. The various excipients were analyzed and finally optimized for construction of pseudo-

ternary phase diagram. Therefore, the effect of the components on formation of nano-emulsion

was chosen as the criteria for optimization [7].

Construction of Pseudo-Ternary Phase Diagram

On the basis of the solubility studies, Oleic acid was selected as the oil phase. Tween 20 and

PEG 200 were selected as surfactant and cosurfactant, respectively. Distilled water was used as

an aqueous phase. Phase diagrams involve the plotting the three components surfactant: co-

surfactant (Smix), oil and water each of them representing an apex of triangle. Ternary mixtures

with varying compositions of the components were formed. For any ternary mixture formed the

total of surfactants, co-surfactants and oil concentrations always added to 100%. The required

amount of the three components were weighed accurately and then sonicated for 3 minutes. The

mixture was then gently heated at 45–50ºC and vortex to form homogenous mixture. To this

mixture distilled water was added drop by drop until a transparent solution was formed. The

surfactant and co-surfactant was varied in mass ratios 1:1, 1:2, 2:1. The different concentration

ratios of oil and mixture of surfactant and cosurfactant were taken as 0.5:9.5, 0.5:9, 2:8, 3:7, 4:6,

5:5, 6:4, and 7:3. Ternary mixtures were formed in these ratios and then quantity of water

forming transparent solution was plotted in the pseudo-ternary phase diagram [8,9]. According to




the mass ratio distribution the following distribution was formed of the formulation out of which

10mg (1%w/w) was API rest formulation. The calculations as per the mass ratio of the various

combinations for construction of pseudo-ternary diagram are shown in tables 1 to 3.

Table No. 1: Smix Ratio 1:1(Oleic acid+Tween 20+PEG 200)

Table No. 2: Smix Ratio 1:2 (Oleic acid+Tween 20+PEG 200)

Oil:S/Cos Formulation

code

Oil

(mg)

Surfactant

(mg)

Co-surfactant

(mg)

0.5:9.5 NA1 0.050 0.470 0.470

0.5:9 NA2 0.056 0.467 0.467

2:8 NA3 0.198 0.396 0.396

3:7 NA4 0.298 0.346 0.346

4:6 NA5 0.396 0.297 0.297

5:5 NA6 0.496 0.247 0.247

6:4 NA7 0.594 0.198 0.198

7:3 NA8 0.694 0.148 0.148


code

Oil

(mg)

Surfactant

(mg)

Co-surfactant

(mg)

0.5:9.5 NB1 0.050 0.314 0.626

0.5:9 NB2 0.056 0.312 0.622

2:8 NB3 0.198 0.264 0.528

3:7 NB4 0.298 0.230 0.462

4:6 NB5 0.396 0.198 0.396

5:5 NB6 0.496 0.164 0.330

6:4 NB7 0.594 0.132 0.264

7:3 NB8 0.694 0.099 0.197




Table No. 3: Smix Ratio 2:1 (Oleic acid+Tween 20+PEG 200)

All above formulations contain 1% (10mg) of salicylic acid

From each phase diagram constructed, different mass ratios were selected according to nano-

emulsion region formed in the diagrams. For the preparation of nano-emulsion aqueous phase

titration method was used. The 1% of API was dissolved in oil and Smix. The prepared mixture

was then sonicated for 3 minutes and then homogenized at 50-60ºC for 5 minutes. Then water

was added drop wise and then vortex to form a clear transparent NE. These were further

characterized to optimize the final formulation.

Characterization of Nanoemulsion

From the pseudo-ternary diagrams it was observed that there are more than one formulation

having transparency and forming nano-emulsion. To eliminate and optimize only one

formulation which could then be transformed into the gel base the following parameters were

analyzed for each ternary mixture which forms a transparent nano-emulsions.

Emulsification

This was the parameter important to determine the spontaneity of the components to form the

emulsion. The ternary mixture forming emulsions were analyzed by adding drop wise the

prepared mixture in a 100ml volumetric flask filled with distilled water. In certain cases it turned

translucent to turbid, indicating the possibility of unstable emulsion formed [10,11].


code

Oil

(mg)

Surfactant

(mg)

Co-surfactant

(mg)

0.5:9.5 NC1 0.050 0.626 0.314

0.5:9 NC2 0.056 0.622 0.312

2:8 NC3 0.198 0.528 0.264

3:7 NC4 0.298 0.462 0.230

4:6 NC5 0.396 0.396 0.198

5:5 NC6 0.496 0.330 0.164

6:4 NC7 0.594 0.264 0.132

7:3 NC8 0.694 0.197 0.099




Visual Assessment

The samples giving good emulsification were further analyzed for turbidity initially and after 24

hours. It would indicate phase separation of the emulsion.

%Transmittance

The samples kept for 24 hrs at room temperature were also analyzed for % transmittance which

would determine the clarity of the solution. % Transmittance was analyzed by using UV

spectrophotometer.

Drug Content

Drug content was the important criterion for optimization of formulation as it indicates the

amount of drug entrapped in the nano-size globules of nano-emulsion. This study was carried out

by diluting each ternary mixture with methanol and then centrifuged for 2 hrs at 1000-1500rpm

which was then analyzed using UV spectrophotometer [12-14].

pH

The topically delivered formulations have a pH generally between 6-8 which is similar to the

skin pH. The samples were analyzed for pH using pH meter. 5 ml of the sample was transferred

into a beaker and the pH meter probe was immersed into the container. Then the pH reading was

recorded. The pH meter was calibrated before using it to measure the pH of the nanoemulsion.

The pH of the freshly prepared formulation was measured and was used to compare the change

in pH of the formulation after specified time intervals at the different temperatures studied.

TEM

This was done to analyze the morphology and structure of the formed nanoemulsion droplets

which was studied using transmission electron microscopy Malvern version 6.30 and it is

capable of point to point resolution. In order to carry out the study a drop of the nanoemulsion

was deposited on the holey film grid and studied after drying.

Droplet Size Analysis

This study is mainly carried out by using photon correlation spectroscopy; the technique

analyzes the fluctuations in light scattering due to Brownian motion of the particles. The

instrument used was a Zetasizer 1000 HS Malvern Instruments, UK (Light scattering was

monitored at temp 25°C and at 90° angle). The studies were performed at a fixed refractive index

that depends on the formulation involved in the study.




FT-IR Studies

FTIR spectrums of drug and mixture of drug and excipients formulation were obtained by means

of a FTIR spectrophotometer. The samples were prepared by the potassium bromide disk method

and measurements were attempted with the accumulation of 20 scans and a resolution of 4 cm-1

over the range of 400–4000cm-1

. After running the spectra, significant peaks relating to major

functional groups were identified; spectra of the subsequent sample of the same compound were

compared with the original.

Optimization of Nanoemulsion Based Gel

After optimization of the nano-emulsion formulation it was given a gel base as for topical

delivery the nano-emulsion has very low viscosity. The gel was formed by varying the

concentration of carbopol 980 (NF) as 0.75%, 1%, 2%. Carbopol 980 forms very good

consistency transparent gel at low concentration. Carbopol 980 is not toxic and does not cause

any irritation on skin. So carbopol 980 was selected as a gelling agent. For the preparation of gel

the required amount of carbopol was dipped in sufficient amount of water for 24 hrs and the

nano-emulsion formulation was added after neutralising the pH of carbopol.

Evaluation of Nano-Emulsion Based Gel

pH

The pH of the gel was determined by using Labindia Pvt. Ltd pH meter. 5 ml of the sample was

transferred into a beaker and the pH meter probe was immersed into the container. Then the pH

reading was recorded. The pH meter was calibrated before using it to measure the pH of the

nanoemulsion. The pH of the freshly prepared formulation was measured and was used to

compare the change in pH of the formulation after specified time intervals at the different

temperatures studied.

Viscosity

Viscosity measurements were carried out using a Brookfield viscometer DV III ultra V6.0 RV

cone and plate rheometer (Brookfield Engineering Laboratories, Middleboro, MA, USA). 20 ml

of nanoemulsion was filled in the cylindrical tube and the dial reading was noted at 10, 20, 50

and 100 rpm. The speed was then successively lowered and the corres-ponding dial readings

were noted. Direct multiplication of the dial readings with factors given in the Brookfield

viscometer catalogue gave the viscosity in centipoises (cp).




In-vitro Drug Release and Permeation Studies

Figure 1: Franz-Diffusion Cell Assembly

In-vitro study is performed using Franz-Diffusion Cell Assembly. The jacked cell embodied two

limb reservoirs that involve a donor compartment, receptor compartment and a sampling port.

The area of donar compartment exposed to receptor compartment i.e. diffusion cross sectional

area is 19.63cm2 and the total capacity of the receptor compartment is 30 ml. the receptor

compartment is stirred throughout the study at 100rpm employing a magnetic stirrer. The

temperature of the receptor medium is maintained at 37±2ºC by circulating hot water in the outer

jacket of cell employing a thermostatic water circulator. The release study was carried out using

Egg membrane cut off range 12-18 kD.

Prior to permeation experiment, membrane was thawed and clamped in donor and the receptor

compartment of the jacketed vertical Franz Diffusion Cell. The receptor compartment was filled

with the Phosphate buffer pH 7.4. The prepared gel formulation was applied on the membrane.

The donor chamber and sampling port were covered by parafilm to prevent evaporation during

the study. Aliquots of 1 ml were withdrawn periodically and replaced with equal volume of the

receptor medium to maintain the receptor phase volume at the constant level. Samples are

suitable diluted and analyzed for salicylic acid in UV-spectroscopy at 296nm. The study was

carried out for 12 hrs.

Comparison with Marketed Preparation

The comparison was carried out to determine that the developed formulation is at power with the

already marketed formulation. For this determination the parameters were selected on the basis

of which the comparison between the two could be carried out. The parameters are described as

follows:

Viscosity




In vitro-studies

The procedures employed for the determination of these parameters are the same as described in

the appropriate places in this chapter.

Kinetic Analysis of Drug Release

To analyze the mechanism of drug release from salicylic acid nanoemulsion based gel the in

vitro dissolution data were fitted to zero order, first order, Higuchi release model, and

Korsemeyer-Peppa’s model and the model with higher correlation coefficient (R2) was

considered to be the best model [15].

Accelerated Stability Studies

The optimized formulation was subjected to accelerated stability test over a period of three

months as per ICH guidelines at a temperature of 40 ± 2ºC, 75% RH, 25 ± 2ºC, 60% RH and 50

± 3ºC. The optimized formulation was analyzed for changes in appearance, pH and drug content.

RESULTS AND DISCUSSION

Solubility Studies

Solubility of salicylic acid in different oil,surfactant and co-surfactant was determined. From the

table 4 it was observed that sunflower oil has certain solubility but oleic acid has the highest

solubility. The possibility of an oil to form a transparent nano-emulsion is based on the carbon

chain length of the oils. From the above data it was concluded that almost all oils have poor

solubility; so oleic acid and sunflower oil which have medium chain length were selected based

on the solubility and for further screening procedure. From the table 5 it was observed that

Tween 20 has the best solubility and was thus selected for further study. From the table 6 it was

observed that PEG 200 has maximum solubility as compared to others.

Table No. 4: Solubility of salicylic acid in various oils

Sr. No Oil Solubility (mg/ml) (Mean±SD)

1 Castor Oil 1.7±0.61

2 Sunflower Oil 9.4±0.46

3 Isopropyl Myristate (IPM) 0.61±0.59

4 Myritol 0.36±1.14

5 Medium Chain Triglycerides

(MCT) Oil 0.4±0.66

6 Oleic acid 89.09±0.39

7 Olive Oil 2.9±0.57




Table No. 5: Solubility of salicylic acid in various surfactants

Sr. No. Surfactant Solubility (mg/ml) (mean±SD)

1 Cremophor RH 40 0.67±0.44

2 Kolliphor ELP 5.81±0.51

3 Tween 80 122.07±0.10

4 Tween 60 112±0.23

5 Tween 20 215.63±0.31

6 Span 80 15.23±0.67

7 Span 20 12.98±0.89

Table No. 6: Solubility of salicylic acid in various co-surfactants

Sr. No. Co-surfactant Solubility (mg/ml) (Mean±SD)

1 Glycerin 16.54±0.78

2 Propylene Glycol 8.61±0.53

3 Ethanol 367.54±0.21

4 PEG 400 314.54±0.40

5 PEG 200 423.63±0.26

6 Propanol 330.54±0.28

Pseudo-Ternary Phase Diagram

The pseudo-ternary phase diagrams were constructed by using water titration method to obtain

the o/w nano-emulsion region, within which the concentration range of the components (oil,

surfactant and co-surfactant) was identified. The mass ratio of surfactant to co-surfactant were

varied as 1:1, 1:2, and 2:1 and the ratio of oil: Smix were varied as 0.5:9.5, 0.5:9, 2:8, 3:7, 4:6,

5:5, 6:4, and 7:3.

Figure 2: TPD of Smix ratio 1:1 (Oleic acid+Tween-20+PEG-200)






Nano-emulsion region: The nano-emulsion region was found only in NA1, NA2, NC1

formulations i.e. clear and transparent liquid this is because in the initial stage of titration with

water the ratio of Oil:Smix is 0.5:9.5 and 0.5:9 means conc. of oil is very less as compared to Smix.

When water is added drop wise due to high conc. of surfactant, the oil and water disperse in each

other properly and no phase separation of oil and water molecules and hence clear and

transparent nano-emulsion was formed.

Coarse emulsion or Turbid region: The coarse emulsion or turbid region was found in all

formulations other than NA1, NA2, NC1 i.e. turbid liquid this is because in these formulation the

ratio of Oil:Smix is 2:8, 3:7, 4:6, 5:5, 6:4, 7:3 means conc. of oil increases as compared to Smix.

When water is added drop wise due to low conc. of surfactant, the oil and water molecule do not




disperse in each other properly and hence phase separation of oil and water molecules takes

place and hence turbid solution (coarse emulsion) was formed.

Characterization of Nanoemulsion Formulation

The construction of pseudo-ternary diagrams the all four combinations which should be included

in the optimum ratio of 1:1 and 2:1. In these also there were two ratios having a clear solution on

visual assessment i.e. ratios 0.5:9.5 and 0.5:9; out of these the selection was done on the basis of

following parameters and the results are tabulated in table 7.

Table No. 7: Results of characterization of nano-emulsion

From the table 7, the formulation NC1 which has oleic acid as an oil phase, Tween-20 as a

surfactant and PEG-200 as a co-surfactant was selected as the final formulation because of

higher transmittance and drug content and it has Smix ratio of 2:1. Oleic acid is unsaturated

medium chain fatty acid moiety containing carbon chain length of 16 and 18, respectively. Oleic

acid has HLB (hydrophilic-lipophilic balance) value 1. Oleic acid, a fatty acid with one double

bond in the chain structure, perturbs the lipid barrier in the stratum corneum by forming separate

domains which interfere with the continuity of the multilamellar stratum corneum and may

induce highly permeable pathways in the stratum corneum.

In general, the surfactant for Nanoemulsion should be very hydrophilic with HLB value of 16.7.

The higher the HLB is, the easier to dissolve in water. Tween 20 is capable to form

nanoemulsion in combination with oleic acid. These results indicate that HLB is important

parameter determining the surfactant’s ability to form nano-emulsion. The structure of the

surfactant also played an important role. Tween 20 is a polyoxyethylene derivative of sorbitan

monolaurate, and is distinguished from the other members in the polysorbate range by the length

of the polyoxyethylene chain and the fatty acid ester moiety. It has branched alkyl structure

whereas polysorbate has linear chain alkyl structure. It was reported that alkyl chain structure of

surfactant impact an effect on penetration of oil onto the curved surfactant film thus resulting in

F.C.

Name of

Compon-

ents

Smix

Ratio

Oil:Smix

Ratio

Emulsi-

fication

Visual

Assessment %T %D.C pH

NA1

O.A+

Tween20

+PEG 200

1:1

0.5:9.5 Slow Clear 98%

17.09 5.5

NA2 0.5:9 Slow Clear 96.3%

11.04 5.0

NC1 2:1 0.5:9.5 Rapid Clear 99.9%

73.18 5.9




the self-nanoemulsion formation. Co-surfactant helps the surfactant to form stable nano-

emulsion. It is amphiphilic with an affinity for both oil and aqueous phases and partitions to an

appreciable extent into the surfactant interfacial monolayer present at the oil-water interface. Co-

surfactant provides very low interfacial tension required for the stability and formation of

nanoemulsion. In this present study, PEG-200 was suitable as co-surfactant.

TEM

Figure 5: Result of TEM

Morphology and structure of the nano-emulsions were studied using a transmission electron

microscope. To perform the TEM observations, 1 ml of the nano-emulsion was diluted to 100 ml

using distilled water and a drop of the resultant system was deposited on the film grid and

observed after drying. Figure 5 showing the droplets of nano-emulsion.

Particle Size Analysis

The particle size of nano-emulsion was analysed to determine whether it is in the range suitable

for a nano-emulsion i.e. less than 100 nm. The result of this study is depicted from the following

figure 6; which clearly depicts the globule size of the formed nano-emulsion.

Figure 6: Graphical representation of particle size distribution




From the Table 8; it was concluded that the peak was shown at the particle size 337.4 d.nm and

the graph depicts that it has a homogenous distribution of the particles. Thus, the result shows

that the particle size of the formed nano-emulsion was in the required range therefore a

transparent nano-emulsion was formulated successfully.

Table No. 8: Results of particle size and distribution

Figure 7: Results depicting the size of particles and their intensity in the region

FT-IR Analysis

Structural Compatibility between drug and excipients were studied with the help of FT-IR

spectra of drug and FT-IR spectra of mixture of drug and excipients.

Figure 8: FT-IR spectra of Salicylic acid

C:\Program Files\OPUS_65\MEAS\Salicylic acid.0 Salicylic acid Powder 19/02/2014

3846

.99

3739

.62

3617

.40

2513

.98

1659

.46

1438

.32

1293

.0212

42.10

888.5

3

755.5

2

100015002000250030003500

Wavenumber cm-1

2040

6080

100

Tran

smitta

nce [

%]

Page 1/1




Table No. 9: FT-IR analysis of drug

Figure 9: FT-IR spectra of Drug+Excipients

Table No. 10: FT-IR analysis of mixture of drug and excipients

Groups Actual Value Observed Value

C=O 1650-1800 cm-1

1638 cm-1

O-H 2400-3400 cm-1

2513 cm-1

C-O 1000-1300 cm-1

1293 cm-1

C-C 1475-1600cm-1

1638 cm-1

C-H 3000 cm-1

3300 cm-1

All the groups of drug in mixture were present at same value or near it hence no structural

incompatibility was found.

Optimization of Nano-Emulsion Gel

The developed nano-emulsion was in liquid state; thus having low viscosity which could not be

applied topically; thus it was incorporated into a hydrogel of carbopol 980NF. Three different

concentration of carbopol 980 NF were selected for optimization of the required amount of

carbopol 980NF to form a hydrogel. The concentration of carbopol 980 NF was optimized on the

basis of the pH obtained and the % drug content achieved by incorporating in the particular

concentrations.

Groups Actual Value Observed Value

C=O 1650-1800 cm-1

1669 cm-1

O-H 2400-3400 cm-1

2513 cm-1

C-O 1000-1300 cm-1

1293 cm-1

C-C 1475-1600cm-1

1659 cm-1

C-H 3000 cm-1

2982 cm-1

C:\Program Files\OPUS_65\MEAS\FORMULATION.0 FORMULATION liquid 18/02/2014

3300

.18

2127

.42

1638

.58

1293

.39

1081

.65

100015002000250030003500

Wavenumber cm-1

2040

6080

100

Tran

smitta

nce [

%]

Page 1/1




Table No. 11: Results of optimization of nano-emulsion based gel

Sr. No %Carbopol

980

Drug

Content

% Drug

Content pH Consistency

1 0.75%

9.863 98.63 7.4 Viscous

2 1%

9.681 96.81 7.3 Less Viscous

3

2% 9.136 91.36 7.3 Highly viscous

From the table 11, it can be concluded that the concentration of 0.75% of carbopol 980 NF

would be optimum for the formation of nano-emulsion gel. This was on the basis of the viscosity

observed, % Drug content and pH.

Table No. 12: Final optimized formulation

Component Quantity(%w/w)

Salicylic acid 1%

Oleic acid (Oil) 5.37

Tween-20+PEG-200 (Smix) 86.27

Water 8.36

Triethanolamine 0.2

Carbopol 980 NF 0.75

Evaluation of Nano-Emulsion Based Gel

pH

The optimized concentration of carbopol 980 NF was 0.75% w/w and the pH of the gel prepared

as 7.0 which is well within the range of skin pH 6-8; it is also a neutral pH. Thus, the optimized

formulation shows the optimum pH.

Viscosity Determination

Viscosity of formulated as well as marketed formulation was measured by using the Brookfield

Viscometer and spindle no.63 was used and the viscosity was determined at spindle speed in

rpm.




Table No. 13: Results of Viscosity of formulated & marketed formulation

Sr. No. Spindle speed in

rpm

Formulated gel Viscosity

(cp)

Marketed gel Viscosity

(cp)

1 10 9756 ± 0.64 9984 ± 0.69

2 20 4235 ± 0.78 6452 ± 0.75

3 50 1960 ± 0.89 2579 ± 0.86

4 100 1047 ± 0.96 1168 ±0.94

Figure 10: Comparison of viscosity of formulated with marketed

From the fig. 10, it can be concluded that compared to the formulated gel the marketed gel had

higher viscosity which helps to determine that the diffusion of formulated gel would be greater

than the marketed gel due to lower viscosity than the marketed.

IN-VITRO STUDIES

Gel of formulated as well as marketed formulation was prepared and their release study through

Egg membrane was studied. The formulated formulation showed 52.19% release in about 12 hr.

But the marketed formulation showed 17.81% release in about 12 hr. The release profile of

optimized as well as marketed formulation is shown in the Table 14 and graph is shown in

Figure 11.

Table No. 14: In-vitro drug release of Marketed and Formulated formulation

Sr. No Time(min.) %CDR of Marketed

formulation

%CDR of Formulated

formulation

1 0 0 0

2 60 1.23 5.24

3 120 2.67 7.87

4 180 3.98 11.10

5 240 5.20 14.63

6 300 7.21 18.40

7 360 9.58 22.46

8 420 10.03 26.78




9 480 11.42 31.38

10 540 13.23 36.21

11 600 15.98 41.28

12 660 16.40 46.61

13 720 17.81 52.19

Figure 11: Results of Drug Release

From the figure 11, depicting the release profile of the formulations compared shows that the

formulated gel showed a greater amount of release as compared to any other formulation studied.

Thus, it could be concluded that the formulated gel showed a better release pattern as compared

to the marketed formulations.

Drug Release Kinetics Model

The release kinetic of Salicylic acid nano-emulsion based gel was studied by various kinetic

release models. The release data of nano-emulsion gel was shown in table 15.

Table No. 15: Drug release data of nano-emulsion based gel of SA

Time

(min)

Log

time

S.R. of

time

%

cumulative

release

log %

cumulative

release

%cumulative

remaining

log %

cumulative

remaining

0 0 0 0 0 100 2

60 1.778 7.74596 5.2470 0.7199 94.7529 1.9765

120 2.079 10.9544 7.8571 0.8952 92.1428 1.9644

180 2.255 13.4164 11.0664 1.0440 88.9335 1.9490

240 2.380 15.4919 14.5685 1.1634 85.4314 1.9316

300 2.477 17.3205 18.3204 1.2629 81.6795 1.9121

360 2.556 18.973 22.3514 1.3493 77.6485 1.8901

420 2.623 20.4939 26.6446 1.4256 73.3553 1.8654

480 2.681 21.9089 31.2159 1.4943 68.7840 1.8374

540 2.732 23.2379 36.0135 1.5564 63.9864 1.8060

600 2.778 24.4948 41.0569 1.6133 58.9430 1.7704

660 2.819 25.6904 46.3535 1.6660 53.6464 1.7295

720 2.8573 26.8328 51.8976 1.7151 48.1023 1.6821




Zero Order Model

Graph was plotted between % cumulative drug release Vs time.

Figure 12: Zero order plots for drug release of SA nano- emulsion based gel

First Order Model

Graph was plotted between log % cumulative remaining Vs time.

Figure 13: First order plots for drug release of SA nano- emulsion based gel

Higuchi Model

Graph was plotted between % cumulative releases Vs square root time

Figure 14: Higuchi plot for SA nano-emulsion based gel




Korsmeyer –Peppas Model

Graph was plotted between log % cumulative release Vs log time.

Figure 15: Korsmeyer –Peppas plot for SA nano-emulsion based gel

Table No. 16: Kinetic equation parameter of SA nano-emulsion based gel

Formulation

Name

Zero order

First order

Higuchi

Peppas

R2

KO

R2

Kf

R2

Kh

R2

N

SA

nanoemulsion

based gel

0.984

0.132

0.965

0.000

0.884

1.994

0.927

0.603

From the data obtained in table 15 were fitted into equation for the zero order, first order and

higuchi and Korsmeyer peppas release models. The interpretation of data was based on the value

of the resulting regression coefficients; as depicted in table 16. The zero order rates describes the

system where the drug release independent of its concentration shows the cumulative amount of

drug release Vs time for zero order kinetics. It was found that the in vitro drug release of SA

nano-emulsion based gel was best explained by zero order equation as the plot showed the

highest linearity. Thus, it can be concluded that; the developed formulation showed a zero order

drug release.

Accelerated Stability Studies

The results of the accelerated stability studies are shown in Table 17 and 18. The appearance of

the gels remained clear and no significant variation in pH was observed after subjecting the

formulations to stability stress for 3 months. Also there was no significant change in the drug

content was observed after the 3-month period.




Table No. 17: Stability study of Nano-emulsion based gel formulations

Formulation

(RC1) Month Appearance pH %Drug Content

25 ± 2ºC,

60 % RH

0 Clear 7.3 89.83 ± 0.31

1 Clear 7.4 88.75 ± 0.64

2 Clear 7.2 87.69 ± 0.46

3 Clear 7.3 87.52 ± 0.73

40 ± 2ºC,

75 % RH

0 Clear 7.4 89.95 ± 0.42

1 Clear 7.2 89.86 ± 0.45

2 Clear 7.3 88.79 ± 0.77

3 Clear 7.1 88.63 ± 0.43

Table No. 18: Data showing stability studies of NE gel formulation at 50 ± 3ºC

Time (days) Appearance pH % Drug Content

0 Clear 7.4 88.65 ± 0.53

15 Clear 7.3 87.96 ± 0.42

30 Clear 7.1 87.59 ± 0.71

CONCLUSIONS

Nanoemulsion gel of salicylic acid is more acceptable for the topical route of administration. The

ingredients used in the formulations are highly stable and safe for the topical delivery. Also the

study confirmed that nano-emulsion based gel are very promising carrier for the topical delivery

of Salicylic acid revealed from TEM and In-Vitro release study. The study revealed that for

preparation of a nano-emulsion the concentration of oil was reduced while selecting the mass

ratio; for development of nano-emulsion. This nano-emulsion formed also showed an optimum

particle size which could easily permeate through the stratum corneum layer of the skin and thus

permeation could be enhanced along with solubility of the API. Thus, from these conclusions it

could be inferred that the objectives are achieved; further animal studies would confirm that the

irritation side effects are eliminated by such formulations or it still requires any improvement.

ACKNOWLEDGEMENT

Authors wish to give thanks to Company Oniosome Healthcare Pvt. Ltd., Mohali, for providing suitable

research laboratory to carry out this project work.




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