Government College of Pharmaceutics-560027. Karnataka India.
Psoriasis is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and impaired barrier function. Topical delivery remains the preferred route for localized management; however, conventional formulations suffer from inadequate penetration through the stratum corneum, poor retention, and low patient compliance. In the present study, thymoquinone-loaded ethosomes were developed using a cold method and systematically optimized through a 2³ full factorial design to evaluate the influence of soya lecithin, ethanol, and cholesterol on particle size, entrapment efficiency, and drug release. The optimized formulation exhibited a vesicle size of 235 nm, polydispersity index of 0.28, zeta potential of –32.5 mV, and entrapment efficiency of 83.1%. In-vitro release studies demonstrated sustained drug diffusion, showing 81.1% cumulative release at 8 h with Higuchi kinetics (R² = 0.997), indicating diffusion-controlled transport. Incorporation of the optimized vesicles into a film-forming gel resulted in a smooth, homogeneous formulation with suitable pH, viscosity, Spreadability, and rapid film formation. The gel provided prolonged residence on skin with 75% drug release at 8 h and non-tacky flexible films, suggesting enhanced patient acceptability. Comparative evaluation showed superior permeation and stability over conventional liposomes. Overall, ethosomal film-forming gel significantly improved topical delivery of thymoquinone, offering a promising strategy for localized, sustained management of psoriatic lesions while minimizing systemic burden.
Psoriasis is a chronic, immune-mediated skin condition that can cause significant morbidity and mortality as a consequence of hyperproliferation and poor epidermal keratinocyte differentiation. The entire body may be affected, ranging from a few isolated red spots to scaly plaques. The symptoms include mild to severe erythema and itchy, scaly, flaky skin. It is also associated with skin lesions that are large, painful, and deformable 1. It is clinically described by the formation of small, limited patches that cover various body surface areas. Psoriasis affects roughly 5% of people in western countries; the severity and course of treatment will depend on how bad the condition is. Usually starting in late childhood or early adulthood, the chronic illness shows symptoms around the age of 40. Psoriasis affects the skin, joints, ligaments, mucous membranes, tendons, and nails in addition to these other body parts 2.
Figure 1: Clinical manifestations of psoriasis showing lesions on the scalp (left) and dorsal surface of the hand (right).
Figure 2: Clinical variants of psoriasis
Thymoquinone (TQ), also referred to as black seed or cumin, is the primary active ingredient of the Nigella sativa, a member of the Ranunculaceae family. TQ is a lipid-soluble benzoquinone that is responsible for the majority of Nigella's biological action. TQ has antifungal, antibacterial, antineoplastic, and anti-inflammatory properties with regard to dermatopharmacological effects. The lack of dosage proportionality, limited bioavailability, and poor solubility of TQ have prevented the oral formulations from becoming successful 3.
As in psoriasis, the stratum corneum becomes thickened and scaly, further complicating drug diffusion. Conventional formulations such as creams, ointments, and lotions may be quickly rubbed off or washed away, require multiple daily applications, and can cause irritation or dryness. Therefore, developing an advanced delivery system capable of overcoming the skin barrier and providing sustained therapeutic levels at the site of inflammation is crucial for improving treatment outcomes.
Since the carrier-based system promises to address these drawbacks, treating psoriasis will need to take a new turn. By interfering with inflammatory cells, carrier-based drug delivery systems can successfully improve drug permeability and increase drug content in skin thus
improving efficacy. Examples of these systems include liposomes, noisome, nano emulsions, nanospheres, microneedles, ethosomes, nanocrystals, liposphere nanostructured lipid carriers, and foams. With this approach, the amount of the medication that is locally targeted to the skin will be maximized while the amount reaching the systemic circulation will be minimized 4.
Ethosomes, which are non-invasive delivery carriers composed of phospholipids, high concentrations of ethanol, and water, facilitate drug delivery deep into the skin or systemic circulation. Despite their advantages, traditional transdermal systems often face challenges with poor skin penetration, primarily due to the stratum corneum, the skin’s outermost barrier.
Ethosomes, containing 20–50% ethanol, were developed to address this issue. Ethanol acts as an effective permeation enhancer by interacting with the lipid molecules of the stratum corneum, reducing its melting point, increasing lipid fluidity, and enhancing cell membrane permeability. Compared to conventional liposomes and hydroalcoholic solutions, ethosomes systems demonstrated superior efficiency in delivering substances through the skin.5
Figure 3; Structure of ethosome
Preparation Methods of Ethosomes 6,7:
There are various methods to prepare ethosome, which are as follows:
1. COLD METHOD: Cold process is the most widely used technique for creating Ethosomal. Phospholipids and other lipophilic substances are dissolved in ethanol at room temperature and rapidly stirred. The organic phase is heated in a water bath to a maximum temperature of 30°C. Before adding the aqueous phase to the organic phase, it is heated to 30°C in a separate vessel and vigorously stirred. The aqueous phase is added, and stirring is continued for an additional time. The aqueous phase may consist of a buffered solution, regular saline, or water.
2. HOT METHOD: To disperse the phospholipid in water and to form a colloidal solution, heat the solution in a water bath at 40°C. Mix propylene glycol and ethanol in another container at 40°C. Add the organic phase into the aqueous phase once both mixtures have reached 40°C. Based on whether the drug is hydrophilic or hydrophobic, it dissolves in either water or ethanol. In an ethosomal formulation, vesicle size can be decreased by probe sonication or extrusion.
3. THIN FILM HYDRATION METHOD: The lipids will be dissolved in an organic solvent in a round-bottom flask, and the organic solvent will be removed using a rotary evaporator above the temperature at which the lipid transition occurs. An ethanolic mixture will be used to hydrate the thin film that forms around the inner walls of the flask. The resultant ethosome suspension will then be dispersed using a probe sonicater.
Topical route of drug delivery is considered as a promising approach as the drugs administered by this route bypass the first pass metabolism. Furthermore,
Film-forming gels (FFG) with ethosomes can be developed in different forms and contains both the medication and additives that form a film in a vehicle, when it comes into touch with the skin, thus evaporating the solvent and leaving the excipients and medication and provide controlled and sustained release of the drug, maintaining therapeutic levels for extended periods and reducing the need for frequent application. This not only enhances the treatment’s effectiveness but also improves patient compliance by improved patient adherence, longer adhesion, transparency, a non-greasy texture, less irritation to the skin, resistance to washing off, and a visually appealing appearance so ethosomal gels become more comfortable and convenient to use compared to traditional formulations 8
Significance combining ethosomes with fil forming gel
1. Enhanced skin penetration
2. Controlled and sustained drug release
3. Improved drug stability
4. Increased retention time
5. Better aesthetic and patient compliance
6. Enhanced bioavailability
7. Suitable for lipophilic drugs
8. Targeted and localized action
9. Moisturizing effect
10. Synergistic effect
Ethosomes v/s Liposomes: Comparative Insights into Drug TQ Delivery Efficiency 9
Topical drug delivery is often limited by the strong barrier function of the stratum corneum. To overcome this, nano vesicular carriers such as liposomes and ethosomes have been extensively studied. This study was designed to compare the performance of optimised ethosomes and liposomes for TQ delivery and to demonstrate that ethosomes offer superior entrapment, permeation, release, and stability.
MATERIALS AND METHODS
MATERIALS
Thymoquinone was obtained from BLD Pharma, Hyderabad (India). Ethanol (S.D. Fine-Chem Pvt. Ltd., Mumbai), Soya lecithin (Sigma-Aldrich, Bangalore), and Cholesterol (Central Drug House Pvt. Ltd., New Delhi) were used as primary formulation components. Propylene glycol,Carbopol® 934 PVP K-30, and Triethanolamine (TEA)were supplied by Loba Chemie, Mumbai. All other reagents were of analytical grade, and double-distilled water was used throughout the study.
The main instruments included a UV–Visible spectrophotometer (Shimadzu UV-1800, Japan), FTIR spectrophotometer (Thermo Fisher Scientific, Mumbai), Digital pH meter (Servewell Instruments Pvt. Ltd.,Magnetic stirrer (REMI Instruments Pvt. Ltd.), Ultracentrifuge (REMI Instruments Pvt. Ltd.), and Nanoparticle size analyzer (Horiba SZ-100, Japan). A Franz diffusion cell (DBK Instruments) was used for drug release and permeation studies.
Method Of Preparation:
A 2³ full factorial design with three centre points was employed to evaluate ethanol, soya lecithin, and cholesterol effects on particle size, entrapment efficiency, and cumulative drug release. Eleven batches were prepared to assess linearity and interaction effects.
Independent Variables (Factors):
Table 1: Independent Variables (Factors)
|
Sn |
Factor |
Unit |
Low |
High |
Reason |
|
1 |
Ethanol |
% |
20 |
40 |
Ethanol contributes to ethosome flexibility, enhances drug solubility, and improves skin penetration. |
|
2 |
Soya Lecithin |
% |
2 |
4 |
Lecithin is the primary phospholipid forming the ethosomal bilayer. |
|
3 |
Cholesterol |
% |
0.2 |
0.5 |
Cholesterol stabilizes vesicles by modulating bilayer fluidity. |
Dependent Variables
Table 2: Dependent Variables (Factors)
|
Sn |
Variable |
Unit |
Goal |
|
1 |
Particle size |
nm |
Minimize |
|
2. |
Entrapment efficiency |
% |
Maximize |
|
3 |
Cumulative drug release |
% |
Maximize |
Table3: Formulation Design for Preparation of TQ Ethosomes
|
Run |
X1;soya lecithin |
X2; ethanol |
X3; cholesrerol |
|
1 |
4 |
20 |
0.5 |
|
2 |
4 |
20 |
0.2 |
|
3 |
2 |
40 |
0.5 |
|
4 |
4 |
40 |
0.2 |
|
5 |
4 |
40 |
0.5 |
|
6 |
3 |
30 |
0.35 |
|
7 |
3 |
30 |
0.35 |
|
8 |
3 |
30 |
0.35 |
|
9 |
2 |
40 |
0.2 |
|
10 |
2 |
20 |
0.5 |
|
11 |
2 |
20 |
0.2 |
Preparation Of Film Forming Gel Loaded with TQ Ethosomes
Step 1: Preparation Of TQ Ethosomes 10,11,12
Ethosomes (20 mL batch) were prepared by well known cold method as the thymoquinone is thermolabile, in this dissolving lecithin and cholesterol in ethanol at 40 °C on magnetic stirrer with 800rpm . Thymoquinone was added and dissolved, followed by slow addition of pre-heated purified water under stirring. The dispersion was cooled, sonicated for vesicle size reduction, and stored at 2–8 °C for 24 h before evaluation.
Evaluation Of Ethosomes 13,14,15
Zeta Potential: Surface charge was measured using a Malvern Zetasizer at 25 °C to assess vesicle stability.
Optimization
Independent variables (ethanol, lecithin, cholesterol) and dependent responses (%EE, particle size, %CDR) were processed using Design-Expert software. Suggested optimized composition was selected and evaluated.
Preparation Of Liposomes 16,17,18
Liposomes were prepared by the thin-film hydration method. Soya lecithin (4%) and cholesterol (0.2%) were dissolved in a chloroform:ethanol mixture (2:1) in a round-bottom flask. The solvent mixture was evaporated using a rotary evaporator at 40 °C under reduce pressure to form a thin lipid film on the flask wall. The film was hydrated with phosphate buffered saline (pH 7.4) containing thymoquinone (25 mg/20 ml). The resulting dispersion was
Step 2: Film-Forming Gel Preparation 19,20,21
Hydrate 0.10 g Carbopol 934 in ~5–6 mL purified water; allow 20–30 min swelling with gentle stirring. Dissolve 0.40 g PVP K-30 and 0.40 g PG in 1.00 g ethanol; add this polymer/solvent solution into hydrated Carbopol under propeller stirring (150–300 rpm) to form a uniform pre-gel. Slowly incorporate 2 g equivalent ethosomal dispersion with gentle stirring (150–200 rpm) to minimize vesicle rupture .Adjust pH to 6.0–6.5 by dropwise addition of 0.08 g TEA; make up to 10 g with purified water (add ≈ 6.10 g), mix gently, degas, and store at 2–8 °C until evaluation.
Evaluation Of Film-Forming Gel 22,23
Drug release data were fitted to zero-order, first-order, Higuchi, and Korsmeyer-Peppas models. Regression (R²) and diffusion exponent (n) values were used to identify the best-fit mechanism.
The optimized gel was packed in aluminium foil and stored at different temperature/humidity conditions (15 °C/45%RH, 30 °C/65%RH, 40 °C/75%RH) for 30 days. Samples withdrawn on days 0, 15, and 30 were analysed for %EE and drug content to determine product stability as per ICH guidelines.
RESULTS
1.Preformulation Studies of the Drug:
Table4: Preformulation Studies of the Drug
|
Properties |
Observation |
|
|
1.1 Organoleptic |
Color |
Yellow |
|
Odor |
Odourless |
|
|
Nature |
Yellow crystalline powder |
|
|
1.2 Melting point (n=3) |
470 C± 2 0C |
|
1.3 Determination of λmax of thymoquinone in PBS: ETHANOL (60:40)
Scan mode: spectrum , λmax = 256 nm
2.Compatibility Studies
FTIR spectra of thymoquinone and physical mixtures with excipients showed no significant shifts or disappearance of major peaks, indicating chemical compatibility. DSC thermograms displayed characteristic endothermic peaks without additional transitions, confirming the absence of drug–excipient interaction.
Preparation Of Thymoquinone Loaded Ethosomes
Table 5; formulation chart for preparation ethosomes
|
Sn |
TQ(API) |
Soya lecithin w/v (%) |
Ethanol (%)v/v |
Cholesterol w/v (%) |
Propylene glycol ml |
Water |
|
F1 |
25 mg |
4 |
20 |
0.5 |
1 |
QS |
|
F2 |
25 mg |
4 |
20 |
0.2 |
1 |
QS |
|
F3 |
25 mg |
2 |
40 |
0.5 |
1 |
QS |
|
F4 |
25 mg |
4 |
40 |
0.2 |
1 |
QS |
|
F5 |
25 mg |
4 |
40 |
0.5 |
1 |
QS |
|
F6 |
25 mg |
3 |
30 |
0.35 |
1 |
QS |
|
F7 |
25 mg |
3 |
30 |
0.35 |
1 |
QS |
|
F8 |
25 mg |
3 |
30 |
0.35 |
1 |
QS |
|
F9 |
25 mg |
2 |
40 |
0.2 |
1 |
QS |
|
F10 |
25 mg |
2 |
20 |
0.5 |
1 |
QS |
|
F11 |
25 mg |
2 |
20 |
0.2 |
1 |
QS |
Figure 10; formulations of ethosomes F1 to F11
3.Characterization of Ethosomes
3.1. Microscopic Examination:
Figure 11; microscopic evaluation of prepared ethosomes
3.2. Particle size, PDI and Entrapment efficiency analysis:
Table 6; particle size, PDI and % entrapment efficiency analysis of various TQ-loaded ethosomal formulations (F1–F11)."
|
Formulation Code |
Particle Size In Nm (N=3) |
PDI Value |
%Entrapment Efficiency (N=3) |
|
F1 |
229.9±2 |
0.31 |
86±.02 |
|
F2 |
210±2 |
0.27 |
83.5±.012 |
|
F3 |
295±5 |
0.36 |
77.7±0.2 |
|
F4 |
235±1 |
0.28 |
83.1±.023 |
|
F5 |
247.1±5 |
0.3 |
88.13±0.12 |
|
F6 |
235.2±6 |
0.29 |
79.6±0.45 |
|
F7 |
235.2±3 |
0.29 |
79.6±.0236 |
|
F8 |
235.2±6 |
0.29 |
79.6±.012 |
|
F9 |
268.2±2 |
0.34 |
73.8±.0124 |
|
F10 |
232.5±2 |
0.32 |
74.62±.210 |
|
F11 |
209± 1 |
0.26 |
68.7±.012 |
3.3 In-vitro release studies:
Anova For Selected Factorial Model
Response 1: Particle Size
Table No 7: Anova model for Particle size
|
Source |
Sum of Squares |
DF |
Mean Square |
F-value |
p-value |
|
|
Model |
5849.51 |
4 |
1462.38 |
67.95 |
< 0.0001 |
significant |
|
A-soya lecithin |
854.91 |
1 |
854.91 |
39.72 |
0.0007 |
|
|
B-ethanol |
3357.90 |
1 |
3357.90 |
156.02 |
< 0.0001 |
|
|
C-cholesterol |
846.66 |
1 |
846.66 |
39.34 |
0.0008 |
|
|
AB |
790.03 |
1 |
790.03 |
36.71 |
0.0009 |
|
|
Residual |
129.14 |
6 |
21.52 |
|
|
|
|
Lack of Fit |
129.14 |
4 |
32.28 |
|
|
|
|
Pure Error |
0.0000 |
2 |
0.0000 |
|
|
|
|
Cor Total |
5978.64 |
10 |
|
|
|
|
Figure 13; predicted v/s actual correlation of particle size
Response 2: Entrapment Efficiency %
Table No 8: ANOVA model for %EE
|
Source |
Sum of Squares |
DF |
Mean Square |
F-value |
p-value |
|
|
Model |
313.37 |
3 |
104.46 |
85.73 |
< 0.0001 |
significant |
|
A-soya lecithin |
263.47 |
1 |
263.47 |
216.22 |
< 0.0001 |
|
|
B-ethanol |
12.28 |
1 |
12.28 |
10.07 |
0.0156 |
|
|
C-cholesterol |
37.63 |
1 |
37.63 |
30.88 |
0.0009 |
|
|
Residual |
8.53 |
7 |
1.22 |
|
|
|
|
Lack of Fit |
8.53 |
5 |
1.71 |
|
|
|
|
Pure Error |
0.0000 |
2 |
0.0000 |
|
|
|
|
Cor Total |
321.90 |
10 |
|
|
|
|
Figure 16; predicted v/s actual correlation of %EE
Response 3: %Cummulative Drug Release
Table No 9: Anova model for %CDR
|
Source |
Sum of Squares |
DF |
Mean Square |
F-value |
p-value |
|
|
Model |
630.61 |
3 |
210.20 |
214.15 |
< 0.0001 |
significant |
|
B-ethanol |
323.34 |
1 |
323.34 |
329.42 |
< 0.0001 |
|
|
C-cholesterol |
277.77 |
1 |
277.77 |
282.99 |
< 0.0001 |
|
|
BC |
29.49 |
1 |
29.49 |
30.05 |
0.0009 |
|
|
Residual |
6.87 |
7 |
0.9816 |
|
|
|
|
Lack of Fit |
6.87 |
5 |
1.37 |
|
|
|
|
Pure Error |
0.0000 |
2 |
0.0000 |
|
|
|
|
Cor Total |
637.48 |
10 |
|
|
|
|
Figure 19; predicted v/s actual correlation of %CDR
Optimization Results
Solution for optimized formulation given by design expert
Table 10; Solution for optimized formulation given by DOE
|
Sl.n |
Soya lecithin |
ethanol |
cholesterol |
Particle size |
%EE |
%CDR |
desirability |
|
|
1 |
4.00 |
40.0 |
0.200 |
229.225 |
84.295 |
80.908 |
.848 |
Selected |
Table 11: Actual and predicted values of optimized formulation
|
Variables |
Predicted |
Actual |
|
Particle size |
229.2 |
235 |
|
%EE |
84.9 |
83.1 |
|
In-vitro drug release at 8 h (%) |
80.9 |
81.1 |
Evaluation Results of Optimized Ethosomes
Figure 22; optimized formulation particle size and PDI report
Figure 23; Optimized Formulation Zeta Potential Report
Table 12; In-vitro drug release of optimized formulation for 8 hours
|
Time (Hour) |
%CDR ± SD (n=3) |
|
0.5 |
20.40± 2.3 |
|
1 |
28.855± 2.7 |
|
2 |
43.17± 1.5 |
|
3 |
52.33± 1.4 |
|
4 |
61.1± 1.3 |
|
5 |
68.1± 2.3 |
|
6 |
74.3± 2.1 |
|
7 |
80.10± 1.6 |
Table 13 : drug release kinetic study of optimized formula
|
Time (HOUR) |
%CDR |
Log CDR% |
%drug remain |
log %remain |
sq T |
LOG T |
|
0.5 |
20.403 |
1.30 |
79.59 |
1.90 |
0.70 |
-0.30 |
|
1 |
28.8 |
1.46 |
71.14 |
1.85 |
1 |
0 |
|
2 |
43.17 |
1.635 |
56.85 |
1.754 |
1.41 |
0.30 |
|
3 |
52.33 |
1.71 |
47.66 |
1.678 |
1.73 |
0.47 |
|
4 |
61.13 |
1.786 |
38.8 |
1.58 |
2 |
0.60 |
|
5 |
68.1 |
1.83 |
31.8 |
1.50 |
2.23 |
0.69 |
|
6 |
74.3 |
1.871 |
25.65 |
1.40 |
2.4 |
0.77 |
|
7 |
80.1 |
1.90 |
19.89 |
1.29 |
2.6 |
0.84 |
|
8 |
81.11 |
1.909 |
18.89 |
1.27 |
2.82 |
0.90 |
Figure 25; Zero order model of optimized TQ Ethosomes
Figure 26; First Order Model of Optimized TQ Ethosomes
Figure 27; HIGUCHI model of optimized TQ Ethosomes
Figure 28; Korsmeyer PEPPPAS model of optimized TQ Ethosomes
Table 14; Data for In-vitro drug release kinetic models of optimized dispersions
|
|
Zero order |
First order |
Higuchi |
Korsmeyer-Peppas |
|
KSLOPE |
9.993 |
-0.089 |
29.92 |
0.506 |
|
R2 |
0.912 |
0.991 |
0.997 |
0.994 |
Evaluation Tests of Optimised Ethosomes and Liposomes
Table 15: comparison data of evaluation tests of optimised ethosomes and liposomes
|
Parameter |
Ethosomes (Optimized) |
Liposomes |
Inference |
|
Particle Size (nm) |
235 ± 10 |
375 ± 15 |
Ethosomes smaller, better permeation |
|
PDI |
0.28 ± 0.02 |
0.39 ± 0.04 |
Ethosomes more uniform |
|
Zeta Potential (mV) |
–32.5 ± 2.5 |
–20.1 ± 1.8 |
Ethosomes more stable |
|
Entrapment Efficiency (%) |
83.1 ± 2.2 |
72.4 ± 2.1 |
Higher in ethosomes |
|
CDR at 24 h (%) |
81.1 ± 2.3 |
61.5 ± 2.5 |
Ethosomes sustained & higher release |
Evaluated Parameters for the Prepared Gel Containing TQ Ethosomes
Table 16: Evaluation result of FFG
|
Test |
Result |
|
Appearance |
Smooth, homogenous, pale-yellow gel |
|
pH (25°C) |
6.1 ± 0.1 |
|
Viscosity (Brookfield, Spindle 64, 10 rpm) |
12,480 ± 320 cP |
|
Spread ability |
7.2 ± 0.3 g·cm/sec |
|
Drug Content Uniformity |
98.2 ± 1.5 % |
|
Drying Time |
5.8 ± 0.4 min |
|
Film Thickness |
0.28 ± 0.02 mm |
|
Folding Endurance |
> 300 folds (no break) |
|
Tackiness |
Non-tacky after drying |
In-Vitro Drug Release of FFG for 8 h
Table 17; Invitro Release Data of FFG
|
Sl. n |
Time (HR) |
%CDR ± SD (n=D) |
|
1 |
0 |
0.0 ±0.0 |
|
2 |
0.5 |
12.5± .0 |
|
3 |
1 |
20.8 ±1.2 |
|
4 |
2 |
34.6 ±1.8 |
|
5 |
3 |
43.2±1.9 |
|
6 |
4 |
51.0±2.1 |
|
7 |
5 |
59.0±2.3 |
|
8 |
6 |
66.2±2.4 |
|
9 |
7 |
71.0±2.5 |
|
10 |
8 |
75.0±2.6 |
Drug Release Kinetic Study Of film forming gel
Table 18; Drug Release Kinetic Study Of FFG
|
Sl.n |
Time (h) |
%CDR |
Log %CDR |
%drug remain |
log %remain |
sq T |
|
1 |
0 |
0 |
|
100 |
2 |
0 |
|
2 |
0.5 |
12.5 |
1.09 |
87.5 |
1.94 |
0.70 |
|
3 |
1 |
20.8 |
1.31 |
79.2 |
1.89 |
1 |
|
4 |
2 |
34.6 |
1.5 |
65.4 |
1.81 |
1.41 |
|
5 |
3 |
43.2 |
1.63 |
56.8 |
1.75 |
1.73 |
|
6 |
4 |
51 |
1.70 |
49 |
1.69 |
2 |
|
7 |
5 |
59 |
1.77 |
41 |
1.61 |
2.23 |
|
8 |
6 |
66.2 |
1.8 |
33.8 |
1.52 |
2.44 |
|
9 |
7 |
71 |
1.8 |
29 |
1.46 |
2.64 |
Figure 30: zero order of FFG formulation
Figure 31: first order of FFG formulation
Figure 32: HIGUCHI MODEL of FFG formulation
Figure 33: korsmeyer peppas model of FFG formulation
Table 19: Data for In-vitro drug release kinetic models of FFG
|
|
Zero order |
First order |
Higuchi |
Korsmeyer-Peppas |
|
KSLOPE |
8.97 |
-0.0724 |
28.092 |
0.615 |
|
R2 |
0.952 |
0.998 |
0.992 |
0.995 |
Invitro Skin Permeation Test (Ex Vivo)
Table 20: invitro release data FFG through skin
|
Sl. n |
Time (hr) |
%CDR ± SD (n=3) |
|
1 |
0 |
0 |
|
2 |
0.5 |
2.5±2 |
|
3 |
1 |
6±1.3 |
|
4 |
2 |
14.5±1.4 |
|
5 |
3 |
24±2.3 |
|
6 |
4 |
32±1.7 |
|
7 |
5 |
40±1.5 |
|
8 |
6 |
46±1.4 |
|
9 |
7 |
50±1.3 |
|
10 |
8 |
52.7±1.8 |
Figure34: Release Data of FFG
Stability Studies
Table 21: data of stability studies
|
Storage condition 400C±20C/75%RH±5%RH |
|||
|
Sample type |
Sampling interval |
% drug content |
Entrapment Efficiency |
|
Ethosomal formulation |
0 day |
81.1 |
83±3.4 |
|
30 days |
79.0 |
81±2.9 |
|
DISCUSIION
The primary goal of this work was to formulate and evaluate a thymoquinone (TQ)-loaded ethosomal film-forming gel (FFG) for topical treatment of psoriasis. This approach aimed to overcome TQ’s limitations of poor aqueous solubility, high lipophilicity, and thermal instability, while ensuring localized drug action, prolonged residence, and minimal systemic absorption. The combination of ethosomal vesicles and film-forming polymers was designed to achieve enhanced skin permeation and sustained drug release.
Preformulation Studies
Organoleptic Properties
The preliminary inspection of TQ confirmed it as a yellow, odorless crystalline powder without any discoloration or foreign odor, indicating chemical integrity. The crystalline nature ensures better physical stability and resistance to polymorphic conversion during formulation processes, confirming the suitability of the raw drug material.
Melting Point
The recorded melting point of TQ was 47 ± 0.02 °C, sharp and within the expected range, confirming purity. However, its low melting point highlighted thermolabile behavior, necessitating cold-processing methods during ethosome preparation to avoid degradation.
UV–Visible Spectrophotometric Analysis
The λmax at 256 nm and the linear calibration curve (R² = 0.9993) demonstrated excellent method reliability for quantifying drug concentration. This method was later employed successfully in determining drug content, entrapment efficiency, and release studies.
Compatibility Studies
FTIR Analysis
FTIR spectra of TQ alone and with excipients displayed all characteristic peaks without any additional or shifted bands. The absence of new peaks confirmed no significant chemical interaction between the drug and excipients, ensuring formulation stability.
DSC Analysis
Differential scanning calorimetry revealed a sharp endothermic peak of TQ at 50.36 °C, consistent with its crystalline nature. The physical mixture retained this peak, indicating that the drug remained stable and compatible within the selected excipients. No evidence of thermal interaction was observed.
3. Ethosome Formulation and Optimization
A 2³ full factorial design was adopted to optimize the composition using soya lecithin (A), ethanol (B), and cholesterol (C) as independent variables. The aim was to minimize vesicle size while maximizing entrapment efficiency (EE%) and cumulative drug release (CDR%).
Effect of Variables
Lecithin acts as the structural backbone of vesicles, forming bilayers capable of encapsulating lipophilic drugs like TQ. Increasing lecithin concentration enhanced EE% (up to ~88.13%) by providing more hydrophobic domains. However, excessive lecithin led to larger vesicle sizes (~295 nm) due to multilamellar formation.
Ethanol played a dual role as a bilayer fluidizer and penetration enhancer. At moderate levels (20–30%), it produced small, deformable vesicles with improved skin permeability. At higher concentrations (40%), ethanol promoted higher release (up to 81.11% at 8 h) but could slightly increase particle size due to bilayer swelling. Ethanol was found to be the most significant factor affecting size and release (p < 0.0001).
Cholesterol provided membrane rigidity and stability. Moderate inclusion reduced drug leakage and improved EE, while excessive levels decreased bilayer flexibility and drug diffusion. The optimized level (0.2%) ensured a balance between stability and permeability.
Optimized Formulation (F4)
The optimized ethosome (F4) containing 4% lecithin, 40% ethanol, and 0.2% cholesterol achieved an average particle size of 235 nm, EE 83.1%, and CDR 81.11% at 8 h. These results closely matched statistical predictions, confirming the efficiency of the design model.
Vesicle Characterization
Microscopic and SEM observations confirmed spherical, smooth, and discrete vesicles with uniform distribution. The small size (<300 nm) favored deep skin penetration while minimizing systemic absorption, ideal for topical therapy in psoriasis.
zeta potential
The zeta potential value of –32.5 mV indicates that the ethosomal vesicles possess a strong negative surface charge, suggesting good electrostatic stability and minimal aggregation tendency.
Entrapment Efficiency (EE%)
Entrapment efficiency varied from 68.7% to 88.13%, with F4 showing 83.1%. Lecithin significantly increased EE, while excessive ethanol slightly reduced it due to drug partitioning into the external phase. Cholesterol enhanced EE by reducing leakage, but excessive amounts limited drug incorporation.
In Vitro Drug Release
Drug release followed a biphasic pattern with an initial burst phase followed by sustained diffusion-controlled release. The optimized batch (F4) released 81.11% of TQ in 8 h, following the Higuchi model (R² = 0.997) and Korsmeyer–Peppas n ≈ 0.506, indicating Fickian diffusion.
Drug Release Kinetics of Ethosomes
The in-vitro release profile of the optimized ethosomal formulation (F4) showed a biphasic pattern with an initial burst followed by sustained release. The burst phase was due to surface-bound drug, while the later phase represented diffusion of TQ through the lipid bilayer. The data best fitted the Higuchi model (R² = 0.997), confirming diffusion-controlled release. The Korsmeyer–Peppas exponent (n ≈ 0.50) indicated Fickian diffusion, where the drug diffused mainly through the ethosomal bilayer without significant structural erosion. Thus, ethosomes provided a controlled, predictable release suitable for maintaining steady topical drug levels.
Comparison with Liposomes
Ethosomes exhibited a smaller vesicle size (≈235 nm) than liposomes (≈375 nm), enhancing skin penetration.The presence of ethanol in ethosomes increased bilayer fluidity and deformability.Liposomes had a higher PDI (0.39) compared to ethosomes (0.28), indicating less uniformity.Zeta potential was higher for ethosomes (–32.5 mV), ensuring better stability and less aggregation.Entrapment efficiency was greater in ethosomes (83.1%) than in liposomes (72.4%).Ethosomes showed higher cumulative drug release (81% at 24 h) than liposomes (61%).Ethanol enhanced skin permeation by disrupting stratum corneum lipids.Liposomes mainly remained on the skin surface, while ethosomes penetrated deeper layers.Ethosomes maintained stability for 30 days with minimal EE loss (<2%), whereas liposomes showed ~15% loss.Overall, ethosomes proved superior for topical delivery, offering better penetration, sustained release, and storage stability.
4. Film-Forming Gel (FFG) Evaluation
Physical Appearance
The formulated gel was pale yellow, smooth, and homogeneous with no phase separation, confirming physical stability.
pH
The gel’s pH (6.1 ± 0.1) was close to the physiological range (5.5–6.5), ensuring non-irritancy upon application.
Viscosity and Spreadability
With viscosity of 12,480 ± 320 cP and spreadability of 7.2 ± 0.3 g·cm/sec, the gel exhibited good flow and easy application while maintaining structural integrity.
Drug Content and Film Properties
Drug content was 98.2 ± 1.5%, indicating uniform distribution. The film dried within 5.8 ± 0.4 min, had a thickness of 0.28 mm, folding endurance > 300, and was non-tacky—characteristics that enhance patient compliance.
In Vitro and Ex Vivo Studies
In Vitro Release
The ethosomal gel released 75% of TQ in 8 h, slightly lower than the ethosomal suspension but desirable for sustained delivery. Release followed first-order (R² = 0.998) and Higuchi (R² = 0.992) models, with n = 0.615, suggesting anomalous diffusion involving both Fickian transport and polymer relaxation.
Drug Release Kinetics of Film-Forming Gel (FFG)
The FFG exhibited a slower and more prolonged release (about 75% at 8 h) compared to the ethosomal suspension due to the polymeric network acting as an additional diffusion barrier. The release followed first-order kinetics (R² = 0.998), indicating a concentration-dependent mechanism, and also fitted the Higuchi model (R² = 0.992), confirming diffusion as the dominant process. The Korsmeyer–Peppas exponent (n = 0.615) suggested anomalous transport, meaning both diffusion and polymer relaxation contributed. This dual mechanism ensured sustained delivery and prolonged skin residence, ideal for psoriasis management.
Ex Vivo Permeation
Ex vivo permeation through rat skin showed 52.7% drug release at 8 h. Ethanol disrupted the stratum corneum, while the deformable ethosomes enhanced penetration. The film-forming gel maintained prolonged contact with the skin, promoting sustained local drug delivery with minimal systemic exposure.
Stability Studies
After 30 days at 40 °C/75% RH, only a minor decrease in EE (from 83% to 81%) and drug content (from 81.1% to 79%) was observed. The slight changes were attributed to mild oxidative degradation or vesicle reorganization. The results confirmed good physical and chemical stability of the optimized formulation.
All preformulation data confirmed the purity and suitability of TQ. Compatibility studies ruled out interactions, and the factorial design effectively optimized vesicle parameters. The ethosomal system improved drug encapsulation, release, and stability, while the film-forming gel enhanced application convenience and sustained skin retention.
Collectively, the TQ-loaded ethosomal film-forming gel demonstrated high potential for topical psoriasis management, offering improved drug penetration, prolonged local action, and excellent formulation stability.
CONCLUSION
The present study successfully developed and optimized a thymoquinone-loaded ethosomal film-forming gel as an advanced topical delivery system for psoriasis. Preformulation and compatibility analyses confirmed the purity of thymoquinone and its stability with selected excipients. Statistical optimization using a 2³ factorial design revealed that ethanol was the key factor influencing vesicle size and drug release, lecithin enhanced entrapment efficiency, and cholesterol contributed to vesicle stability
The optimized formulation (lecithin 4%, ethanol 40%, cholesterol 0.2%) produced nanosized vesicles (≈235 nm) with high entrapment efficiency (83.1%) and controlled drug release (81.1% at 8 h). Comparative evaluation clearly established the superiority of ethosomes over liposomes in terms of smaller vesicle size, greater stability, higher drug loading, and sustained release characteristics.
Incorporating the optimized ethosomal dispersion into a film-forming gel resulted in a smooth, stable, and non-tacky preparation with acceptable pH and viscosity suitable for topical use. The formulation demonstrated controlled in-vitro and ex-vivo release with enhanced skin permeation, suggesting effective localized delivery and reduced systemic exposure. Stability testing under accelerated conditions confirmed the robustness of the system with minimal variation in drug content and entrapment efficiency.
Overall, the developed thymoquinone-loaded ethosomal film-forming gel presents a stable, patient-friendly, and effective platform for topical therapy. Its enhanced penetration, sustained release, and superior performance over conventional liposomes highlight its potential as a promising carrier system for managing psoriasis and other inflammatory skin disorders.
ACKNOWLEDGEMENT
The authors extend sincere thanks to all the teaching staff of Government college of Pharmacy, Bengaluru for their help and guidance. Finally, a heartfelt gratitude to our batch mates.
REFERENCES
Jayashree Halluru*, Dr. Sangeetha S. S., Formulation and Evaluation of Film Forming Gel Loaded with Thymoquinone Ethosomes for Treating Psoriasis, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 11, 2808-2832 https://doi.org/10.5281/zenodo.17646420
10.5281/zenodo.17646420
