Investigating the Impact of Preparation Conditions and Formulation on the Accelerated Stability of Tretinoin (RA) Loaded Liposomes Prepared by Heating Method

Objective: The aim of this study was to investigate the most appropriate ingredients and the ideal conditions for preparing a stable all-trans retinoic acid or tretinoin (RA) loaded liposome formulation with a high encapsulation efficiency intended for immediate application on the skin using a simple preparation method.
Practical Procedures and Methods: The study was divided into three stages:
Stage 1: The optimization of the preparation conditions.
The Formulas were prepared using a heating method. The effect of formula variables on liposome properties was investigated.
Formula variables included:
- Phospholipid quantity (Soybean-L-
-phosphatidylcholine (SPC) was used during this stage).
- Water-miscible solvent type.
- Temperature of adding the active substance.
- Stirring speed.
- Duration of homogenizing size with a sonicator.
- pH of phosphate buffer.
- Ionic strength of phosphate buffer.
Liposome properties included:
- Mean hydrodynamic diameter (Z-average)
- Zeta potential (Zp)
- Polydispersity index (PdI)
- Encapsulation efficiency (EE%).
All properties were measured before and after sonication. The formulations prepared at different values of pH and ionic strength of the phosphate buffer were left at room temperature for a period of time and then the measurement of properties was repeated in addition to measurement of pH.
Dynamic Light Scattering Process (DLS) and the electrophoretic mobility process were used to determine the mean size and zeta potential, respectively, with a Malvern Zetasizer Nano-series. Polydispersity index which indicates the homogeneity of particle size was also obtained with a Malvern Zetasizer Nano-series.
The total concentration and unentrapped concentration of tretinoin were determined spectrophotometrically at wavelength 352nm to calculate the EE%. Acidified isopropyl alcohol was used as a blank.
Based on the results at this stage, it was selected the conditions that exhibited the RA-loaded liposome properties, which expected to show the best stability with time.
Stage 2: Preparation of final formulas.
According to the selected conditions at stage 1, six liposome formulas (Fs) were prepared. Three types of phospholipid were used.
Soybean-L-
-phosphatidylcholine (SPC) was used to prepare F1 and F2, F3 was prepared with a hydrogenated soybean- L-
-phosphatidylcholine (HSPC) whereas F4 was prepared with a 1, 2-Dimyristoyl-rac-glycero-3-phosphocholine (DMPC). F5 and F6 were prepared by replacing 20% and 40% of SPC in the formula 2 with cholesterol, respectively.
Sodium cholesteryl sulfate (SCS) was added to all formulas except F 1.
A topical ethanolic solution of tretinoin F7 was also prepared for comparison.
Stage 3: Studying the accelerated stability of the final formulas
The final formulas (F1 to F7) were stored at 40 °C±2 °C/ 75% RH±5% RH for 6 months. All properties of liposome particles were determined as stage 1 except EE%, which was determined by a validated H. P. L. C method . Chromatographic separation was achieved in the reverse phase C18 column (EC 250/4.6 Nucleodur 100-5 C18, Macherey-Nagel, Germany) as a stationary phase while the mobile phase was (acetonitrile: TFA 0.01%) in a gradient elution mode in the ratios of (70:30,65:35,70:30 v/v) for (24, 4,4 min), respectively. Other parameters were: flow rate 1 ml/min, injection volume 20
l, oven temperature 30 °C and wavelength 354nm utilizing a PDA detector. Acetonitrile was used to dilute all standard and sample solutions.
Results:
Stage1: After sonication, 100 mg of SPC showed the best homogeneity (PdI= 0.459±0.044) and the highest zeta potential (-63.2±2.83). A slight difference in EE% was noticed between three amount (100,150,200 mg) of SPC.
The formula that was prepared by adding 3%(v/v) of glycerol showed the most homogeneous size distribution (PdI= 0.459±0.044) and the smallest mean size (194.56±16.74nm), which was located within the targeted range (100-300 m).
The lowest temperature (30°C) showed both the smallest mean size (194.56±16.74) and PdI (0.459±0.044). Besides, there was no significant difference in EE% between three temperatures (30°C, 45°C and 65°C).
1300 rpm showed a higher EE% (87.27±3.38%) and a smaller mean size (194.56±16.74) than 600 rpm. No significant difference in zeta potential was noticed.
Stage2:
The mean size of prepared particles was within the targeted range (100-300 nm) with 15 min of sonication, which also showed a relatively small decrease in EE% (87.27±3.38%) and a high zeta potential (-63.2±2.83).
pH= 6.5 of phosphate buffer, 0.05M showed the lowest change in pH with time and the highest EE% (64.64±0.132%) and no significant change in both zeta potential and mean size was noticed with time.
Substantial decrease in zeta potential and EE% was noticed by increasing the ionic strength. The lowest ionic strength, 0.025 M of phosphate buffer (pH=6.5), showed both the highest EE% (79.98±0.044%) and zeta potential(-45.4±4.03). The mean size was also relatively stable with time.
Stage3:
pH: By comparing F1 with F2 it was noticed that adding sodium cholesteryl sulfate (SCS) had a negligible effect on the change of pH observed with time.
HSPC (F3) and DMPC (F4) showed less noticeable change in pH with time in comparison with SPC (F2).
By increasing the ratio of cholesterol, the change in pH became less noticeable.
Mean Diameter and Zeta Potential: Zeta potential decreased simultaneously with the decrease of pH in all formulas.
SCS showed a positive effect on the stability of liposomes with time, since the F2 prepared with SCS showed less change in mean size in comparison with F1 prepared without SCS.
The best stability of mean size was noticed with HSPC (F3)which has a phase transition temperature (Tm= 50-60°C) higher than the storage temperature (40°C), where it showed the lowest increase in mean size with time in comparision with SPC (F2) and DMPC (F4).
By comparing F5 and F6 (prepared with cholesterol 20%, 40%, respectively) with F2 (prepared without cholesterol), no deficiency in the tendency of particles to fusion was observed, where the three formulas showed more noticeable fusion after three months.
EE%: EE% was obviously lower in F 4 (DMPC) (56.52±0.81%) in comparison with F2 and F3. Adding sodium cholesteryl sulfate to formula 2 showed a slight decrease in EE% in comparison with F1. The cholesterol added to the formulas F5 and F6 didn,t improve EE%, but the leakage of RA from liposomes was not observed then.
The Total Percentage: The liposome formulas (F1 to F6) didn't show a noticeable improvement in the stability of active substance in comparison with ethanolic solution (F7). F2 and F3 showed a little more improvemet in the stability of active substance than F4. F2 and F5 showed a little more improvemet in the stability of active substance than F6.
Conclusion: The pH of buffer had a noticeable effect on the change of pH with time. The ionic strength of buffer had a noticeable effect on zeta potential and EE%. It is necessary to add a charge inducing substance to a neutral phospholipid to improve its stability with time.
The phospholipid that had a phase transition temperature (Tm) above the storage temperature was the best choice to improve the stability of particles and decrease the fusion of them with time and to prevent the leakage of the active substance without needing to add cholesterol.
It is necessary to add cholesterol to phospholipid that had phase transition temperature (Tm) lower than the storage temperature to prevent the leakage of the active substance.
Liposome formulations didn't show a significant improvement in the stability of active substance in comparing with ethanolic solution.