P.A. Panmand1*, P. R. Mahaparale2, V. P. Thorat3
1Indrayani Institute of Pharmaceutical Education and Research, Talegaon, Dabhade.
2Government College of Pharmacy, Osmanpura Aurangabad, Maharashtra, India – 431005.
3Siddhi College of Pharmacy Chikhali, Pune, Maharashtra 411062.
*Corresponding Author E-mail: priyankapanmand92@gmail.com
ABSTRACT:
In order to understand the in vitro drug release of the produced gel, the goal of the current investigation was to construct and characterize. Acitretin loaded Solid Lipid Nanoparticles (ActSLNs). Act SLNs were created utilizing the Box Benhken design and the hot homogenization procedure. Act SLN's average diameter and surface morphology were assessed. Act SLNs were lyophilized, then they underwent stability testing, powder X-Ray diffraction, Differential Scanning Calorimetry (DSC), and Fourier Transform Infrared (FT-IR) tests to characterize them. The SLNs were added to a 0.25% w/w Carbopol 940 gel base the Stability study, ex vivo drug release in vitro drug releases in rat skin were carried out. The optimized Act SLNs had a spherical form, an entrapment efficiency of 78.82% to 85.73%, and an average particle size of 123.24nm to 409nm. The generation of SLNs was confirmed by DSC, FTIR, and XRD data. ActSLN gel (0.056mg/cm2) significantly increased the amount of accutane deposited in rat skin compared to Act plain gel (0.012mg/cm2).No discernible change was found in the stability studies, according to stability studies.
KEYWORDS: Acitretin, Solid lipid nanoparticles, Hot homogenization technique.
INTRODUCTION:
Psoriasis is a non-contagious, dry, inflammatory, and unsightly skin condition that can impact a person's entire system. The scalp, tips of fingers and toes, palms and soles, umbilicus, gluteus, under the breasts and genitalia, elbows, knees, shins, and sacrum are the area’s most frequently afflicted. The nature of this illness is chronic. Due to the rapid and excessive growth of epidermal cells in this condition, the skin keeps scaling as flakes known as psoriatic plaques, which eventually peel off as exfoliation. Psoriatic arthritis is a form of joint inflammation brought on by psoriasis. Dandruff and some types of arthritis, regrettably, are associated with psoriasis.1, SLNs have been included into the formulation of topical medications and cosmetics.
The stratum corneum is in intimate touch with them thanks to their small particle size, and the lipid in them allows for targeted drug administration to the skin's layers. Because of their solid matrix, SLNs have the ability to control drug release over an extended period of time with a lower rate of systemic absorption.Another advantage of SLNs for topical distribution of active chemicals is how quickly these medicines can be brought to market.2 Acitretin (Act), also known as 13 cis-trans retinoic acid, is a vitamin A metabolite that has a wide range of physiological effects, including the control of collagen synthesis, sebum production, and epithelial cell proliferation and differentiation.
Although oral acitretin is now approved for the treatment of severe psoriasis in adults, its usage is constrained by teratogenicity and systemic adverse effects.3 Acitretin may have a lower potential for systemic toxicity when applied topically, while its local bioavailability in the skin is increased. Despite these intriguing characteristics, the material's usefulness is severely constrained by a number of drawbacks, including skin irritation, a very low water solubility, and a significant degree of instability in the presence of air, light, and heat.4
Acitretin has a poor photostability and a low solubility (0.0729mg/L), which may make it difficult to incorporate into a suitable vehicle and render the topically applied medication ineffective.5 Additionally, the topical use of Acitretin frequently results in local irritation including erythema, peeling, and burning at the application site as well as increased vulnerability to sunlight, which frequently limits patients' willingness to accept it. The current study's goal was to investigate if SLNs could enhance the topical administration of Acitretin.6
MATERIALS AND METHOD:
Materials: Compritol ATO 888 and Acitretin were gifts from Colorcon Asia Pvt. Ltd., Goa and Glenmark Pharmaceuticals, Nashik, respectively. Tween 80 was acquired from Research Lab Fine Chem. in Mumbai. All additional compounds were of the analytical variety.
Preparation of Acitretin loaded SLN:
Acitretin loaded Solid lipid nanoparticles were prepared by the hot homogenizationtechnique, In the hot homogenization approach, the medication was introduced to the lipid at a temperature that was 5–10 0C above the lipid melting point, and the melted substance was subsequently stirred in an aqueous surfactant solution at the same temperature. Following homogenization of the pre-emulsion, a hot o/w microemulsion was created, which was cooled to room temperature or a lower temperature to form SLN.7
Characterization of Acitretin loaded SLNs:
1. Particle size analysis:
An optical microscope and software (Pixel Pro) were used to measure the particle size. The term "nm" was used to represent the average particle size. SLNs were mounted on slides and positioned above the micrometer stage for image processing using the software (Pixel Pro). A minimum of 100 particles were used for each determination, and the mean value was reported.8
2. Drug entrapment efficiency:
The centrifugation method was used to determine the SLN dispersion's entrapment efficiency. To get the supernatant liquid, the SLN dispersion was centrifuged at 11000rpm for 30 minutes in a cooling centrifuge. After appropriate dilution with a phosphate buffer pH 7.4, the collected 1ml liquid was filtered to quantify the free drug concentration. In a UV Spectrophotometer, the absorbance was determined at 353 nanometers.9
The following formula was used to compute the entrapment efficiency:10
Entrapment efficiency %) = weight of drug incorporated /weight of drug initially taken x 100
3. Scanning electron microscopy:
Scanning electron microscopy (SEM) was used to examine the surface morphology of the particle. SLNs were coated with a thin gold-palladium layer using a sputter-coated unit, placed on double-faced adhesive tape, and examined using a scanning electron microscope (JEOL JSM-6360 A).11
4. FT-IR spectrum:
For characterization purposes, FT-IR investigations on the SLNs optimized formulation were conducted. The scanning was done between a range of 4000 cm-1 and 400 cm-1. (Japan, JASCO 4100)12
5. Differential Scanning Calorimetry (DSC):
The lyophilized SLN powder underwent DSC tests. In a DSC aluminium cup, precisely weighed samples were carefully introduced, and heating curves were recorded between 40 and 280°C at a heating rate of 10 °C/min in an inert atmosphere. Utilizing a differential scanning calorimeter, the study was conducted. (Japan's Shimadzu DSC 60)13
Table No.1: Box Behnken Design for Optimization
|
Formulation No. |
Factor 1 |
Factor 2 |
Factor 3 |
Response 1 |
Response 2 |
|
A :Stirring Speed (RPM) |
B:Stirring time (Hrs) |
C: Tween 80(ml) |
Entrapment efficiency (%) |
Particle size (nm) |
|
|
1 |
8000 |
1 |
1.5 |
85.28±1.065 |
133±1.159 |
|
2 |
7000 |
0.5 |
1.5 |
82.13±1.457 |
217±1.032 |
|
3 |
8000 |
1.5 |
1 |
85.33±2.608 |
176.88±1.220 |
|
4 |
7000 |
1 |
1 |
82.18±1.110 |
260.54±1.136 |
|
5 |
8000 |
1 |
0.5 |
81.77±1.972 |
232±1.566 |
|
6 |
7000 |
1 |
1 |
81.65±1.110 |
262.05±1.136 |
|
7 |
7000 |
1 |
1 |
80.45±1.110 |
261.86±1.136 |
|
8 |
7000 |
1 |
1 |
81.97±1.110 |
261.9±1.136 |
|
9 |
6000 |
1 |
0.5 |
79.07±1.565 |
358±1.42 |
|
10 |
6000 |
1.5 |
1 |
82.63±1.861 |
210.41±1.825 |
|
11 |
8000 |
0.5 |
1 |
81.73±1.157 |
310.66±1.164 |
|
12 |
6000 |
1 |
1.5 |
82.58±1.654 |
181.53±1.727 |
|
13 |
6000 |
0.5 |
1 |
79.03±1.521 |
344.19±1.205 |
|
14 |
7000 |
1 |
1 |
82.88±1.110 |
260.34±1.136 |
|
15 |
7000 |
1.5 |
0.5 |
82.22±1.083 |
289.41±1.557 |
|
16 |
7000 |
1.5 |
1.5 |
85.73±1.683 |
123.24±1.301 |
|
17 |
7000 |
0.5 |
0.5 |
78.82±1.099 |
409±1.019 |
6. X-ray diffraction study :
To determine if the Acitretin loaded SLNs were crystalline or amorphous, an XRD investigation was conducted. Lyophilized SLNs powder was subjected to an X-ray powder diffraction research utilising an advance X-ray diffractometer (Bruker AXS D8 Advance XRD, Germany).14
Stability study:
SLN formulations were kept at 2-80C and 250C (room temperature) in sealed amber-colored glass vials that were kept in a dark setting. After being stored for three months, the formulations' particle size and drug entrapment effectiveness were examined, and they were contrasted with newly created formulations. Three duplicates of the experiments were run.15
Preparation of Acitretin loaded SLN gel:
Carbopol 940, a gel-forming polymer, was soaked in a combination of SLN dispersion for two hours before being agitated with a digital mechanical stirrer to obtain a smooth dispersion. Triethanolamine was used to bring the resulting viscous solution's pH value down to 7 while slowly agitating it. Using a digital pH-meter (model EQ-614 EQIP-TRONICS) and a Brookfield viscometer (Model DVII + Viscometer, respectively), the pH and viscosity of the formulation were determined. 16
Evaluation of SLN loaded gel:
In-vitro release studies:
Artificial cellophane membrane with a molecular weight of 12000 was used for in-vitro release tests. This experiment made use of a vertical Franz diffusion cell. Between the two parts of the diffusion cell, the artificial membrane was firmly positioned. Phosphate buffer (pH 7.4) is present in the receptor compartment, which is continually agitated with a magnetic stirrer at a temperature of 37±0.50C. On the donor side, a predefined amount of SLN loaded with Acitretin containing 0.2mg of Acitretin was injected. At regular intervals, one ml of the sample was taken out of the receptor compartment and replaced with a fresh volume of receptor fluid. The aliquots were properly diluted with receptor media before being examined with a UV spectrophotometer. Measurements were performed three times, and the means were recorded. The release of Acitretin loaded solid lipid nanoparticles was compared with plain 0.25% Acitretin gel.17
Ex-vivo drug deposition study:
Wister rat skin that had been removed was used for the ex-vivo diffusion research. After being carefully positioned on the Franz diffusion cell with the epidermal side facing the donor compartment and the dermal side in touch with the receptor solution, the rat's abdomen skin was shaved. The donor compartment received the sample. After eight hours, the diffusion cell was disassembled. The skin was carefully removed, then rinsed with distilled water to eliminate any gel that might have been on the skin's surface. Skin was broken up into tiny pieces and homogenised with 10 millilitres of DMSO. Remove solvent, then phosphate buffer is used to dilute. With the use of absorbance measurements made using UV spectroscopy, the concentration of the medicine within the extract was appropriately diluted. Measurements were performed three times, and the means were recorded.18
Stability study:
A stability analysis of an improved formulation was done in accordance with the recommendations of the International Conference on Harmonisation (ICH). The accelerated stability conditions of temperature and relative humidity (40±200C/75%±5%RH) were used to monitor the optimized batches of SLNs gel with accutane loaded for up to one month. Following a month, samples were removed and evaluated for appearance, pH, and viscosity. Acitretin loaded SLNs gel's long-term stability must be predicted by selecting the proper storage conditions during the accelerated stability research.19
RESULTS AND DISCUSSION:
1. Particle size analysis:
A range of 123.24nm to 409nm was discovered for the particle size of the SLNs formulation.
Figure No. 1: Particle size of F1, F3 and F16 Formulations
2. Drug entrapment efficiency:
Entrapment efficiency for all SLNs was discovered to be between 78.82% and 85.73%.
Scanning Electron Microscopy:
Figure No. 4 shows the SEM of SLNs that have been loaded with acitretin.The SLNs' uniformity and spherical shape were noted.
Figure No. 2: Scanning Electron Microscopy (SEM) of formulation (Hot homogenization technique
4. FTIR Study of SLNs:
Figure No.3: FTIR Study of SLN
Figure No. 7 displays the SLN's FTIR spectrum. Some peaks that are seen in drug spectrums were seen to be masking in the spectra, it was discovered.The development of SLN and appropriate drug entrapment are indicated.
5. Differential Scanning Calorimetery (DSC):
Figure No. 4: Differential Scanning Calorimetery (DSC) of SLNs
The differential scanning calorimetry (DSC) profile of Acitretin and Compritol ATO 888 was displayed in Figures 8 and 9.The melting temperatures for Acitretin and Compritol ATO 888 were shown to be 227.33°C and 73.93°C, respectively, in a strong endothermic peak. The drug's refined form was visible in its sharp peak. The DSC peak intensity of peak was significantly altered, and the decrease in peak intensity indicated that SLN was forming.
6. X-ray diffraction study(XRD) of SLNs
Figure No.5:X-ray diffraction of SLNs
The X-ray diffraction profiles of Acitretin and Compritol ATO 888 were displayed in Figures 11 and 12.Acitretin's X-ray diffractogram showed a peak with a high intensity, indicating that it was crystalline. Low peak intensities in Compritol ATO 888 suggested the amorphous nature of the lipid.The study shows that after the synthesis of nanoparticles with the surfactant, the crystalline structure of the Acitretin and Compritol ATO 888 somewhataltered. It was seen that the peak's strength dropped, which suggested that proper entrapment of acitretin in the SLNs' lipid matrix had no effect on the structure of the lipid matrix.
7. Stability study:
The results in Table No. 2 demonstrated that the SLNs formulation's particle size and entrapment efficiency did not significantly change when stored at 40C and 250C
Table No. 2: Stability study
|
Evaluation parameter |
Before |
1 Month |
2 Month |
3 Month |
|||
|
40C |
250C |
40C |
250C |
40C |
250C |
||
|
Particle size (nm) |
123.24±1.301 |
125.11±1.981 |
124.75±1.530 |
125.43±1.091 |
127.98±1.214 |
128.01±1.362 |
129.65±1.036 |
|
Entrapment efficiency (%) |
85.73 ±1.683 |
85.02±1.097 |
84.10±1.954 |
84.96±1.076 |
83.43±1.542 |
81.67±1.321 |
80.91±1.456 |
Evaluation of SLN loaded gel:
1. In-vitro release studies
Table No.3: Comparative In-vitro release studies
|
Sr No. |
Formulation |
Cumulative Drug release (%) after 8 hrs |
|
1. |
SLN gel (Hot homogenization method) |
86.83% |
|
2. |
Plain gel (After 4 Hrs) |
92.5% |
Act SLN's in vitro release investigation revealed that the drug released initially more quickly, within 8 hours. This can be caused by an unequal medication distribution over the first several hours in the SLN. Later, Act SLN demonstrated sustained drug release throughout the duration of the research.
2. Ex –vivo Drug Deposition test: The amount of drug left on the rat skin after 8 hours was calculated.
Table No.4: Ex –vivo drug deposition study
|
Sr No. |
Formulation |
Drug Deposition after 8 Hrs (mg/cm2) |
|
1. |
SLN gel (Hot homogenization method) |
0.056 |
|
2. |
Plain gel (after 4Hrs) |
0.012 |
After 8 hours, the amount of acutance deposited in the skin from the SLN-loaded gel was higher than it was with the simple gel. This shows that SLNs increased skin's ability to withstand drugs.
3. Stability study:
Studies on accelerated stability were done on the improved formulation. There was no noticeable change in the gel's stability testing after three months.
CONCLUSION:
The ActSLN gel formulation was modified using a Box Benhken design in order to provide the optimal particle size and greatest entrapment effectiveness. The stirring time, rate, andconcentration of the surfactant were chosen. As seen under SEM, the resulting SLNs were nearly spherical and smooth. Act SLNs were found to be present in an amorphous or molecular dispersion state, according to studies conducted using FTIR, DSC, and XRD. ActSLN's in vitro release study demonstrated a faster initial release of the drug within 8 hours. Which might result from an uneven distribution of the drug during the first few hours in the SLN Later, ActSLN demonstrated sustained drug release throughout the entire study period. Studies on in vitro skin deposition revealed that ActSLN gel significantly increased the amount of accutane deposited in the skin compared to Plain Act gel.
ACKNOWLEDGMENT:
Authors are thankful to Alembic Pharmaceuticals Ltd, Baroda for providing a gift sample of the drug.The authors are thankful to Principal, Dr D Y Patil College of Pharmacy, Akurdi Pune for providing the facility to carry out the research work
Table No.5: Stability study of SLN loaded gel
|
Months |
Evaluation parameter of gel |
||||
|
Visual examination |
pH |
Viscosity |
Spreadability (Gm.cm/sec) |
In-vitro release studies after 8 hrs |
|
|
Before |
Transparent, smooth and Homogenous |
6.9 |
2618±1.654 |
18.9±0.732 |
86.83±1.806 |
|
After 1 month |
Transparent, smooth and Homogenous |
7.0 |
2607±1.547 |
18.7±0.183 |
86.01±1.340 |
|
After 2 month |
Transparent, smooth and Homogenous |
6.8 |
2595±1.297 |
17.9±0.235 |
85.50±1.953 |
|
After 3 month |
Transparent, smooth and Homogenous |
6.8 |
2586±1.166 |
17.1±0.217 |
84.67±1.095 |
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Received on 02.06.2023 Modified on 28.09.2023
Accepted on 02.01.2024 © RJPT All right reserved
Research J. Pharm. and Tech. 2024; 17(3):1015-1020.
DOI: 10.52711/0974-360X.2024.00157