Performance evaluation of non-ionic surfactant based tazarotene encapsulated proniosomal gel for the treatment of psoriasis

Materials Science and Engineering C 79 (2017) 168–176

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Performance evaluation of non-ionic surfactant based tazarotene encapsulated proniosomal gel for the treatment of psoriasis Vure Prasad a,b,⁎, Sundeep Chaurasia a a b

Research and Development, Virchow Biotech Pvt. Ltd., Survey No. 172 Part, Gagillapur (V), Quthbullapur (M), R.R. Dist, 500 043 Hyderabad, Telangana, India Department of Pharmaceutics, School of Pharmacy, Anurag Group of Institutions, Ghatkesar-501 301 Hyderabad, Telangana, India

a r t i c l e

i n f o

Article history: Received 25 August 2016 Received in revised form 25 April 2017 Accepted 7 May 2017 Available online 8 May 2017 Keywords: Tazarotene Proniosomes Topical delivery Skin histology

a b s t r a c t The study aims to explore the potential of non-ionic surfactant based proniosomal gel (PNG) in improving the topical delivery of tazarotene by in vitro and in vivo studies. The PNG was prepared using coacervation phase separation method composed of span, stearylamine, cholesterol, and lecithin. The PNG demonstrated favorable vesicle size (3.26±0.22μm) and percent encapsulation efficiency (49.50±2.3%). The PNG was evaluated for viscosity which indicated that the ratio of span:cholesterol:stearylamine (64.5:30.5:5 mM) demonstrated no any fluctuations in viscosity. The scanning electron micrographs exhibited spherical vesicles with sharp boundaries. The in vitro drug release through cellulose membrane and rat's skin were found to be in the following order of the formulation code A2NA4NA3NA5 and A4NA2NA3NA5, respectively, which showed the prolonged release of entrapped tazarotene. Further, in vitro drug permeation and retention studies revealed that formulations A2 and A4 showed the higher percent of drug permeation whereas formulations A3 and A5 showed the higher percent of drug retention through rat's skin. Moreover, PNG A2 and A4 formulations demonstrated good stability characteristics at different temperature conditions. The stability in the presence of detergent revealed that no any abrupt change in turbidity. The skin irritation studies performed with formulations A2 and A4 showed no erythema compared with the plain PNG. The male Albino NMRI mice tail model was used to performed in vivo skin histological examination which revealed that an increase in the orthokeratosis strengthened. Thus, all the results concluded that surfactant, Span 60 based PNG formulations have shown a good ability to increase drug accumulation in the various skin layers and more potential carrier for topical delivery of tazarotene for an effective therapy of psoriasis. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Psoriasis is a chronic, autoimmune skin disorder characterized by relapsing episodes of inflammatory lesions, localization of rash, hyperkeratotic plaques, guttate, pustular and erythroderma with the worldwide occurrence of around 2–5% [1]. It is a lifelong disorder which affects mainly knees, elbows, and scalp with unpredictable remissions and relapses, thereby patients have been disturbing physically, psychologically and socially [1,2]. This is further supported by the results of national psoriasis foundation survey indicating moderate to large negative impact on the quality of life of psoriasis patients [3]. Recent, evidence suggests that psoriasis has a genetic basis dominated by the Th1Abbreviations: PNG, proniosomal gel; A2 A3 A4 and A5, formulation code; S20, Span 20; S40, Span 40; S60, Span 60; S80, Span 80; μm, micrometer; ml, milliliter; mm, millimeter; cm, centimeter; mg, milligram; min, minute; mM, millimoles; h, hour; kg, kilogram; °C, degree centigrade; g, gram; nm, nanometer; rpm, rotation per minute. ⁎ Corresponding author at: Product Development, Virchow Biotech Pvt. Ltd., Survey No. 172 Part, Gagillapur (V), Quthubullapur (M), R.R. Distt., 500 043- Hyderabad, Telangana, India. E-mail address: [email protected] (V. Prasad).

http://dx.doi.org/10.1016/j.msec.2017.05.036 0928-4931/© 2017 Elsevier B.V. All rights reserved.

immunological pathway that can be triggered by environmental factors such as trauma, drugs, infection, smoking, alcohol consumption, diet, and stress with prevalence ranging between 0.6% and 4.8% but its accurate origin is still not known [4]. Psoriasis is a common condition with an increased risk of morbidity and mortality compared to the general population [5]. The treatment of psoriasis varies depending on disease severity and spread. However, topical treatments remain the approach of psoriasis treatment for majority patients. The third-generation topical retinoid is frequently used for the management of psoriasis for decades. Tazarotene, the topical retinoid is widely used in the treatment of psoriasis. It modulates there key factors in psoriasis: keratinocyte differentiation, keratinocyte proliferation, and inflammation [6]. Tazarotene has selective affinity for the β and other subtypes of retinoic acid receptors (RARs) like RARg and does not bind to retinoid X receptors [7]. The predominant type of RAR expressed in human epidermis is RAR-Ɣ, suggesting that it is a major mediator of retinoid action. As with other topical retinoids, local irritation is the most common adverse effect, including mild to moderate pruritis, erythema, burning, and desquamation. Systemic absorption of tazarotene appears to be very low; leading to no systemic side effects [8]. So, it can be suggested that the topical

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delivery of tazarotene is a promising drug candidate in the treatment of psoriasis. Currently, researches are being focused toward the development of new approach in order to get enhanced benefit of using tazarotene for the management of psoriasis. Vesicular systems have been widely studied as vehicles for dermal and transdermal drug delivery. Their benefits in enhancing drug permeation have been well established [9]. Vesicular system, both liposomes, and niosomes are uni- or multi-lamellar spheroidal structures composed of amphiphilic molecules assembled into bilayers. They are considered primitive cell models, cell-like bioreactors, and matrices for bio-encapsulation. In the recent years, non-ionic surfactant vesicles known as niosomes received great attention as an alternative potential drug delivery system to conventional liposomes. Moreover, compared to liposomes, niosomes offer higher chemical and physical stability [10] with lower cost and greater availability of surfactant classes [11]. Niosomal vesicles can encapsulate both lipophilic and hydrophilic drugs [12]. It has been reported to enhance the residence time of drugs in the stratum corneum and epidermis [13] while reducing the systemic absorption of the drug and improve penetration of the trapped substances across the skin. In addition, these systems have been reported to decrease side effects and to give a considerable drug release. They are thought to improve the horny layer properties both by reducing trans-epidermal water loss and by increasing smoothness via replenishing lost skin lipids [9]. Moreover, it has been reported in several studies that compared to conventional dosage forms, vesicular formulations exhibited an enhanced cutaneous drug bioavailability [14]. However, there may be problems of physical instability in niosome dispersions during storage like vesicles aggregation, fusion, leaking or hydrolysis of encapsulated drugs, which affected the shelf life of the dispersion [15]. So far, proniosomes vesicular system could be a potential delivery carrier for improving solubility and enhancing the therapeutic concentration of tazarotene to the target tissue. Proniosomes allows the prolonged release of the drug at the target and minimizes dose-dependent side effects. It overcomes the disadvantage of vesicular instability associated with niosomes [16–18]. Therefore, several researchers have been investigated proniosomes based drug carrier such as tenoxicam loaded proniosomal formulation proved to be non-irritant, with significantly higher anti-inflammatory and analgesic effects compared to that of the oral market tenoxicam tablets [19] and transdermal delivery of ketorolac encapsulated proniosomes based drug carrier [20]. So far, exploring the potential of proniosomes loaded with tazarotene appears valuable. The aim of the present investigation was to develop proniosomes gel (PNG) using coacervation phase separation method. The prepared PNG evaluated for physicochemical characterizations, viscosity, vesicle size, vesicles morphology, in vitro drug release, in vitro permeation and retention studies, skin irritation, and vesicles stability at a different temperature. Furthermore, the in vivo study of the prepared PNG was evaluated using mouse tail model to examine the skin histology at the end of the treatment.

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2. Material and methods 2.1. Materials Tazarotene was obtained as gift sample from Dr. Reddy's Laboratories Ltd., Hyderabad, India. Soya lecithin was purchased from Sadguru Chemicals Pvt. Ltd., Hyderabad. The Span 20, Span 40, Span 80, Span 60, Tween 20, Tween 80, and absolute ethanol were obtained from SD Fine Chemicals, Mumbai, India. All others chemicals and reagents used in the study were of analytical grade and ultra-pure Milli-Q (SG LABOSTAR TWF-UV, Germany) water was used throughout the experiments. 2.2. Preparation of PNG The PNG was prepared using coacervation phase separation method with little modification [21]. Briefly, 0.1%w/w of tazarotene with a surfactant, lecithin and cholesterol were mixed with 2.5 ml absolute ethanol in a wide mouth glass tube. The compositions of additives are listed in Table 1. Then the open end of the glass tube was covered with aluminum foil and warmed in a water bath at 65± 3°C for 15 min. A 1.6 ml, phosphate buffer pH 7.4 was added and still warmed on the water bath for about 5 min till the clear solution was observed. The mixture was allowed to cool down at room temperature till the dispersion was converted into PNG. 2.3. Encapsulation efficiency The PNG (0.5 g) was reconstituted with 10 ml of phosphate buffer, pH 7.4 in a glass tube. The aqueous suspension was sonicated using bath sonicator (CITIZEN Digital Ultra-sonicator CD-4820, Mumbai, India) for 30 min. The tazarotene encapsulated proniosomes were separated from unentrapped drug by centrifuging at 20,000 rpm at 20°C for 30 min (REMI Cooling Centrifuge TR-01, Mumbai, India) [22]. The supernatant was taken and diluted with methanol, and drug concentration in the resulting solution was assayed by UV‐visible spectrophotometer (UV 1601, Shimadzu Corporation, Germany) at 351 nm. The percentage of drug encapsulation was calculated by the following equation: % Encapsulation efficiency ¼ ½ðCt −C f Þ=Ct   100 where, Ct is the concentration of total tazarotene, and Cf is the concentration of free tazarotene. 2.4. Viscosity measurement Measurement of the viscosity is important when the fluid is being studied, since the Stokes' law asserts that the rate of phase separation (v) between liquid 1 (ρ1) and liquid 2 (ρ2) depends on gravity (g), on

Table 1 Composition of various PNG formulations and their percent encapsulation efficiency. Prinosomal code

S20 (mg)

PNG F1a PNG F2a PNG F3a PNG F4a PNG F5a

180

S60 (mg)

S80 (mg)

S40 (mg)

T80 (mg)

Cholesterol (mg)

Lecithin (mg)

Absolute ethanol (ml)

PBS pH 7.4 (ml)

Encapsulationb efficiency (%)

180

20 20 20 20 20

180 180 180 180 180

2.5 2.5 2.5 2.5 2.5

1.6 1.6 1.6 1.6 1.6

23.84±1.4 49.50±2.3 23.84±1.6 28.84±2.0 17.24±1.9

180 180 180

Where, S; span and T; tween a Stearylamine is added in all formulations at a constant quantity b All data are show as mean±SD; n=3.

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the radius of the particles (r) and on the viscosity (η) of the medium. v ¼ 2r 2 ðρ1−ρ2Þ g=9η

Table 2 PNG formulations with various ratios of sorbitan fatty acid esters and lecithin. Sl. no

Proniosomal code

Ratios

Tazarotene (mg)

Vesicle size (μm)

7.46 3.70 8.84 3.28

3.28±0.22 6.70±0.29 10.84±0.86 17.28±0.92

Lecithin

where; ‘v’ is the particles'/vesicles settling velocity (m/s); ‘r’ is the stokes radius of the particle/vesicle (m); ‘g’ is the standard gravity (m/s2); ‘ρ’1 is the density of the particles/vesicles (kg/m3); ‘ρ2’ is the density of the fluid (kg/m3) and ‘η’ is the (dynamic) fluid viscosity (Pa.s). Rheological parameters were calculated and analyzed using viscometer (Bohlin Visco 88 Viscometer, New Delhi, India) equipped with Bohlin software. Cone and plate geometry (cp 5.4°C/30) was employed with 0.05 mm gap. The samples were centrifuged free of bubbles and an amount sufficient to fill the cone and plate gap completely was carefully scooped out and spread over the cone (~1 ml). The ambient temperature was maintained at 25± 1°C. 2.5. Morphological evaluation The surface morphology of proniosome vesicles in the dispersion was evaluated in the terms of shape, texture, formation of aggregates and size distribution by using scanning electron microscopy. Briefly, 1 g of the PNG in a glass tube was diluted with 10 ml of phosphate buffer, pH 7.4. The proniosomes were mounted on an aluminum stub using double-sided carbon adhesive tape. Then the vesicles were sputtercoated with gold palladium (Au/Pd) using a vacuum evaporator (Edwards, Crawley, United Kingdom) and examined using a scanning electron microscope JSM-5510 (JEOL Ltd., Tokyo, Japan) equipped with a digital camera at 20 kV accelerating voltage. 2.6. In vitro release study The release of tazarotene from PNG formulations was determined using membrane diffusion technique [23]. The PNG formulations equivalent to 5 mg of tazarotene was converted to niosomal suspension and taken in a glass tube having a diameter 2.5 cm with an effective length of 8 cm that was previously covered with soaked osmosis cellulose membrane, which acts as a donor compartment. The glass tube was placed in a beaker containing 75 ml of phosphate buffer, pH 7.4, which acts as receptor compartment. The whole assembly was fixed in such a way that the lower end of the tube containing suspension was just touched (1–2 mm deep) the surface of diffusion medium. The temperature of receptor medium maintained at 37± 1°C and the medium was agitated at 100 rpm speed using magnetic stirrer for 12 h. Aliquots of 5 ml sample were withdrawn periodically and after each withdrawal, the same volume of medium was replaced. The collected samples were analyzed at 351 nm in a double beam UV‐visible spectrophotometer using phosphate buffer, pH 7.4 as blank. 2.7. In vitro skin permeation and retention studies 2.7.1. Permeation study The permeation of tazarotene from PNG formulations was determined by using Franz (vertical) diffusion cell with the diffusion area of 2.54 cm2 [24]. The Wistar rats (7–9 weeks old) skin was mounted on the receptor compartment with the stratum corneum side facing upwards into the donor compartment and the top of the diffusion cell was covered with paraffin paper. The donor compartment was filled with the 1 g of PNG formulations. A 30 ml aliquot of phosphate buffer, pH 7.4 was used as receptor medium to maintain a sink condition. The temperature of receptor compartment was maintained at 37± 1°C using a thermostatic hotplate temperature controller available on a magnetic stirrer [25]. The receptor fluid was stirred at 600 rpm by a magnetic bead on a magnetic stirrer. The samples were analyzed at 351 nm in a double beam UV‐visible spectrophotometer using phosphate buffer pH 7.4 as blank.

1. 2. 3. 4.

A (S60) B (S40) C (S80) D (S20)

2 2 2 2

1 1 1 1

Where, S; span; all data are show as mean±SD; n=3.

2.7.2. Retention study The drug retained in skin is measured after permeation experiments, for which the skin was removed from the diffusion cells after completion of experiments. The surface of skin specimens was washed 10 times with 1 ml distilled water and dried with filter paper. The effective surface area of the skin was separated and minced with a surgical sterile scalpel then finally homogenized in a vial filled with methanol by using homogenizer (Remi RQT-124A/D, Mumbai, India) at 4000 rpm for 5 min in an ice bath. The tissue suspension was centrifuged (Refrigerated Centrifuge RC 4100F, Elteck, Mumbai, India) at 9000 rpm for 15 min and then the supernatant was filtered [26]. Further, the supernatant tissue suspension was extracted with methanol and filtered. The supernatant from receptor solution and tissue suspension as well as washing solution were assayed for tazarotene content by double beam UV‐visible spectrophotometer using phosphate buffer 7.4 as blank at 351 nm. 2.8. Stability study Stability study of the prepared PNG formulations was carried out at various conditions such as refrigeration temperature (2–8 °C), room temperature (25±0.5°C) and elevated temperature (45±0.5°C) from a period of one month to three months. The variation in percent encapsulation efficiency and average vesicles diameter were periodically recorded. 2.9. Stability in presence of detergent The stability of niosomal dispersion (surfactant vesicles) obtained from PNG has been subjected to the turbidity of their 10-fold diluted suspensions in different concentrations of sodium deoxycholate (0‐ 20mM in PBS pH 7.4) solutions at 37°C after incubating for 24 h. The results were interpreted by measuring percentage turbidity (defined in terms of optical density) using a UV‐visible spectrophotometer and measured at a wavelength 400 nm [27]. % Turbidity ¼ 1−%Transmittance: The turbidity was measured at room temperature initially and after 24 h. Bile salt solution without added vesicles was used as blank for each specific concentration (0–20 i.e. 0, 2.5, 5, 7.5, 10, 15 and 20 mM). All these studies are carried for all non-ionic surfactant based formulations, in order to understand the stability of vesicles in the presence of detergents and conversion of vesicles into micelles.

Table 3 Percent encapsulation efficiency and vesicle size of the different formulations. Sl. no

Proniosomal code

1. 2. 3. 4.

A2 A3 A4 A5

Ratios S 60

Lecithin

3 1 2 1

1 2 1 3

Encapsulation efficiency (%)

Vesicle size (μm)

65.0±1.9 57.4±1.6 61.2±2.1 57.2±1.5

3.26±0.286 6.70±0.542 4.84±0.322 8.28±0.442

Where, S; span; all data are show as mean±SD; n=3.

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Table 4 Viscosity of the different vesicular PNG formulations. Formulation code

S 40:Ch:SA

Viscosity (cp)

Coefficient (γ 2)

Torque (Nm)

Newtonian model fit

Regression (R2)

NV1a NV1b NV1c NV1

30.5:64.5:5 mM 47.5:47.5:5 mM 55.0:40.0:5 mM 64.5:30.5:5 mM S 60:Ch:SA 30.5:64.5:5 mM 47.5:47.5:5 mM 55.0:40.0:5 mM 64.5:30.5:5 mM S 80:Ch:SA 30.5:64.5:5 mM 47.5:47.5:5 mM 55.0:40.0:5 mM 64.5:30.5:5 mM

5.502±0.025 5.603±0.035 5.997±0.028 6.038±0.036

8.89 6.86 1.963 2.252

0.02048 0.02047 0.02047 0.01738

Passes Passes Passes Passes

0.9518 0.9183 0.9598 0.9723

6.159±0.046 6.058±0.053 6.232±0.074 6.295±0.056

1.143 3.186 1.202 1.429

0.02047 0.02048 0.02046 0.01862

Passes Passes Passes Passes

0.9679 0.9295 0.9259 0.9746

4.951±0.051 4.414±0.059 4.402±0.064 5.118±0.026

1.607 2.813 3.222 1.567

0.02047 0.02048 0.02047 0.02048

Passes Passes Passes Passes

0.9292 0.9333 0.9722 0.9576

NV2a NV2b NV2c NV2 NV3a NV3b NV3c NV3

Where; Ch: cholesterol; SA: stearylamine; S; span; all data are show as mean±SD; n=3.

2.10. Skin irritation study

2.11. In vivo studies

Three albino rabbits of either sex (weighed 2–2.5 kg) with intact skin were used for skin irritation test [28]. The skin from the back of each rabbit was depilated 24 h prior to application of the patch. Two areas of the back of each rabbit, approximately 10 cm2 apart were designated for the position of the patches. One area was used for application of plain PNG and the other was used for drug loaded PNG. The animals were immobilized using rabbit holder during 24 h exposure. After washing the applied gel, the resulting reaction was evaluated using weighed scores. The skin was observed for any visual change, such as erythema at 24, 48 and 72 h after the application of formulations. The mean erythemal scores were recorded (ranging from 0 to 4), depending on the degree of erythema, as follows: no erythema = 0; slight erythema (barely perceptible-light pink) = 1; moderate erythema (dark pink) = 2; moderate to severe erythema (light red) = 3; and severe erythema (extreme redness) = 4 grade.

All the in vivo study protocols were approved by the Animal Ethical Committee of Anurag Group of Institutions, Jawaharlal Nehru Technological University, Hyderabad, India (No. Dean/14-15/CAEC/311). Studies were performed according to the guidelines compiled by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Ministry of Culture, and Government of India). All the experimental mice were housed in cages, with access to food and water ad libitum until use. The normal mouse tail contains regions of ortho and parakeratosis stratum corneum. Parakeratosis, an abnormal form of an epidermal keratinization pattern, is characterized by the absence of a granular layer in the epidermis. In the rat tail skin, parakeratosis sets in by days 7–9 after birth and the scales start developing. During this period, small depressions are observed in the epidermis, which further develops and forms the orthokeratosis area (i.e., the normal pattern of keratinization),

Fig. 1. Scanning electron micrographs of various PNG formulations (a) A2, (b) A3, (c) A4 and (d) A4.

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Fig. 2. The in vitro release rate of tazarotene across (a) cellulose membrane and (b) rat skin; all data are show as mean±SD; n=3.

while the area between these notches shows an abnormal pattern of keratinization, known as the parakeratosis area. Any agent that causes the inhibition of parakeratosis and the restoration of orthokeratosis can be used for treating psoriasis. Hence, the mouse tail model is commonly employed for screening drugs for the treatment of psoriasis [29]. In this study, three groups of five male Albino NMRI mice (weighed 25–27 g) [30–31], each was treated once daily, 5 times a week, for 4 weeks with saline, PNG A4, and PNG A2. One group of five animals acted as a control (i.e., no treatment). The 0.5 g of PNG formulations were applied with a paint brush to the proximal half of the mouse tail. For the contact time of 2 h, a plastic cylinder is slipped over the tail and fixed with adhesive tape. At the end of contact time, the cylinders were removed and washed. Twenty-four hours after the last application of formulations, the mice were killed by cervical dislocation, and the tail skin was cut by longitudinal dissection of about 5 mm3 thickness with a scalpel and stripped from the underlying cartilage. The skin samples obtained were appropriately processed with fixation in 4% formalin, embedded with paraplastic and stained with hematoxylin and eosin. The processed skins were examined under light microscopically (Eclipse 4000, Nikon, Tokyo, Japan) for the presence of granular layer in the scale regions and epidermal thickness [32]. Induction of orthokeratosis in those parts of the mouse tail, which have normally a parakeratotic differentiation, was quantified measuring the length of the granular layer (A) and the length of the scale (B). Ten sequential scales were examined for each skin section. The proportion, that is, A/B ∗ 100 represents the percent orthokeratosis per scale. The drug activity (DA) was calculated by the following equation [33]: DA ¼

was found to be much higher than S20, S40, T80, and S80. Therefore, S60 shows the maximum percent encapsulation efficiency and small vesicle size as shown in Tables 1 and 2. This was due to the fact that S60 is solid at room temperature, exhibited higher phase transition temperature and low permeability. Further, this could be attributed to relatively high hydrophobicity with small critical packing behavior of S60. Therefore, it requires only small amount of cholesterol to obtain the optimum membrane curvature for lamellar (vesicular) structure [35]. In addition, cholesterol concentrations play an important role in the encapsulation efficiency of drugs in the PNG formulations, which may be due to intercalation of cholesterol in the bilayers [36,37]. Moreover, it is wellknown fact that S40 and S60 have the same head group but different alkyl chain [38]. Among these surfactants, only S80 in formulation ‘PNGF3’ has an unsaturated alkyl chain. The introduction of double bonds affects bending of alkyl chain and thus fluidity. This means that adjacent molecule cannot be tight enough to form the niosomes. This causes the membranes to be more permeable due to lose packing which possibly explains the lowest percent entrapment efficiency. The S60 has the longest saturated alkyl chain and shows the highest percent entrapment efficiency. Therefore, the alkyl chain is the crucial factor for the permeability of long chain products. Moreover, the S60 and S40 are solid at room temperature and exhibit the higher phase transition

Mean percentage of orthokeratosisðOKÞof treated group−Mean OK of control  100 Total length of scale−Mean OK of control

2.12. Statistical analysis All the results were expressed as the mean ± standard deviation (SD) for in vitro studies and mean ± standard errors mean (SEM) for in vivo studies. 3. Results and discussion The PNG was prepared by coacervation phase separation method using non-ionic surfactants of alkyl ester including span (sorbitan esters) and tween (polyoxyethylene sorbitan esters) for topical/localized delivery of tazarotene. These formulations were developed for greater skin retention of drugs. Delivery, penetration, and localization of the drug in the different skin layers are greatly affected by the composition of the PNG system [34]. The percent encapsulation efficiency using S60

Fig. 3. Flux and release rate of various PNG formulations across cellulose membrane and rat skin; all data are show as mean±SD; n=3.

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Fig. 4. (a) Percentage drug permeated and (b) retained in the rat skin after completion of in vitro permeation experiments; all data are show as mean±SD; n=3.

temperature (Tc) that reflects the effect of phase transition temperature on the percent entrapment efficiency. Therefore, S60 was selected for preparation of PNG with varying S60 and lecithin ratios and further evaluated as shown in Table 3. Further, it has been reported that the HLB value of S40, S60 and S80 are 6.7, 4.7 and 4.3, respectively. It is evident from the literature when increases the hydrophobicity of surfactant monomer leads to decreases vesicle size [35]. This may be due to the fact that surface free energy decreases with increasing the hydrophobicity i.e. S80bS60bS40. Therefore, the size of the vesicles obtained from S60 is less than S40. The S80 do not form vesicles by itself; the, however, certain amount of cholesterol is needed to form vesicles. The sizes of the vesicles are found in the order of S 80bS60bS40. Furthermore, it is surprisingly the percent entrapment efficiency was determined in formulations with different ratios of S60 and lecithin were almost same with a not much significant difference, but the formulations having surfactant in large quantitatively have higher percent entrapment efficiency than those with higher lecithin ratio as shown in Table 3. Therefore, the formulations containing S60 and lecithin ratios were further characterized and evaluated in vitro as well as in vivo. The viscosity of various formulations (without the addition of tazarotene) was recorded and effects of shear rate on viscosity are shown in Table 4. From the data, it was observed that there was a gradual increase in viscosity of the formulations starting from S40 to S60 and when different molar ratios of Spans: Cholesterol: Stearylamine were recorded. Further, it has been observed that consistent increase in viscosity with an increase in the molar ratio of span and decrease in cholesterol quantity. As this may be one of the reasons where the quantum of vesicles increases there would be more resistance to flow. No significant difference in viscosity was observed between any formulations made either from S40, S60, and S80 having different composition and the viscosity was in the range of 4–6 cp. Table 4 represents the effect of shear rate on viscosity, from which it was lucid that, the ratio of span:cholesterol:stearylamine (64.5:30.5:5 mM) exhibits a very near to straight line evinced by (regression is 0.9746), without any fluctuations in viscosity, when shear rate was increased [35].

Scanning electron micrographs revealed the formation of well identified spherical niosomal vesicles with sharp boundaries after hydration of proniosomal gel. The SEM micrographs of the different PNG formulations are depicted in Fig. 1. Moreover, the high HLB values of S60 (6.7) results in a reduction in surface free energy which allows forming vesicles of smaller size. The vesicle size was found to be in the range of 3–10 μm. The vesicle size of formulation A2 and A4 was found to be smaller than the formulations A3 and A5. This was due to the chemical nature of the surfactants and lecithin imparts a high negative residual charge on the vesicular system. Negative charge maintains the small size and vesicular stability of these systems provided by the electrostatic repulsion [39]. The in vitro drug release studies were carried out of all PNG formulations. The percentage of drug release of all formulations through cellulose membrane after 12 h was found to be 75.88±3.974% (A2), 67.73 ±3.386% (A3), 75.63±3.781% (A4) and 62.34±3.117% (A5) as shown in Fig. 2(a) and percent drug release through rat skin were found to be 57.67±2.883% (A2), 56.23±2.811% (A3), 59.78±2.989% (A4) and 55.22±2.761% (A5), respectively as illustrated in Fig. 2(b). The drug release through the cellulose membrane was found to be maximum in order of A2NA4NA3NA5, this was due to the higher concentration of surfactant to lecithin ratio which has higher drug enhancer properties. Moreover, the drug release through rat skin was found to be maximum in order of A4NA2NA3NA5, this was happened because of surfactant present in the PNG which may also incorporate as well as mix with skin lipids to release their structure by distressing the lamellar arrangement of the lipids [40]. Furthermore, the flux and release rate of tazarotene from various PNG formulations across rat skin and cellulose membrane are illustrated in Fig. 3. We found that the release rate of tazarotene was significantly higher than its flux across rat's skin, depicting the permeation negativity or barrier properties of skin. The trends of tazarotene permeation from various formulations across rat's skin and cellulose membrane were quite different and more of drug release was seen using the cellulose membrane when compared with rat's skin because of the barrier properties of the skin. More of all our

Table 5 Change of vesicle size and percent encapsulation efficiency of the PNG formulations after storage. Formulation code

Vesicle size (μ) Initial

A2 A3 A4 A5

13.70±1.09 17.46±1.13 18.54±1.23 13.28±0.97

All data are show as mean±SD; n=3.

Encapsulation efficiency (%) After 3 months

Initial

Room temp.

Refg. temp.

12.33±0.89 15.78±0.78 16.56±1.03 12.12±0.91

13.20±1.21 16.88±1.33 17.64±1.42 12.58±0.88

65.0±1.9 57.4±1.6 61.2±2.1 57.2±1.5

After 3 months Room temp.

Refg. temp.

61.23±1.6 53.68±1.8 56.19±2.4 53.34±1.9

63.68±2.0 54.32±1.5 58.43±1.9 55.62±1.3

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hypothesis of drug permeation into the skin, but not into blood circulation has provided a strong evidence of drug permeation into the skin, which may always be an ideal delivery system for the treatment of psoriatic therapy. In order to assess the skin targeting capability of the PNG formulations, the permeation ability of tazarotene through the hairless rat skin was examined by conducting in vitro skin permeation and retention studies. In permeation study, data obtained from PNG formulations (A2, A3, A4, and A5) were compared with each other. We found that formulations A2 and A4 showed the higher percent of tazarotene permeation as compared with formulations A3 and A5 as illustrated in Fig. 4(a). The higher percent drug permeation through the Wistar rat's skin may be attributed to high concentration of S60 to lecithin ratio, act as permeation enhancers as evident from other literature. In retention study, the percentage of tazarotene accumulated into the viable skin for all PNG formulations at the end of the experiments has been depicted in Fig. 4(b). A significantly higher percent of drug retention was obtained from PNG formulations, A3 (52.34±2.617%) and A5 (61.05±3.052%) as compared with formulations A2 (49.39±2.469%) and A4 (43.13±2.156%), respectively. The percent drug retention for all the PNG formulations was found to be maximal in the rat's skin. Moreover, formulation A5 produced a significantly higher percent drug accumulation in the viable skin, compared with other PNG formulations. The possible reasons for a higher percent of tazarotene penetration and accumulation into the skin layers following PNG applications are surfactant: lipid ratio, small vesicle size, and penetration enhancer effect of surfactants and also the interaction between PNG with the stratum corneum lipids, providing a deposit effect of the drug in the skin. Lipidic compositions of the PNG are more similar to stratum corneum lipids, in contrast to gel which may promote the accumulation of the encapsulated tazarotene moiety into the upper skin layers, thus creating a reservoir which may prolong the skin residence time. Moreover, a surfactant present in the PNG may be responsible for the higher drug retention and penetration effect which may also integrate as well as mix with skin lipids to loosen their structure by disturbing the lamellar arrangement of the lipids [41,42]. Further, the permeation and retention of PNG formulations also correlates with in vitro release studies of the various proniosomal formulations, which found that release of various proniosomal formulations through rat's skin is less as compared to cellulosic membrane because several barriers are present in the skin layer

Fig. 6. Relative stability of vesicular formulation of ‘BL’ as a function of different concentration of (sodium deoxycholate) SDC in PBS, pH 7.4; incubated at 37°C for 60 min revealed as turbidity at 400 nm; all data are show as mean ± SD; n=3.

which retain the drug for long time. So far, percent of drug retention through rat's skin is more as compared to cellulosic membrane. Stability studies supported that there was no significant change in the vesicle size and percent encapsulation efficiency of the drug in various PNG formulations at room temperature and refrigeration conditions are shown in Table 5. This was due to the small vesicle size, better encapsulation efficiency and higher percent of drug retention in the rat's skin. However, the formulations were found to be unstable at elevated temperature and drug leakage was seen. Vesicles (niosomes) stability against solubilization by micelle forming detergents (such as bile salts) has been studied using turbidimetry method [27]. This method was selected for the present study because it is simplest of all other techniques like fluorescence probe and gel chromatography. The vesicle-micelle transition takes place in three phases: the first phase includes the association of detergents with outer bi-layers and saturation of them without any significant change in turbidity. The second phase involves solubilization of outer bi-layers and finally solubilization of remaining bi-layers. In all studied formulations, transferring from phase I to phase II did not show the sharp increase in turbidity as shown in Fig. 5, may be due to multilamellar of vesicles or the type of used solubilizer [43]. As shown in Figs. 5 and 6, there was a sharp change in turbidity at initial concentrations of sodium desoxycholate, which is in agreement with the initial phase of detergent interactions with outer bi-layers. On further, increasing the concentration above 10mM abrupt decline in turbidity was observed in almost all formulations, which could be due to solubilization of outer bi-layers. The concentration of bile salt solution at the onset of phase II in the case of S80, S20, and S40 was 2.5, 10, and 15mM, respectively. S60 niosomes did not display any abrupt change in turbidity at the studied detergent

Table 6 Mean erythemal scores and PII observed for PNG formulations obtained at the end of 24, 48 and 72 h (n=3). Formulation code

Fig. 5. Relative stability of vesicular formulation of ‘PNGF2’ as a function of different BS concentration in PBS, pH 7.4; incubated at 37°C for 60 min revealed as turbidity at 400 nm; all data are show as mean±SD; n=3.

Plain PNG PNG A4 PNG A2

Erythemal scores 24 h

48 h

72 h

PII

3 0 0

2 0 0

2 0 0

2.33 0 0

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Fig. 7. Skin histology (a) non-treated mouse tail scale region; (b and c) skin treated with PNG A4 and PNG A2 and (d) arrows indicated Orthokeratotic regions.

concentrations; we postulated this is due to high gel to liquid crystal transition temperature and rigidity of S60 bi-layers. This points out the particular resistance of S60 vesicles to lysis by bile salt so that the final percent of turbidity of its dispersion (60.8±3.04%) on exposure to 20mM of sodium desoxycholate is significantly higher than all other dispersions. This is similar to finding that the rigidity of gel-state Palmitoyl phosphatidylcholine bilayers in the presence of high level of cholesterol slows down the solubilization rate [44]. The turbidity of S40 also remained high (48.3±2.41%) compared to S20 (21.3±1.06%) and S80 (19.2±0.96%), respectively after exposure to 20mM of sodium desoxycholate. Parallel to this hypothesis, we found that on the incorporation of bile salt as integral component up to 10mM, the turbidity was maximum (89.8±4.49%), on exposure to 15mM bile salt showing optimum stability. On the contrary, if we omit incorporation of bile salt in vesicles the turbidity was lowest (68.0±3.40%), which showed the least stability. Higher incorporation of bile salt to 15mM again results in a slight decrease in turbidity; which could be due to the destabilizing property of detergent beyond critical concentration. The relevant justification can be given according to body concentration of bile salt. The stable formulations were subjected to visual assessment on the application of tazarotene is associated with skin irritation, which limits its applicability and acceptability by the patients. It is necessary to study the irritation caused by with and without tazarotene loaded PNG formulations to ensure that the topical preparations are safe and innocuous. The skin-irritation studies indicated that tazarotene loaded PNG

Table 7 Percent othrokeratosis and drug activity. Treatment group

Orthokeratosis (%)

Drug activity (%)

Non-treated PNG A4 PNG A2

22.0±2.5 76.60±3.1 78.22±2.8

81.32±4.3 87.77±6.6

All data are show as mean±SEM; n=5.

exhibited minimum to no irritation, as compared with plain PNG, even after 72 h of the application as showed in Table 6. The primary irritation index (PII) was found to be 0.00 for PNG, showing no irritation. Therefore, the developed tazarotene loaded PNG formulations resulted in no erythema and were safe, as compared with the plain PNG. Furthermore, in psoriasis, lymphocytes may change the growth of epidermis leading to keratinocytes proliferation and abnormal differentiation of apoptotic cells. The mouse tail model was used for studying the efficacy of the PNG formulations in vivo. In mouse skin, parakeratosis is seen 7–9 days after birth and after that scales start developing. The normal pattern of keratinization is called orthokeratosis and retention of nuclei and abnormal maturation of epidermal keratinocytes is called parakeratosis [45]. In this process, granular layer is also decreased. Fig. 7(a) indicates the histological appearance of untreated mouse tail skin after staining with hematoxylin and eosin, showing alternate scale and interscale epidermal regions. The granular layer was only apparent in the orthokeratosis interscale regions and not in the parakeratotic scale regions. The treatment was done for 4 weeks. Fig. 7(b) indicates the treatment with PNG formulations and the most common keratosis is orthokeratosis; in only one point nuclei are caught in the keratin (parakeratosis). Fig. 7(c) shows hyperplasia of epidermis with multiple layers of cells especially the basal cells. The top most layer of keratin shows parakeratosis. The epidermis is totally orthokeratosis and has continuous granular cell layer. The most common reasons for parakeratosis are increased in mitosis and decrease in transit time of differentiating keratinocytes at the time of cornification. The transverse section of rat tail skin was treated with PNG formulations for 4 weeks and the percentage of orthokeratosis calculated in drug activity is shown in Fig. 4(d) and Table 7. The control group showed orthokeratosis, that is 22% only in the notches or no scaly regions, while the scaly regions showed parakeratotic differentiation. Whereas, PNG A4 and PNG A2 showed 76.60% and 78.22% orthokeratosis, respectively (i.e., the presence of granular layer per scale). This shows the effectiveness of proniosomal gel therapy to induce normal differentiation pattern in the epidermis of the skin.

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4. Conclusion This invention presented the successful fabrication of tazarotene encapsulated PNG based on lipid bi-layers using coacervation phase separation technique. The prepared PNG having favorable vesicle size and good percent encapsulation efficiency. Further, Formulations based on S60 has been represented an excellent vesicle size as well as higher percent encapsulation efficiency as compared to other span based formulations. The morphological studies confirm spherical vesicles with sharp boundaries after hydration of proniosome gels. Moreover, in vitro permeation of tazarotene from PNG formulations of various ratios and the effect of various types of non-ionic surfactants have been studied and evaluated. The prepared PNG exhibited good stability at different temperature conditions. In conclusion, the experimental results and supportive theoretical analysis suggest that either fusion of the vesicles with the intercellular lipid of the stratum corneum and direct transfer of drug from vesicles to the skin and the penetration enhancement effect of the nonionic surfactants may contribute to the mechanism of drug permeation enhancement by proniosomal formulations. The prolonged release of the entrapped drug at the local site enhancing the residence time of the drug for a longer duration, less irritancy, and increase in the Orthokeratotic region observed by in vivo studies facilitated the advantage of tazarotene formulation as a PNG for the treatment of psoriasis. Conflict of interest statement The authors report no conflicts of interest. Acknowledgements The first author is thankful to Dr. Reddy's Laboratories Ltd., Hyderabad, India for providing Tazarotene as a gift sample for this study. We also thankful to Dr. Koti Reddy and Dr. Praveen Kumar from Aptus Biosciences Pvt. Ltd., Mahboobnagar, India for providing research facilities to carry out an evaluation of in vivo studies. Dr. Rajeswar Reddy, Chairman, Anurag Group of Institutions, Hyderabad, India for providing infrastructure and research facilities. References [1] M.A. Lowes, A.M. Bowcock, J.G. Krueger, Pathogenesis and therapy of psoriasis, Nature 445 (2007) 866–873. [2] Tsuruta, NF-B links keratinocytes and lymphocytes in the pathogenesis of psoriasis, Recent Patents Inflamm. Allergy Drug Discov. 3 (2009) 40–48. [3] K. Reich, K. Kruger, R. Mossner, M. Augustin, Epidemiology and clinical pattern of psoriatic arthritis in Germany: a prospective interdisciplinary epidemiological study of 1511 patients with plaque-type psoriasis, Br. J. Dermatol. 160 (2009) 1040–1047. [4] L. Naldi, Epidemiology of psoriasis, Curr. Drug Targets 3 (2004) 121–128. [5] D.J. Pearce, A.E. Morrison, K.B. Higgins, The co-morbid state of psoriasis patients in a university dermatology practice, J. Dermatol. Treat. 16 (2005) 319–323. [6] T. Esgleyes-Ribot, R.A. Chandraratna, D.A. Lew-Kaya, J. Sefton, M. Duvic, Response of psoriasis to a new topical retinoid, J. Am. Acad. Dermatol. 30 (1994) 581–590. [7] R.A.S. Chandraratna, Tazarotene: first of a new generation of receptor-selective retinoids, Br. J. Dermatol. 135 (1996) 18–25. [8] J. Sun, W. Dou, Y. Zhao, J. Hu, A comparison of the effects of topical treatment of calcipotriol, camptothecin, clobetasol and tazarotene on an imiquimod-induced psoriasis-like mouse model, Immunopharmacol. Immunotoxicol. 36 (2014) 17–24. [9] R. Schreier, J. Bouwstra, Liposomes and niosomes as topical drug carriers: dermal and transdermal drug delivery, J. Control. Release 30 (1994) 1–15. [10] B. Vora, A.J. Khopade, N.K. Jain, Proniosome based transdermal delivery of levonorgestrel for effective contraception, J. Control. Release 54 (1998) 149–165. [11] M. Manconi, Niosomes as a carrier for tretinoin III. A study into the in vitro cutaneous delivery of vesicle-incorporated Tretinoin, Int. J. Pharm. 311 (2006) 11–19. [12] H. Yoshida, Niosomes for oral delivery of peptide drugs, J. Control. Release 21 (1992) 145–154. [13] H.E.J. Hofland, Estradiol permeation from nonionic surfactant vesicles through human stratum corneum in vitro, Pharm. Res. 11 (1994) 659–664. [14] S. Mura, Liposomes and niosomes as potential carriers for dermal delivery of minoxidil, J. Drug Target. 15 (2007) 101–108. [15] A. Pardakhty, J. Varshosaz, A. Rouholamini, In vitro study of polyoxyethylene alkyl ether niosomes for delivery of insulin, Int. J. Pharm. 328 (2007) 130–141.

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