INTRODUCTION:

Isotretinoin is an anti acne agent. It is mainly used to treating severe type of acne. A new vesicular derivative “Transfersomes” has paved the way to minimize the defective transdermal permeation of a number of low and high molecular weight drugs which has been found to be one of the major advancement in vesicle research. Transfersome is highly adaptable, stress responsive, complex aggregate. Its preferred form is an ultra deformable vesicle possessing an aqueous core surrounded by the complex lipid bilayer. Interdependency of local composition and shape of the bilayer makes the vesicle both self-regulating and self optimizing. This enables the transfersomes to cross various transport barriers efficiently and then act as a drug carrier for noninvasive targeted drug delivery and sustained release of the therapeutic agent.

The ultraflexible carrier’s hydration sensitivity and its unique driving force create an unprecedented opportunity to control the depth of the carriers’ migration, by selecting different drug dose and/or carrier dose per area. Transfersomes can effectively protect the drug against undesired rapid clearance into cutaneous blood vessels and are capable of retaining the drug long enough, on, in and below the skin barrier. Furthermore, they can cross the stratum corneum independent of drug concentration. The aim of the present study was to refine the formulation of ultraflexible lipid vesicles for enhanced skin delivery of a model lipophilic drug, Isotretinoin.

MATERIALS AND METHODS:

Materials

Isotretinoin was obtained as gift sample from Intas Pharmaceuticals, Ahemdabad, India. Phospholipon® 90 H was obtained as a gift sample from Lipoid, Germany. Span 80 and chloroform was purchased from S. D. Fine chem. Pvt. Ltd. Methanol was purchased from Central drug house, Gujarat, India. Tritin X-100 was purchased from Hi Media Labs. Pvt. Ltd., Mumbai, India, Carbopol 980 was purchased from Sulab laboritries, India. Tri ethanolamine purchased from sulab laboratories, India. All buffering agents were purchased from Finer chemicals Ltd., India. All other reagents and chemicals used in the study were of analytical grade.

Methods:

1. Formation of lipid film

Transferosomes were prepared using Phospholipon 90 H, Surfactant, Chloroform and Methanol by thin film Hydration method.

Table 1 Formulation Table

Concentration

of Phospholipon 90 H (mg)

Concentration of Span 80

Isotretinoin

(mg)

Weighed amount of drug, lipid and surfactant was added to organic solvent ratio of chloroform and methanol (2:1% v/v). This mixture was added to the round bottom flask and a thin film was formed in rotary evaporator maintained at temp 40⁰ C and rotation at 80 rpm.

2. Hydration of lipid film

The lipid film formed which was hydrated with phosphate buffer pH 7.4 for the period of 1 hr.

3. Sonication

Transferosome formed were then sonicated by using probe sonicator (2 cycle, Pulse=15 sec. on and 5 sec. off). The transferosomes thus obtained were stored in amber colored glass vials.

4. Preparation of transferosomal gel

Transferosomal suspension was incorporate into Carbopol gel (2% w/w) with triethanolamine by gentle levigation. The specified amount of Carbopol 980 powder was slowly added to Distilled water and kept at 100 °C for 20 min. Triethanolamine was added to it drop‐wise. Appropriate amount of transferosomal suspension was dispersed in gel.

5 Evaluation of transferosomes

5.1 Vesicle size, size distribution and zeta potential analysis

The vesicle size, size distribution profile and zeta potential of transferosomal suspension were determined employing Malvern Zetasizer (Malvern Instruments Ltd.)

The polydispersity index (PDI) was used as a measurement of the width of the size distribution.

The zeta potential was measured after appropriately diluting the vesicle suspension with distilled water in zeta cuvette. Analysis was carried out for 60 s, at a detection angle of 173⁰, at a temperature of 25±1⁰C with electric field strength around 23.2 V/cm and an average of 20 zeta counts was obtained.

5.3 Entrapment efficiency

Transferosomal suspensions (2 ml) were ultracentrifuged at 20,000 rpm and 10̊ C for 30 min. After centrifugation, 1 ml of supernatant was diluted with the addition of 9 ml phosphate saline buffer (pH 7.4) and then the absorbance was measured using UV–Visible spectrophotometer by measuring absorbance at 348 nm.

The drug entrapment efficiency was calculated as below,

DEE= (WT -WF) =WT × 100%

Where, DEE is the drug entrapment efficiency,

WT is the total amount of insulin in transferosomal suspensions

WF is the free amount of insulin that was found in the supernatants

5.4 In vitro drug release by diffusion study

The permeation Transfersomes through an artificial cellophane membrane was performed in Franz-type diffusion cells with a diffusion area of 1.77cm². The receptor medium was 7.5 ml phosphate buffer (pH 7.4) which was constantly stirred at 100 rpm with a small magnetic bar. The receptor compartment was maintained at 37±0.2⁰ C by a circulating water jacket. An amount of Transfersomes equivalent to 600μg drug was placed in the donor compartment. Samples were withdrawn from the receptor compartment via the sampling port at 0.5, 1, 2, 4, 6, 8, 9, 10, 11, 12 and 24, and immediately replaced with an equal volume of fresh receptor solution to maintain sink condition. All samples were analyzed for drug content spectrophotometrically at a wavelength of 248 nm. The obtained data were kinetically treated to determine the order of release.

6. Evaluation of transfersomal gel

6.1 pH

Direct measurements were made using a digital pH meter (LI-601 Malvern Elico Ltd)

6.2 Viscosity determination

Viscosities were determined using Brookfield Viscometer (DV- I Prime Brookfield Pvt. Ltd)

6.3 Spreadability

Spreadability was determined by applying weight to glass slides into which formulation was placed, and time in seconds required to separate the slides was noted. Spreadability of each formulation was reported in seconds. Spreadability was then calculated by using the formula:

S = M.L/T

Where, S = spreadability, M = weight tide to upper slide, L = length of glass slide, and T = time taken to separate the slide completely from each other.

6.4 Skin Irritation study

For the skin irritation study of the transferosomal gel formulation, rats were divided into four groups, each group containing three rats. Hairs were depleted from the back side of rats with the help of depilatories, and the area was marked on both sides. One side served as the control while the other side served as the test. Gel was applied once a day for three days, and skin irritation from the formulation was determined by observations of any skin sensitivity and reactions such as redness, edema, and skin rash. The skin irritation effect of the gel was graded as: A-no reaction; B-slight, patchy erythema; C-moderate but patchy erythema; D-moderate erythema, and E-severe erythema with or without edema.

6.5 In vitro skin permeation study by Franz diffusion cell

In vitro skin permeation studies were carried out using Franz diffusion cell. The cell consists of two chambers, the donor and the receptor compartments with a diffusion area of 1.43 cm². The donor compartment was open at the top and was exposed to atmosphere. The abdominal skin was mounted between the compartments of the diffusion cell with stratum corneum facing the donor compartment and clamped into position. Magnetic stirrer bars were added to the receptor chambers and filled with the receptor phase. Phosphate buffer, pH 7.4 was used as receptor medium.

The entire setup was placed over magnetic stirrer and the temperature was maintained at 37± 0.5⁰C. The skin sections were initially left in the Franz cells for 2 hrs in order to facilitate hydration of the skin samples. After this period, 5 ml of the appropriate formulation was applied onto the surface of the skin. 5 ml of medium was collected from the receptor compartment at predetermined intervals over study period and replaced with the same amount of fresh buffer.

The amount of permeated drug was measured using UV-Visible spectrophotometer by measuring absorbance at 348 nm.

6.6 Skin deposition study

At the end of the permeation study (24 hrs), the skin was removed from the Franz cell and the formulation remaining on the skin surface was washed five times with warm receptor medium (45⁰C), then with distilled water. The permeation area of the skin was then excised and soaked in alcohol for 6 hrs to extract the deposited drug. The mixture was centrifuged (at 3000 rpm) and the drug content was measured spectrophotometrically.

6.7 Photomicroscopic analysis and Transmission Electron Microscopy (TEM)

The vesicles were examined under a binocular optical microscope and photographed at a magnification of 100× by means of a fitted camera. For morphological examination of the vesicles, TEM is used. The sample was negatively stained by placing a drop of 1 μ/ml of the vesicular suspension on a carbon coated grid. The suspension was left for 2 min, to allow its absorption in the carbon film, and the excess liquid was drawn off with filter paper. Subsequently, a drop of 1% phosphotungstic acid was placed on the grid. The excess was removed with distilled water and the samples were examined by TEM at 20 and 25 kV

6.8 Physical stability studies

Selected Transfersomal suspensions were stored in glass vials at 4⁰C for up to 6 month. Samples from each Transfersomal formulation were withdrawn at definite time intervals and characterized for their vesicle size, Zeta potential and Size distribution.

RESULT AND DISCUSSION:

1. Evaluation of transferosomes Suspension.

The particle size, size distribution and zeta potential of the transferosomes in aqueous solution were determined by dynamic light scattering (DLS) and the results are displayed in Table 2

The average particle size analyzed by Malvern particle size analyzer was found to be between 100-300 nm which in desired range.

The poly dispersity index (PDI) is± measurement of molecular mass in a given sample. The PDI is the weight average molecular weight divided by number average molecular weight. The PDI has value less than 0.5, this indicates good dispersion.

It shows that as the concentration of lipid increases the particle size increases but with the increases in the concentration of surfactant the particle size also get decreased.

Zeta potential is an indication of the stability of the transferosomes and indicates charge present on the vesicular system.

Here zeta potential was found to be -58±0.68 (±mv) which indicates a high negative surface charge on transferosomes.

This shows higher stability because of the anticipated surface repulsion between similar charged particles hence inhibiting aggregation of transferosomes.

% Entrapment efficiency was found tobe 64.18 % while % Drug release was 81.43 after 24 hours

Table 3 Cumulative % Drug release

Time (Hrs)	% Cumulative Drug release
0	0
0.5	11.95
1	16.04
2	20.03
3	24.45
4	28.86
5	33.83
6	39.1
7	44.51
8	50.50
9	57.27
10	63.69
11	70.68
12	76.05
24	81.43

4. Kinetic model fitting

Figure 2- Higuchi release

5. Kinetic analysis:

The diffusion profile of the Transferosomes of Isotretinoin prepared is fitted in to zero order, first order, Higuchi, and Korsmeyer-Peppa’s release kinetics as shown in Figure 2. The R² value is considered as the tool for repressing the best fitting into a kinetic model. According to this analysis it shows a highest R² value in Higuchi’s model. The ‘n’ value in the Korsmeyer-Peppa’s model was found between 0.5 and 1 that suggest that the mechanism of drug release follows fickian transport.

6. Evaluation of transferosomal gel

pH, Spreadability and Viscosity

The value of pH of Transferosomal gel was measured by using digital pH meter (ELICO LI 610 pH meter) at the room temperature.

Viscosities were determined using Brookfield Viscometer (DV- I Prime Brookfield Pvt. Ltd)

Table 4- pH, Spreadability and viscosity of transferosomal gel

Transferosomal Gel

(Mean ± SD)

Spreadability

gm.mm/min

Viscosity

6.9±0.05

24.58

13050

7. Transmission electron microscopy (TEM) –

TEM image showed that Transferosomes are nearly spherical in shape.

No reaction, erythema with or without edema was observed after 24 hour. Thus no irritation was observed.

9. Comparison of Ex-vivo drug release with marketed preparation shows that Transferosomal gel has sustained drug release as compare to marketed formulation.

Table 5- Comparative ex-vivo permeability profile

Time (hrs)	Cumulative Drug Release (%)
Time (hrs)	Transferosomal gel	Marketed Formulation
0	0	0
1	12.0	28.05
2	21.01	45.85
3	26.10	53.47
4	29.52	62.12
5	33.60	70.40
6	38.87	81.10
7	44.32	90.60
8	49.12	99.52
9	54.62	-
10	59.85	-
11	64.47	-
12	69.12	-
24	79.05	-

Figure 6- Comparison with Marketed preparation

Table 6- Amount of drug permeated and deposited in the skin

Formulation

Amount of drug Permeated μg/ml

Amount of drug deposited μg/ml

Steady state flux

Transferosomal gel

12.571

4.43

0.525

Marketed formulation

6.337

1.74

0.351

Transferosomal gel exhibited sustained permeation as compared to conventional gel as shown in Figure 9. This indicates the effect of vesiculation. Thus gel acts as a depot system, hence offers mean of sustained release. The steady state flux of transferosomal gel was found higher than the marketed conventional gel.

Figure 7- Steady state fluxes of Transferosomal gel and Marketed formulation

10. Stability study

Stability study was carried out for the one month and particle size, PDI and Zeta potential was analyzed.

Table 7- Effect of temperature on stability of Transferosomal gel

Temp (⁰C)

Particle Size (nm)

PDI

Zeta potential (mV)

4⁰C

117.7

0.347

-58

Room Temp.

124.4

0.350

-57

The suspension was stored at 4 ⁰C and room temperature for 6 month and their particle size was evaluated. There was no sedimentation at 4 ̊C. Slight increase in particle size at room temperature due to aggregation.

CONCLUSION:

The Transferosomal gel was compared with marketed preparation for skin permeation and skin deposition study which concludes that optimized formulation showed better results than marketed preparation. Thus it was better than the conventional gel. The stability study indicates that transferosomal gel was stable.

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Received on 20.12.2014 Accepted on 28.12.2014

Res. J. Topical and Cosmetic Sci. 5(2): July –Dec. 2014 page 39-45