Advanced Pharmaceutical Bulletin, 2012, 2(2), 141-147
doi: 10.5681/apb.2012.022 Preparation and Evaluation of Tretinoin Microemulsion Based on
Pseudo-Ternary Phase Diagram

Eskandar Moghimipour 1,2, Anayatollah Salimi 1,2*, Fatemeh Leis 2
1 Nanotechnology Research Center, Jundishapur University of Medical Sciences, Ahvaz, Iran. 2 Department of Pharmaceutics, Faculty of Pharmacy, Jundishapur University of Medical Sciences, Ahvaz, Iran.
Article Type:
Purpose: The aim of the present research was to formulate a transparent microemolsion
Research Article as a topical delivery system for tretinoin for the treatment of acne. Methods:
Microemulsion formulations prepared by mixing appropriate amount of surfactant Article History:
including Tween 80 and Labrasol, co-surfactant such as propylene glycol (PG) and oil Received: 20 May 2012 phase including isopropyl myristate – transcutol P (10:1 ratio). The prepared Accepted: 10 June 2012 ePublished: 15 June 2012 microemolsions were evaluated regarding their particle size, zeta potential,
conductivity, stability, viscosity, differential scanning calorimetry (DSC), scanning
electron microscopy (SEM), refractory index (RI) and pH. Results: The results showed
that maximum oil was incorporated in microemolsion system that was contained surfactant to co-surfactant ratio (Km) of 4:1. The mean droplets size range of microemulsion formulation were in the range of 14.1 to 36.5 nm and its refractory Differential scanning calorimetry index (RI) and pH were 1.46 and 6.1, respectively. Viscosity range was 200-350 cps. Drug release profile showed 49% of the drug released in the first 8 hours of experiment belong to ME-7. Also, Hexagonal and cubic structures were seen in the SEM photograph of the microemulsions. Conclusion: physicochemical properties and in
vitro release were dependent upon the contents of S/C, water and, oil percentage in formulations.Also, ME-7 may be preferable for topical tretinoin formulation.

The word microemulsion w a s originally proposed by Hoar and Schulman in the earliest of the 1940s. They concentration is chosen on the basis of the marketed were generated a clear single-phase solution by tretinoin formulations. The ability of microemulsion to thermodynamically investigated in the present study. isotropic clear colloidal dispersion of oil, water and surfactant, frequently in combination with a co- Materials and Methods
surfactant which have high stability, ultra low Tretinoin was purchased from Sepidaj Company (Iran). interfacial tension, large interface area , low viscosity Isopropyl myristate, Tween 80 and PG were obtained microemulsions can be classified oil- in -water, (o/w), macrogoglycerides (Labrasol) and Diethylene glycol water- -in- oil (w/o) and bicontinuous phase monoethyle ether (Transcutol P) were gift from Gattefosse Company (France). All chemicals and microemulsions include improvement in poorly drug solvents were of analytical grade. Freshly distilled solubility, enhancement of bioavailability, protection of water was used in the experiments. Minitab15 software the unstable drugs against environmental conditions was used for experimental design and the evaluation of and a long shelf life. the effect of variables on responses. Sigma plot Tretinoin is trans-retinoic acid and related to retinol software was applied for providing tertiary phase (vitamin A). The drug in use decreases proliferation of acute promyelocytic leukemia APL cells in culture and in vivo. The drug is used for treatment of various skin Tretinoin assay
disorders including acne, photo aged skin and severe The quantitative determination of tretinoin was condition like psoriasis, squamous cell carcinoma.4,5 spectrophotometry The main aim of our investigation was to develop and (BioWaveII,WPA) at λmax = 362 nm. evaluate a microemulsion based tretinoin (0.05%, w/w) *Corresponding author: Anayatollah Salimi (PhD), Nanotechnology Research Center, Jundishapur University of Medical Sciences, Ahvaz,
Iran. Tel: 09163130905, E-mail: [email protected] Copyright 2012 by Tabriz University of Medical Sciences Moghimipour et al. Solubility of tretinoin
Microemulsion samples were exposed in a temperature Solubility of tretinoin was determined in different oil ranging from +30 0C to - 50 0C (scan rate: 100C/min). (oleic acid, isopropyl myristate, transcutol P), In order to ensure accuracy and repeatability of data, surfactants (Span 80, Labrasol, Tween 80) and co- DSC instrument was calibrated and checked under the surfactant (Propylen glycol) by dissolving an excess conditions of use by indium standard. Changes of amount of tretinoin in 3ml of oil, and other components Enthalpy quantities (∆H) were calculated from using a stirrer at 37 0C ± 0.5 for 72 h.6 The equilibrated samples were then centrifuged at 10000rpm for 30 min thermograms by Eq1:10-12 to remove the undissolved drug. The solubility of ∆H= peak area/sample weight tretinoin was determined by analyzing the filtrate spectrophotometrically using nanospecterophotometry Scanning electron microscopy (SEM)
(Biochrom WPA Bioware) after dilution with methanol Scanning electron microscopy (SEM) was used to characterize microstructure of emulsions. SEM of samples were measured by LED 1455VP, Germany. Pseudo-ternary phase diagram construction
To investigate concentration range of components for Zeta potential determination
the existing boundary of MEs, pseudo-ternary phase Zeta potential of samples were measured by Zetasizer diagrams were constructed using the water titration (Malvern instrument 1td ZEN3600, UK). Samples were method. Three phase diagrams were prepared with the placed in clear disposable zeta cells and results were 2:1, 3:1, and 4:1 weight ratios of (Labrasol /Tween 80) recorded. Before putting the fresh sample, cuvettes Propylen glycol respectively. Oil phase (Oleic acid- were washed with methanol and rinsed using the Transcutol P) and the surfactant mixture were then sample to be measured before each experiment. mixed at the weight ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1.7 These mixtures were diluted Particle size measurements
dropwise with double distilled water, under moderate The average droplet size of samples was measured at 25 °C by SCATTER SCOPE 1 QUIDIX (South Korea) microemulsions when they appeared as clear liquids.8 and their refractory indices were also calculated. Several parameters influence on final properties of microemulsions. Full factorial design was used Determination of viscosity
concerning with 3 variables at 2 levels for formulations The viscosity microemulsions were measured at 25 °C Major variables take part in determination of with a Brookfield viscometer (DV-II+Pro Brookfield., USA) using spindle no. 34. With shear rate 100 rpm.13 surfactant/cosurfactant ratio (S/C), percentage of oil (% oil) and water percentage (%w). Eight different Conductivity measurements
formulations with low and high values of oil (5% and The electric conductivity of ME was measured with a 30%), water (5%, 15%), and S/Co mixing ratio (3:1, conductivity meter (Metrohm Model 712). This was 4:1) were prepared for preparing of microemulsion done by using conductivity cell (with a cell constant of 1.0) consisting of two platinum plates separated by desired distance and having liquid between the Preparation of tretinoin microemulsions
platinum plate acting as a conductor. Various MEs were selected from the pseudoternary phase diagram with 3:1, and 4:1 weight ratio of Determination of pH
Labrasol /Tween 80/Propylen glycol. Tretinoin(0.05%) The pH values for microemolsion was determined at was added to oil phase , then adding S/ CoS mixture 25°C by pH meter (Mettler Toledo seven easy, and an appropriate amount of double distilled water Switzerland). All measurements were carried out in was added to the mixture drop by drop and the MEs containing tretinoin were obtained by stirring the mixtures at ambient temperature.9,10 Surface tension measurement
The surface tension of microemulsion was measured at Differential scanning calorimetry (DSC)
25°C with a Torsion balance (WHITE ELEC Model DSC measurements were carried out by means of a Metller Toldo DSC1 starR system equipped with Stability study
Approximately 10-15mg of microemulsion samples The stability of microemulsions was studied regarding were weighted into hermetic aluminium pans and quickly sealed to prevent water evaporation from Microemulsions were kept in various temperature microemulsion samples. Simultaneously an empty ranges (4 °C, 25 °C and 37 °C) and observed for phase hermetically sealed pan was used as a reference. Microemulsions were centrifuged by high speed 142 Advanced Pharmaceutical Bulletin, 2012, 2(2), 141-147
Copyright 2012 by Tabriz University of Medical Sciences

Tretinoin Microemulsion brushless centrifuge (MPV-350R, Poland at 10000 rpm (Table 2). PI is a measure of particle homogeneity and for 30 minute at 25 °C and inspected for any change in it varies from 0.0 to 1.0. The closer to zero the PI value their homogeneity. the more homogenous are the particles. Release study
Franz diffusion cells (area 3.4618 cm2) with a cellulose
membrane were utilized to determine the Release rate of tretinoin from different microemulsion formulations. The cellulose (molecular weight G12 000) membrane was first hydrated in the distilled water solution at 25 0C for 24 hours. The membrane was then clamped between the donor and receptor compartments of the cells Diffusion cell was filled with 25 ml of phosphate buffer (pH = 7.4) and methanol (1:2). The receptor fluid was constantly stirred by externally driven magnetic bars at 600 rpm throughout the experiment. Tretinoin microemulsion (5 g) was accurately weighted and placed in donor compartment. At 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h time intervals, 2 ml sample was removed spectrophotometric determination and replaced immediately with an equal volume of fresh receptor solution. Samples were spectrophotometer (BioWaveII,WPA) at 362 nm. The results were plotted as cumulative released drug percent versus time. Statistical methods
All the experiments were repeated three times and data
were expressed as the mean value ± SD. Statistical data
were analyzed by one-way analysis of variance
(ANOVA) and P < 0.05 was considered to be
significant with 95% confidence intervals.
Results and discussion
The maximum solubility of tretinoin was found in
Isopropyl mirystate: Transcotol P (10:1) (4.85 ± 0.09)
as compared to other oils (Table 1). In addition, the highest drug solubility of tretinoin in surfactants were found in Labrasol (3.232 ± 0.23), and Tween 80 (4.27 ± 0.12). Based on the solubility studies of tretinoin in oil, surfactant and co-surfactant and the preformulation studies we found IPM-Transcutol P, labrasol, Tween 80 and propylene glycol could be the most appropriate combination for preparation of microemulsion. Pseudo-ternary phase diagrams of the investigated quaternary Transcotol P (10:1)/ Labrasole -Tween 80/ PG is showed in figure 1. Microemulsions were formed at ambient temperature. The phase diagrams clearly indicated increased with increase in the weight ratio of Figure 1. The pseudo-ternary phase diagrams of the oil-
surfactant/cosurfactant (km=2 - 4). surfactant/cosurfactant mixture–water system at the 2:1, The ME formulations had the mean particle size in the 3:1, and 4:1 weight ratio of labrasol /Tween 80/ range of 14-60 nm (Table 2). Particle size of selected Propylene glycol at ambient temperature, dark area show microemulsions zone. ME and drug loaded ME were determined and there was no significant difference observed in average The PI indicated that ME formulation had narrow size particle size after loading the drug. The ME 6 distribution. Analysis of variance is shown that formulation had the lowest average particle size 14.1 ± correlation between mean particle size, PI and 0.6 nm with polydispersity index (PI) of 0.362 ± 0.010 independent variables are not significant (p > 0.05). Copyright 2012 by Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin, 2012, 2(2), 141-147 143
Moghimipour et al. The refractive index (RI) of the ME formulation was The ME formulations had the average conductivity in found 1.45 that is near to oil phase which indicates the range of 0.0447-0.0900 ms/cm. MEs formulations have water -in-oil structures. The ME formulation had appropriate observed pH Analysis of variance is shown that correlation between value (6.84 ± 0.11) that is best for topical application. RI and independent variables (%w) are significant (p < Incorporation of tretinoin did not significantly affect 0.05). Linear equation which indicates all the main the observed pH value of the ME formulations (Table 3). effects for RI is: RI=1.45 + 0.0012s/c-0.000124 (%oil)-0.000625 (%water). Table 1. Solubility of Tretinoin in different oils, surfactant and co-surfactant (mean ± SD, n = 3)
Phase type
Solubility (mg/ml)
Isopropyl Myristat Oleic acid + Transcutol P IsopropyMyristat +Transcutol P surfactant
Propylene glycol Table 2. Compositions of Selected Microemulsions (% w/w) and Particle Size (mean ± SD, n=3)
S/C Oil S+C
water Particle size(nm) Polydispersity
+: high level; - : low level Table 3. pH, Refractive index,Conductivity and Zeta potential of selected Tretinoin microemulsions (mean ± SD, n = 3)
Refractive index Zeta potential(mv)
144 Advanced Pharmaceutical Bulletin, 2012, 2(2), 141-147
Copyright 2012 by Tabriz University of Medical Sciences

Tretinoin Microemulsion The ME formulation had the average viscosity range accumulative release percent is: %Release = 39.9- (215.5 ± 5.2cps - 343.5 ± 8.3cps) (Table 4). The 2.75S/C + 0.0206 (%oil) - 0.349(%water). highest viscosity belongs to ME-8 formulation with bicontinueous structure. Multivariate regression was applied for the analysis of correlation between independent variables and MEs viscosity. The percent of water and s/c had more positive effect and negative effect on viscosity, respectively. Table 4. Surface tension and viscosity of selected
microemulsions (mean ± SD, n=3) Surface tension
Figure 2. In vitro release profile of MEs formulation of tretinoin.
Figure 3 shows the SEM images of ME-7 and ME-8. DSC result implies important information about water state in microemulsions.15 When water is mixed in to a microemulsion system it can be either bound (interfacial) or free (bulk) water depending of its state in the system. In cooling curves of the samples (ME1-ME 2), DSC thermogram showed one exothermic peak at around -10 to -14 0C that indicate the freezing of Linear equation which shows all the main effects for internal or bound water in these formulations. The viscosity is: Viscosity = 336-34.3s/c-0.24 (%oil) + other exothermic peak at around – 19 to – 25 0C belong 4.57(%water). There was no significant difference to oil phase freezing.14,15 In cooling curves of ME-3 found between the viscosities of free drug and drug and ME-4 , DSC thermograms showed one exothermic loaded MEs (p > 0.05). peak at -330C (ME-3) and two exothermic peak at -1 The ME formulation had the zeta potential average (- and -34.40C for ME -4 which indicates bulk water(-10 0.0674-6.41mv) (Table 3). The highest zeta potential C) and bound water (-33, -34.4 0C). DSC thermograms belongs to ME-6 formulation with micellar and of ME-5 and ME-6 showed two exothermic peak at - bicontiuous structure the lowest belong to ME-8 with 20C (bulk water) and 39.8-40.9 0C (bound water. bicontinuous structure. Multivariate regression was science the freezing temperature is very low, the water used for the analysis of correlation between must be strongly bound or interacts with surfactants.16 independent variables and MEs zeta potential. The S/C In cooling curves of ME-7 and ME-8, DSC had more positive effect on zeta potential. There was thermogrms showed two exothermic peaks of around -9 no significant difference found between the zeta to -15 0C and -22 to -24 0C, which indicates bound potential and independent variables (p > 0.05). water and oil phase freezing, respectively. Figure 4 The MEs formulation had the surface tension average shows DSC cooling thermogram of ME-8 and ME-7 (37.83 ± 1.25 to 40 ± 2.17 dyne/cm) (Table 4).The formulations. Pure oil peaks could be observed at -14 and -28 0C, and became smaller when the water microemulsions because surface tension amounts of concentration in creased. The peaks disappeared at 15% MEs is nearby to oil phase surface tension. wt/wt water, suggesting that oil phase changed from Figure 2 shows the release profiles of microemulsions external to internal phase. of tretinoin MEs formulations. The cumulative amount The visual inspection test was performed for 3 months of tretinoin that had permeated through the cellulose by drawing ME sample at weekly interval for the first membrane (%) was plotted as a function of time month and monthly interval for the subsequent months. (hours). In this study, ME-7 and ME-8 have the highest The visual observation showed no evidence of phase and lowest accumulative release percent, respectively. separation or any flocculation or precipitation. It was observed that there is a strong correlation These samples also revealed no sign of phase between the water percent, S/C ratio and release percentage value of tretinoin (p < 0.05). Multivariate centrifugation at 10000 rpm for 30 min. The regression was used for the analysis of correlation centrifugtion tests revealed that microemulsions were between independent variables and MEs release. The remained homogenous without any phase separation percent of water and S/C ratio had more negative throughout the test indicates good physical stability of accumulative release percent, respectively. Linear both preparations. equation which shows all the main effects for Copyright 2012 by Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin, 2012, 2(2), 141-147 145
Moghimipour et al. authors are very thankful to Faratin company Executive manager (Taheri, M, Iran) for providing gratis samples of Labrasol and Transcutol P from GATTEFOSSE (France) and also GATTEFOSSE company (France). LY-T7-COOL, 23.11.2011 10:50:24 LY-T7-COOL, 8.0000 mg LY-T8-COOL, 23.11.2011 11:07:40 LY-T8-COOL, 8.0000 mg Figure 3. SEM photographs of of ME-7 and ME-8.
Figure 4. DSC cooling thermograms of ME-7(A) and ME-8(B)
Conflict of Interest
properties and in vitro release were dependent upon the There is no conflict of interest in this study. contents of S/C, water and, oil percentage in formulations. Phase diagrams indicated more width References
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