Effective topical delivery systems for corticosteroids: dermatological and histological evaluations.

http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–12 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.960981

RESEARCH ARTICLE

Effective topical delivery systems for corticosteroids: dermatological and histological evaluations

Drug Delivery Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.

_ Ipek Erog˘lu1, Erkan Azizog˘lu1, Mine _Ilgen Ertam4, Idil _ U¨nal4, and O¨zgen

O¨zyazıcı1, Merve Nenni2, Hande Gu¨rer Orhan2, Seda O¨zbal3, I¸sıl Tekmen3, O¨zer1

1 Department of Pharmaceutical Technology, 2Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey, 3Department of Histology and Embryology, Faculty of Medicine, Dokuz Eylul University, Inciralti, Izmir, Turkey, and 4Department of Dermatology, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey

Abstract

Keywords

Atopic dermatitis (AD) is a chronic and relapsing skin disease with severe eczematous lesions. Long-term topical corticosteroid treatment can induce skin atrophy, hypopigmentation and transepidermal water loss (TEWL) increase. A new treatment approach was needed to reduce the risk by dermal targeting. For this purpose, Betamethasone valerate (BMV)/Diflucortolone valerate (DFV)-loaded liposomes (220–350 nm) were prepared and incorporated into chitosan gel to obtain adequate viscosity (13 000 cps). Drugs were localized in stratum corneum + epidermis of rat skin in ex-vivo permeation studies. The toxicity was assessed on human fibroblast cells. In point of in-vivo studies, pharmacodynamic responses, treatment efficacy and skin irritation were evaluated and compared with previously prepared nanoparticles. Liposome/ nanoparticle in gel formulations produced higher paw edema inhibition in rats with respect to the commercial cream. Similar skin blanching effect with commercial creams was obtained via liposome in gels although they contain 10 times less drug. Dermatological scoring results, prognostic histological parameters and suppression of mast cell numbers showed higher treatment efficiency of liposome/nanoparticle in gel formulations in AD-induced rats. TEWL and erythema measurements confirmed these results. Overview of obtained results showed that liposomes might be an effective and safe carrier for corticosteroids in skin disease treatment.

Atopic dermatitis, corticosteroids, liposomes, nanoparticles, skin permeation

Introduction Atopic dermatitis (AD) is a complex, chronic and relapsing skin disease with severe itching and eczematous lesions. This cutaneous pathology requires intensive skin care and pharmacological therapy since it has a huge effect on patients’ quality of life. Corticosteroids are commonly used for the current treatment of AD and prolonged application of corticosteroids can cause dermal atrophy as a side effect (Pople & Singh, 2010; Lei et al., 2013). Novel drug delivery systems can provide more effective and targeted therapy strategies. Nanotechnology is frequently used for developing new delivery and targeting systems (Reis et al., 2013). Especially liposomes and nanoparticles are promising formulation alternatives for AD therapy (Fattal et al., 1991; Gautam et al., 2011). Liposomes are vesicular carrier systems, composed of phospholipid layers. These phospholipids have many advantages due to their similarity with skin lipids, like very low toxicity, biodegradability and strong tissue affinity (Liu & Hu, ¨ zgen O ¨ zer, Department of Pharmaceutical Address for correspondence: O Technology, Faculty of Pharmacy, Ege University, 35100, Bornova, Izmir-Turkey. Tel: +90 232 3113981. Fax: +90 232 3885258. E-mail: [email protected]

History Received 11 July 2014 Revised 28 August 2014 Accepted 29 August 2014

2007). Drugs can be encapsulated in the aqueous core and/or the membrane of liposomes, which are well tolerated by the skin and also have a strong affinity for the stratum corneum (Santos Maia et al., 2002). Higher cellular uptake has been reported when active pharmaceutical ingredient (API) is encapsulated in liposomes (Sezer et al., 2004). Topical liposomes may increase solubility of the drug and act as a local depot for sustained release of dermally active compounds. Therefore, they can be used as permeation enhancer across the skin in cosmetic and dermatologic fields (Choi & Maibach, 2005). Targeted drug delivery through the appendageal pathway (hair follicles and sweat ducts) can also be provided by liposomes (El Maghraby et al., 2008). Nanoparticles have great potential in topical drug delivery fields because of their increased surface area and ability to enhance the permeation of drugs into the skin (Tan et al., 2011). Polymeric nanoparticles are generally made of biocompatible and biodegradable polymers, which increase drug bioavailability, provide controlled drug release, target drug to specific tissues, reduce toxicity and side effects (Rosado et al., 2013). Lipid nanoparticles, especially solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were described in the 1990s, and since then lipid nanoparticles have drown a lot of interest. Lipid nanoparticles


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_ Erog˘lu et al. I.

for dermal applications include many advantages such as occlusion, drug targeting and modulation of drug release. Chemically unstable compounds that are sensitive to light, oxidation and hydrolysis can be protected in the form of SLNs (Gokce et al., 2012). Betamethasone 17-valerate (BMV) is used in the treatment of skin disorders, and has a moderate potency. BMV is a standard drug for the studies of developing new topical corticosteroid formulations. Diflucortolone valerate (DFV) is also commonly used in the treatment of skin disorders. DFV is a highly potent topical corticosteroid; however, in the classification of topical corticosteroids, potency is linked to side effects. Therefore, DFV as a high potent corticosteroid, has many side effects (Senyigit et al., 2012). BMV- and DFV-loaded lecithin/chitosan nanoparticles were developed in our previous work. In this study, liposome formulations were prepared and characterized as an alternative formulation. The anti-inflammatory, skin-blanching effect and TEWL measurements were studied as in-vivo. Cell culture studies were also performed to observe the cytotoxicity of formulations. Efficacy of treatment of the liposomes was compared with previously prepared nanoparticle formulations in terms of dermatological and histological evaluations in atopic dermatitis induced rats for the first time in literature.

Materials and methods Materials BMV and DFV were generous gifts from GlaxoSmith Kline (Turkey) and Roche (Turkey), respectively. Phosphatidylcholine from soybean (Lipoid S100) was supplied from Lipoid (Ludwigshafen, Germany). Phospholipon 90 G and cholesterol were purchased from Sigma-Aldrich (St Louis, MO). Medium MW chitosan (specifications: MW 190–310 kDa, deacetylation degree 75–85%, viscosity 200–800 cP for 1% w/v solution in 1% v/v acetic acid) was obtained from Sigma-Aldrich (St Louis, MO). Polyethylene glycol 400 (PEG-400) was provided by Merck KGaA (Darmstadt, Germany). The commercial products of BMV (BetnovateÕ cream, Brentford, UK) and DFV (TemetexÕ cream, Istanbul, Turkey) were used as control formulations. All other chemicals used were of analytical grade. Preparation of formulations Liposomes were prepared by thin-film hydration method in which Lipoid S100 and Phospholipon 90 G were used as lipid components. Briefly, phospholipid (50 mg), cholesterol (6.25 mg) and APIs (0.1% BMV or DFV) were dissolved in chloroform (12.5 mL) in a round-bottom flask. Then the organic solvent was evaporated using a rotary evaporator (RV 10 digital, IKA Werke GmbH&Co., Staufen, Germany) in a 40 C water bath (HB 10 digital, IKA Werke GmbH&Co., Staufen, Germany) under vacuum. The dried thin lipid film was kept under reduced pressure for 2 h to remove traces of the solvent. The lipid film was then hydrated with 25 mL bidistilled water for 30 min at 100 rpm to obtain a crude dispersion of the liposomes without using any vacuum. The liposomal suspension was sonicated (Bandelin

Drug Deliv, Early Online: 1–12

Sonopuls HD 2070, Berlin, Germany) for 15 min and 120 s/ 2 cycle at 50% of amplitude (Choi & Maibach, 2005). As a control group, liposomes without APIs were also prepared with the same method. The lecithin/chitosan nanoparticles were prepared according to the method published in our previous article (Ozcan et al., 2013a, b) via direct injection of soybean lecithin alcoholic solution into aqueous chitosan solution. Drug-loaded liposomes and nanoparticles were incorporated (10% w/w) in chitosan gel (2% chitosan in 1.5% w/v acetic acid solution) for further ex-vivo and in-vivo studies (Ozcan et al., 2013a, b). For comparison studies, chitosan gel containing API was prepared. The drug was dissolved at a concentration of 10% (w/w) in PEG-400 and then added into the chitosan gel. The final concentration of BMV/DFV was 0.1% as in commercial products (w/w). Characterization of formulations Liposomes and nanoparticles were characterized in terms of particle size (PS) and polydispersity index (PI) at 25 C by dynamic light scattering method using a Malvern NanoZS (Malvern Instruments Ltd, Worcestershire, UK). The surface charge of the formulations was determined by zeta potential (ZP) measurements using the Malvern NanoZS. The measurements were conducted in six replicates. Transmission Electron Microscope (TEM Libra 120, Carl Zeiss, Oberkochen, Germany) was used for morphological analysis of vesicles. The sample was stained with 2% w/v uranyl acetate solution for 20 s, immobilized on a copper grid coated with FormvarÕ (AGAR Scientific, Stansted, England) and dried for imaging by TEM. Encapsulation efficiency (EE%) of liposomes (48 000 g, 4 C, 30 min) and nanoparticles (3000 g, 4 C, 10 min) were determined by centrifugation method. The nanoparticle suspension was also ultra-centrifuged at 150 000 g for 2 h to separate the free drug from encapsulated drug. Amount of free drug separated from formulations was analyzed by previously validated HPLC technique. The HPLC system (Agilent Series 1100, Waldbronn, Germany) consisted of a C18 reverse phase column (ACE 5-C18 250 mm 4.6 mm). The mobile phase was a mixture of acetonitrile:water (55:45/v:v) with the flow rate of 1 mL/min. The wavelength of UV detector was set at 240 nm. The stability of the formulations was investigated in capped glass vial at 25 C and 60% relative humidity for 3 months. Samples were further evaluated in terms of physicochemical properties (PS, PI, ZP and EE). Drug-loaded gel formulations including liposome or nanoparticle were also evaluated in terms of the possible changes in pH (pH-meter, Mod pHI 71, Beckman, Brea, CA) and the viscosity (rotational viscometer, DV III, Brookfield, WI), for their stability evaluation. Skin permeation experiments The ex-vivo permeation experiments for API-loaded liposome, liposome in gel, chitosan gel and commercial cream formulations across rat skin were performed using the validated Franz diffusion cells and equipment. A mixture


DOI: 10.3109/10717544.2014.960981

of ethyl alcohol:phosphate buffer saline (PBS) at pH 7.2 in the ratio of 3:7 (v/v) was used as receptor compartment. The available diffusion area between donor and receptor compartments was 0.63 cm2. Infinite dose regimen was applied in all experiments. 300 mL samples were withdrawn from the receptor compartment at specified time intervals and immediately replaced with the same volume of fresh solution to maintain sink conditions. Permeation rates were calculated by taking samples from the receptor phase at various time points. At the end of the 6-h permeation experiments, the excess amount of the formulation was removed and the skin surface was cleaned with dry paper and isopropyl alcohol. Stratum corneum + epidermis was separated from dermis with heat application. All samples were weighed and inserted in vials. The drug was extracted from the two skin layers under sonication for 15 min using 1 ml acetonitrile:water (60:40) mixture. The amount of drug in the permeation medium and accumulated in skin layer sample was determined by the HPLC method. Skin concentration of drug was calculated by normalizing the amount of drug recovered in the SC + epidermis and dermis by the weight of tissues and expressed as mg of drug per mg of tissue. All experiments were done in six replicates. The enhancement ratio was also calculated, to demonstrate the accumulation capacity of formulations for drug, by dividing the amount accumulated with the test formulation (Q-test) to the amount accumulated with the commercial cream (Q-cream) (Ozcan et al., 2013a, b). Cell culture studies For cell attachment, human dermal fibroblast cells (Invitrogen C-013-5C) were cultured in 96-well plates at a density of 5 103cells/cm2 and incubated at 37 C in a humid atmosphere containing 5% CO2 for 24 h. The cells were treated with varying concentrations of the nanoparticles and liposome formulations (0.07–2 mM final API concentration in the media) for 24 h. Control (medium only) and positive control (1.5 mM Triton X-100) were included in every experiment. After completion of exposure period the medium was removed, cells were washed with PBS and then incubated with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at a final concentration of 0.5 mg/ml for 4 h at 37 C. The medium was then removed and the formazan crystals were dissolved in 100 ml of dimethyl sulfoxide. The absorbance was recorded at 550 nm on a microplate reader. Each experiment was repeated on four separate days. The % ratio between the absorbance of treated samples and the absorbance of control was expressed as % cell viability (Rosado et al., 2013). In-vivo studies Male Albino Wistar rats were treated during in-vivo studies according to the experimental protocol approved by the Local Animal Ethical Committee of Dokuz Eylul University (Approval No. 12/2009). The dorsal hair was removed 24 h before the experiment. Similar experiments (anti-inflammatory activity, skin blanching effect and TEWL

Corticosteroid-loaded liposomes for topical delivery

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measurements) in the previous study, were performed to compare the effect of different formulations (Ozcan et al., 2013a, b). Treatment and skin irritation properties of formulations were examined for the first time in this study. All studies were done in six replicates. Pharmacodynamic efficacy of formulations Carrageenan-induced rat paw edema model were used for evaluation of the anti-inflammatory activity of formulations. 100 mL of a 1% (w/v) -carrageenan (Sigma-Aldrich) solution in saline was injected into the plantar surface of the left hind paw of the rats. Formulations were applied to the plantar surface (0.125 mg/cm2) of the hind paw of the rats by gentle rubbing using the index finger for each treatment, 1 h before induction of inflammation. The increase in paw volume (edema) was measured using digital water plethysmometer, Taipei, Taiwan (PV-01; SINGA Corporation, Taipei, Taiwan) at 0, 1, 2, 3, 4 and 5 h after carrageenan administration. Skin blanching studies were assessed by Chroma Meter (CR-400; Konica Minolta, Ramsey, NJ) to evaluate vasoconstrictor effect of corticosteroids at different time intervals before and after application of the formulations. Treatment efficacy of formulations Animals were divided into three groups to evaluate the treatment efficacy of formulations as control group (healthy), untreated atopic control group and treated group. For induction of atopic dermatitis, 150 mL of 0.5% 2.4-dinitrofluorobenzene (DNFB) in acetone/olive oil (3:1) was repeatedly applied on the shaved skin of the dorsal area two times a week for 4 weeks (Wu et al., 2011). After the induction of atopic dermatitis, formulations (liposome in gel, nanoparticle in gel, commercial cream) were topically applied on the skin lesion two times a day during 12 days. On the last day of the experiment, the uniformity of the skin barrier and severity of the skin lesions were measured and scored. Animals were sacrificed under anesthesia (urethane 1 g/kg and alfa-chloralose 50 mg/kg) and the skin sample of 0.5 1.0 cm2 in size was taken from the lesion. At the end of treatment period, the skin barrier recovery in anesthetized rats was examined by Tewameter (TM 300, Courage and Khazaka, Germany) equipped with a TEWL probe. Mexameter (MPA 5, Courage – Khazaka, Germany) measurements were also studied to observe the recovery in terms of erythema. Intensity of AD was determined according to the SCORAD scale (Scoring Atopic Dermatitis), based on the following criteria: 0 points for showing no symptoms, 1 point for mild indication of symptoms (mild), 2 points for moderate symptoms (moderate), and 3 points for severe symptoms (severe). The five symptoms considered in scoring were edema, oozing/crusting, excoriation, erythema and lichenification (Jung et al., 2011). Skin lesions were investigated histologically and for this purpose, initially rat skin sample was fixed with 10% buffered formaldehyde solution and than embedded in paraffin. Sections of the skin at 5 mm thickness were stained with either hematoxylin–eosin to observe the pathological changes

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or toluidine blue to see mast cells infiltrating the lesion. The number of mast cells was counted in five randomly chosen sites. The slides were examined using a light microscopy (Olympus BH-2, Tokyo, Japan) and the histological appearances of tissues in the different groups were compared. Skin irritation studies


For the skin irritation studies, healthy animals were divided into two groups as (i) control group and (ii) formulation applied group. Anesthetized rats were shaved and after 24 h, formulations were topically applied on dorsal area, twice a day for 12 days. TEWL and erythema were evaluated by Tewameter and Mexameter, respectively. Possible structure changes of skin sections and cell filtrations were also evaluated histologically. Statistical analysis Statistical data analyses were performed on all data using a one-way ANOVA followed by a multi-parametric Tukey’s post hoc test with p50.05 set as the minimal level of significance.

Results and discussion Preparation and characterization of formulations A wide variety of lipids can be used for preparation of liposomes. Several studies have demonstrated that the vesicle composition and hence its physicochemical properties can have a significant effect on drug permeation (HoneywellNguyen & Bouwstra, 2005). Phosphatidylcholine from soybean or egg yolk is the most common composition although many other potential ingredients have been evaluated (Benson, 2005). Phospholipon 90 G and Lipoid S100 were used for preparation of liposomes. API-loaded liposomes and nanoparticles were characterized in terms of PS, PI, ZP and EE% as shown in Tables1 and 2, respectively. Influence of the composition of the formulations was assessed on the liposome characteristics. The PS of liposomes prepared with Lipoid S100 was found higher than liposomes prepared with Phospholipon 90 G for both drugs. It could be attributed to the phosphatidylcholine amount in the lecithin fraction (Upadhyay et al., 2012). The results of the surface characterization showed that introduction of the negatively charged phospholipid resulted in higher negative zeta potential values as expected (Naderkhani et al., 2014). On the contrary, nanoparticles with high positive surface charge were attributed to the presence of chitosan chains on the particle surface.

The EE% of formulations was found within the range 39.5–58.8%. Unsaturated phospholipids are relatively more flexible in nature and thereby provide less hindrance for drug to be retained in lipid bilayer. The EE of nanoparticles was found higher than the EE of liposomes. It was considered that BMV or DFV, due to their lipophilic structure, was located in lipid core of lecithin/chitosan nanoparticles whose lipid composition has been modified by the presence of isopropyl myristate (Ozcan et al., 2013b). Cholesterol added to the composition tends to stabilize the structure thereby generating rigid liposomes. Cholesterol localizes to the hydrophobic chain of the phospholipids in the liposome bilayer. Therefore, the addition of cholesterol to liposome bilayers prevents a lipid exchange, and has an additional stabilizing effect. Similarly, Shivhare et al. (2009) showed that, the stability and rigidity of liposomes are linked to the concentration of cholesterol. Our experiments revealed that Phospholipon 90 G liposomes showed good stability under test conditions (25 C and 60% relative humidity) for 3 months. In spite of addition of cholesterol as a stabilizer drug-loaded Lipoid S100 liposomes were less stable. Due to obtained results, Phospholipon 90 G liposomes were used for further studies. The TEM images of drug-loaded liposomes prepared with Phospholipon 90 G are presented in Figure 1. The image showed that the liposomes were spherical, which ranged in size from 200 to 300 nm. Liposomes and nanoparticles have a low viscosity due to their preparation techniques and that makes them unable to stay on the application area. Thus, they were incorporated into the chitosan gel as an adequate vehicle, to obtain semi-solid consistency while preserving their original structure (Pavelic et al., 2005). Nanocarrier in chitosan gel system (10%) showed adequate viscosity (13 000 cps) and spreadability for topical application as shown in our previous study (Ozcan et al., 2013b). In the current study, viscosity values were measured as 13 156–13 200 cps for BMV- and DFV-loaded liposomes or nanopartcile containing gels, at the end of 3 months. The pH values of prepared formulations were also found suitable for topical application (between 4.5 and 5.5). Skin permeation experiments Liposomes enhance the penetration of drugs through the stratum corneum and localize them in the epidermis and dermis. They can penetrate stratum corneum lipid lamellae and fuse with endogenous lipids hence the systemic absorption and side effects can be reduced (Fresta & Puglisi, 1997; Benson, 2005; Liu & Hu, 2007; Zhang et al., 2011). In order to prove that, the ex-vivo permeation experiments were performed using Franz diffusion cells across rat abdominal

Table 1. Physicochemical properties of liposomes.

Lipid

Drug

Particle size (PS) (nm)

Zeta potential (ZP) (mV)

Polydispersity index (PI)

Encapsulation efficiency (EE) (%)

Phospholipon 90 G

BMV DFV BMV DFV

223.7 ± 1.4 331.9 ± 5.7 764.6 ± 10.7 455.8 ± 3.1

27.4 ± 0.5 29.4 ± 0.6 24.5 ± 0.6 28.9 ± 0.8

0.485 ± 0.02 0.543 ± 0.03 0.547 ± 0.02 0.479 ± 0.09

46.3 ± 0.5 39.5 ± 1.2 53.4 ± 0.4 58.8 ± 2.2

Lipoid S100


DOI: 10.3109/10717544.2014.960981

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Table 2. Physicochemical properties of nanoparticles.

Drug

Particle size (PS) (nm)

Zeta potential (ZP) (mV)

Polydispersity index (PI)

Encapsulation efficiency (EE) (%)

BMV DFV

274.6 ± 14 204.2 ± 10.1

40.8 ± 2.80 41.4 ± 1.60

0.241 ± 0.02 0.149 ± 0.02

88.12 ± 0.5 86.8 ± 0.3

Figure 1. TEM images of (A) BMV-loaded; (B) DFV-loaded liposomes.

skin for liposomes, liposome in gels, API in gel and commercial cream. At the end of 6 h, drug was never determined in the receptor compartment. This indicated that there is no permeation rates and transdermal transport through the skin are provided. The enhancement ratio of the liposomes versus commercial cream was calculated to demonstrate the accumulating capacity. Liposomes showed 2.68- and 3.22-fold higher retention when compared with commercial cream for BMV and DFV in stratum corneum + epidermis, respectively (Figures 2 and 3). It could be attributed to the similar skin lipid structure and unique characteristics of the prepared liposomes as well as their large surface area. Correspondingly, Jung et al. showed that, drug-loaded liposomes enhanced skin permeability about 17-fold compared with carbopol gel formulations. van Kuijk-Meuwissen et al. (1998) assessed the localization of fluorescent-labelled liposomes in skin layers. Confocal laser scanning microscopy results suggested that fluorescence intensity increased with these formulations. Egbaria et al. (1990) evaluated liposomes with respect to skin delivery for localizing effect. The deposition of cyclosporin-A into the stratum corneum of hairless mice was increased, whilst transdermal absorption reduced, from liposomal formulations compared with drug solutions and oil/water emulsion. Liposomal formulation developed by Mura et al. (2007) localized benzocaine in skin for improving topical anesthetic effectiveness. This series of investigations emphasized the localizing effects of liposomes. The release can be extended and the side effects can be reduced with liposomes. Lipid-based vesicular and particular systems induced

Figure 2. Amount of BMV accumulated from formulations in the stratum corneum + epidermis (dark bars) and dermis (light bars). *Significantly different from commercial cream and chitosan gel (p 5 0.05).

epidermal targeting, which may increase the benefit/risk ratio of topical therapy with steroids (Santos Maia et al., 2002). The lipid-based vesicles have high interaction with human skin but they have low stability and viscosity for topical usage (Sinico et al., 2005). Usually, liposomes are applied to the skin in hydrogels, since stable liposomal creams are difficult to formulate. For topical application of liposomes, hydrophilic polymers are suitable thickening agents, since they make the formulations convenient for application (Gabrijelcˇicˇ & Sˇentjurc, 1995). In our experiments, although 10 times less drug was incorporated in liposome-in-gel formulation, the similar dermal accumulation have been observed with chitosan gel and commercial cream (Figures 2 and 3). In addition to dermal targeting advantages of liposomes, the

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optimum viscosity of chitosan gel showed synergistic effect. Finally, the extended retention time and higher accumulation of drug were observed via liposome in gel formulations. As shown in our previous study, the nanoparticle in gel formulations showed 2.26- and 1.75-fold higher retention compared to commercial cream for BMV and DFV in stratum corneum + epidermis, respectively, even though they contain 1/10 of drug with respect to commercial formulation.


Cell culture studies The MTT assay relies on the ability of live but not dead cells to reduce a water-soluble yellow dye, MTT, to water-insoluble purple formazan crystals. Since the substrate and product absorb at very different wavelengths, no washing step is required which is a clear advantage of this assay and makes it a useful tool for drug screening studies (Rosado et al., 2013). As can be seen in Figure 4, all formulations were found to be cytotoxic to human fibroblast cells at their highest concentrations tested in the present study (2 mM). However,


this can be attributed to the decreased amount of cell culture medium instead of the cytotoxicity of biologically active molecules, BMV or DFV. In order to test the 2 mM active pharmaceutical ingredients, stock formulation was added at 10% (v/v) to the medium ending with less nutritious medium ingredients which might affect the viability of cells. BMV and DFV nanoparticles and liposome formulations were found to have highest proliferative potential at their 0.2 mM concentrations. This proliferative effect of all formulations was found to be statistically significant. Comparing the two formulations of BMV and DFV, liposome formulations appeared to have higher proliferative effect then their nanoparticles (Bruge` et al., 2013). In-vivo studies In-vitro results suggest that liposomes are effective candidates for AD treatment due to their retention ability in the skin layers. In our previous work, similar in-vitro results were obtained with lecithin/chitosan nanoparticles in gel formulations (Ozcan et al., 2013b). Pharmacodynamic and treatment efficacies of formulations were investigated with male albino Wistar rats as in-vivo studies. Skin irritation efficacy was also assessed histologically. Pharmacodynamic efficacy of formulations

Figure 3. Amount of DFV accumulated from formulations in the stratum corneum + epidermis (dark bars) and dermis (light bars). *Significantly different from commercial cream and chitosan gel (p 50.05).

Figure 4. Potential effect of nanoparticles and liposomes on viability of human dermal fibroblast cells using MTT assay. Concentrations are the final concentration of active pharmaceutical ingredient in the incubation media. Bars represent ‘‘mean ± SD’’ from four seperate studies (n ¼ 4). The numbers present on top of the bars are % cell viability compared to the control values. *p50.05; **p50.005 compared to the control values.

Corticosteroids suppress virtually every component of the inflammatory process, such as synthesis of cytokines, production and activity of leukocytes and cell-mediated immunity (Cronstein et al., 1992). There are various methods for testing inflammation like, UV radiation-induced erythema, vascular permeability, oxazolone or croton-oil induced ear edema, formalin or carrageenan paw edema and granuloma pouch technique. The most commonly used and rapid one is carrageenan-induced paw edema method (Schleyerbach et al., 2002). In this study, liposome in gel and commercial



DOI: 10.3109/10717544.2014.960981

cream formulations were applied by gentle rubbing 1 h before carrageenan injection. The increase in hind paw volume was measured using digital water plethysmometer. API-loaded liposome in gel produced an inhibitory effect on paw edema especially after 4th hour. Pharmacodynamic experimental data demonstrated that the anti-inflammatory activity of API-loaded liposome in gel was found higher than commercial cream, even though gel formulation contained 10 times less drug (p40.05) (Figure 5). It could be attributed that, liposomes in gel formulation enhanced the API permeation into the skin, while decreasing the clearance of drug by minimizing its absorption into the systemic circulation. The small particle size of liposome ensures close contact to the stratum corneum, thus encapsulated drug permeation into the viable skin can be increased. In addition, chitosan gel obtained an adequate viscosity, increased drug retention time in skin layers, which also improved anti-inflammatory activity. The application of corticosteroids to the skin, induces skin blanching effect due to their vasoconstrictive activities on vascular smooth muscle. The degree of skin blanching is linked to the skin concentrations of steroids. In this study, Chroma Meter, which provides accurate measurement of color, has been used for determining the skin blanching effect of liposome/nanoparticle in gel and commercial formulations. The Chroma Meter uses the L* a* b* color system [white (100) black (0), L*; green (60) red (+60), a*; blue (60) yellow (+60), b*]. In this study, only a* was used as an index of skin blanching. The Da*sample was determined from the difference between the skin surface color before (a*sample pre) and after (a*sample, post) the formulation application (Equation 1) (Ishii et al., 2012). Dasample ¼ asample, post asample, pre

ð1Þ

According to the Chroma Meter data, the skin blanching effects of formulations on rats are produced by vasoconstriction in the skin. The Da*4h value was found as + 0.65 for liposome in gel formulations although including 10 times less

Figure 5. Changes in the paw thickness of the rats with respect to time after formulation application.

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drug from marketed product (Da*4h: 1.82). However, the Da*16 h values for the gel and commercial product was found as 2.77 and 3.39, respectively. Data also obtained from nanoparticle in gel formulations were also found similar (Da*16 h: 3.14). At the end of 16 h of experiment, it can be concluded that liposome and nanoparticle in gel formulations both maintained sustained release of drug. The values were found closely related to commercial cream even it contains 10 times less API. The ex-vivo permeation results of current study and in our previous study with nanoparticle in gel formulations showed a better accumulation in stratum corneum + epidermis layer, which is fitted to the results of those produced by Chroma Meter measurements (Ozcan et al., 2013b). A good correlation was also reported between the steroidal drug concentration in skin and the degree of skin blanching (Ishii et al., 2012). Treatment efficacy of formulations Atopic dermatitis model was successfully induced by DNFB, which induces an immune response and elicits an allergic reaction including pruritus as assessed by scratching (Nojima & Carstens, 2003). Treatment efficacy of formulations was evaluated in terms of uniformity of skin barrier function, visual scoring and histological parameters. TEWL gives valuable information on the maintenance of skin barrier function. A lack of barrier substance is indicated by a reduction in skin hydration due to humidity loss and an increase in TEWL (Kircik, 2012). AD is defined by dysfunction of the stratum corneum that results in excessive TEWL and the infiltration of allergens into the skin (Kang et al., 2010). In atopic control group, TEWL values increased from 8 to 11 g/h/m2, while the skin barrier of the treated group with liposomes in gel (8 g/h/m2–6 g/h/m2) and nanoparticle in gel (8 g/h/m2–7 g/h/m2) were uniform at the end of the examinations. Thus, the recovery of the skin barrier was accomplished by both liposome and nanoparticle in gel formulations.


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Figure 6. The morphological changes of different groups in rat dorsal skin. (A) healthy control, (B) atopy control, (C) BMV-loaded commercial cream, (D) BMV-loaded liposome in gel, (E) BMV-loaded nanoparticle in gel, (F) DFV-loaded commercial cream, (G) DFV-loaded liposome in gel, (H) DFV-loaded nanoparticle in gel.

Mexameter results were in accordance with TEWL results. The erythema value of healthy control was found as 130 g/h/m2. Erythema values increased after DNFB application (244 g/h/m2). At the end of the treatment period, the erythema values of the liposome in gel (133 g/h/m2) and nanoparticle in gel (156 g/h/m2) applied groups were decreased. In many studies, visual examination of skin lesions by a dermatologist gives reliable results about recovery process (Jung et al., 2011). In the SCORAD scale, higher scores indicate more symptoms. In this model, the liposome and nanoparticle in gel-treated groups showed lower dorsal skin scoring (average 0.6) compared to commercial cream-treated group (average 2.1). Erythema, edema, scabs and scratching behaviors were observed in the atopy control rats stimulated with DNFB (Figure 6B). As shown in Figure 6 (D, E, G and H), after administration of gel formulations including liposome or nanoparticle formulations, damaged skin started to get better and scab grew. Then scabs exuviated and the healing skin exposed. New hair began to grow at most of damaged skin surface. Recovery and new hair growth did not

yet complete during the same period for commercial cream (Figure 6C and F). Liposomes or nanoparticles incorporated in chitosan gel displayed more effective treatment than commercial cream because of their high retention times in epidermis and dermis. The results were in good accordance with ex-vivo permeation findings. Skin lesions were also investigated histologically to support the scoring data. Sections of the skin were examined in terms of pathological changes and mast cells infiltrating. Histological images were evaluated regarding four prognostic histological parameters: (i) vasodilatation, (ii) edema and non-uniform appearance in epithelial cell, (iii) edema in papillary dermis, (iv) lymphocyte infiltration. All parameters were observed markedly in the atopy control group at the end of the 4 weeks long application of DNFB. On the contrary, all parameters were disappeared in developed liposome/nanoparticle in gel formulations, vasodilatation and lymphocyte infiltration have been existed in commercial cream-treated group skin lesions (Figure 7). Mast cells are believed to be involved in the pathogenesis of AD. The role of mast cells in AD is suggested by an


DOI: 10.3109/10717544.2014.960981


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Figure 7. The histological changes of different groups in rat dorsal skin. (A) healthy control, (B) atopy control, (C) BMV-loaded commercial cream, (D) BMV-loaded liposome in gel, (E) BMV-loaded nanoparticle in gel, (F) DFV-loaded commercial cream, (G) DFV-loaded liposome in gel, (H) DFV-loaded nanoparticle in gel.

increase in the mast cell number (Kawakami et al., 2009). Therapeutic agents for AD can affect mast cells number. Therefore, the number of infiltrating mast cells in the tissue sections of the skin lesion was counted in five sites chosen randomly. API-loaded liposome/nanoparticle in gel formulations suppressed the numbers of mast cells infiltrating in the skin lesions of the AD rat by up to 50% (Figure 8). Skin irritation studies Factors that contribute to skin irritation include changes in the physiological pH of the skin, disruption of the stratum corneum barrier, physiological reactions and pharmacological features of the drug or vehicle. Many features of topical systems contribute to skin irritation including the API, formulation (including excipients and skin permeation enhancers) and occlusion of the skin (Paudel et al., 2010). The type of formulation used for drug delivery can influence the degree of skin irritation. For instance, it has been reported that gel formulations reduce skin irritation by absorbing moisture from the skin’s surface and also controlled-release systems like liposomes might be useful in decreasing irritation induced by

topically applied drugs. The mechanisms for reduction of skin irritation by liposomes include hydration of the epidermis and the sustained release of drugs, hence avoiding the buildup of toxic drug concentrations in the skin (de Leeuw et al., 2009). In this study, it can be possible that using a prepared liposome in gel formulation may decrease the skin irritation potential of the formulation applied to the skin’s surface. For skin irritation studies, observed adverse effects were swelling of the stratum corneum and disruption of the skin barrier function, as indicated by an increase in TEWL. Alteration of skin hydration can be correlated with uniformity of skin and the barrier function. In many studies, TEWL measuring has been found to be a very useful technique for studying skin irritation induced by various physical and chemical effects. Exposure of the skin to chemicals and physical conditions generally results in an increase of TEWL. The possibility of irritation effect of corticosteroids and excipients in formulations was evaluated with Tewameter. In this study, after TEWL measurements, it was recorded that there was no significant difference between basal and post-application values (p40.05). As shown in Figure 9, although an increment was observed after 2 h in liposome in


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Figure 8. Images of infiltrated mast cells from tissue sections. (A) healthy control, (B) atopy control, (C) BMV-loaded commercial cream, (D) BMVloaded liposome in gel, (E) BMV-loaded nanoparticle in gel, (F) DFV-loaded commercial cream, (G) DFV-loaded liposome in gel, (H) DFV-loaded nanoparticle in gel.

Figure 9. TEWL value changes of the groups with respect to time.

gel groups, it was not found as statistically significant with respect to the zero time point values (p40.05). Skin erythema values measured by Mexameter, before and after application of the formulations were compared at the same time intervals. Erythema values of the formulationapplied group were analogous to the control group. Histopathologic patterns associated with external agents give information about morphological changes, such as epidermal liquefaction, edema of collagen fibers and cell infiltration (Requena et al., 2012). Histological analysis of the rat skin did not reveal morphological tissue changes

after application of the commercial creams or gels, since the structure of the stratum corneum, epidermis and dermis were preserved. Identically histological safety studies realized by Pople et al., the entrapment of tacrolimus in lipid nanoparticles could avoid direct contact of the drug with skin. So, this phenomenon can reduce skin irritation alleviating the drug-related local side effects. Skin irritation study in rabbit showed noticeable signs of irritation with reference ointment (Pople & Singh, 2010). Briefly, TEWL, erythema and histological evaluations bring to light, liposome and nanoparticle in chitosan gel

DOI: 10.3109/10717544.2014.960981

formulations have favorable dermal safety profile without any irritant effect.


Conclusion Nanoparticles made from lecithin and chitosan were previously shown to enhance dermal targeting or improve tolerability of active substances. In this study, BMV- and DFVloaded liposomes were prepared and compared with developed nanoparticles and also commercial creams. APIs were localized and accumulated in the desired skin strata for both liposomes and nanoparticles. Skin permeation studies and skin blanching were showed that drug release was also maintained. Higher inhibition of edema and erythema in ADinduced rat model showed good agreement with dermal scoring and histological results. It is a remarkable point that recovery of skin was achieved with only 10% of API used in commercial creams. Thereby, minimized possible side effects and decreased dosing intervals can be provided with liposome in gel formulations. Last but not the least, cell culture studies, TEWL measurements and histological observations confirmed that liposome/nanoparticle in gel formulations were safe to use and could be valuable alternatives for topical treatment of skin diseases. As a conclusion, liposome formulations were regarded as effective and promising delivery systems for treatment of AD with corticosteroids.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by The Scientific and _ ¨ BITAK Technological Research Council of Turkey-TU (Project number: 109S433).

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