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.
Author(s)
Details:
Razan Solayman Awad,
Quality
Control Department, Faculty of Pharmacy, Aleppo University, Syria.
Wassim
Abdelwahed,
Pharmaceutics
Technology Department, Faculty of Pharmacy, Aleppo University, Syria.
Yaser Bitar,
Quality Control Department, Faculty of Pharmacy, Aleppo University,
Syria.
Please see the link here: https://stm.bookpi.org/ACPR-V8/article/view/14043
