Artificial Cells, Nanomedicine, and Biotechnology An International Journal
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Flexosomes for transdermal delivery of meloxicam: characterization and antiinflammatory activity Abdullah H. Alomrani & Mohamed M. Badran To cite this article: Abdullah H. Alomrani & Mohamed M. Badran (2016): Flexosomes for transdermal delivery of meloxicam: characterization and antiinflammatory activity, Artificial Cells, Nanomedicine, and Biotechnology, DOI: 10.3109/21691401.2016.1147452 To link to this article: http://dx.doi.org/10.3109/21691401.2016.1147452
Published online: 28 Feb 2016.
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Date: 02 March 2016, At: 11:04
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016 http://dx.doi.org/10.3109/21691401.2016.1147452
Flexosomes for transdermal delivery of meloxicam: characterization and antiinflammatory activity Abdullah H. Alomrania,b and Mohamed M. Badrana,c Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; bNanomedicine Unit (NMU-KSU), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; cDepartment of Pharmaceutics, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
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a
ABSTRACT
ARTICLE HISTORY
The study aims to investigate the impact of flexosomes (FLs) on transdermal delivery of meloxicam (MLX). FLs are composed of phospholipid, Tween 80, and ethanol which were prepared by film hydration method. The prepared FLs were characterized for particle size, zeta potential, and entrapment efficiency (EE). Ex vivo skin penetration studies were perfomed, and the best formulation was further evaluated using in vivo antiinflammatory activity test. FLs were in nano-size scale carring negative charge and observed high EE% and enhanced skin penetration of MLX compared to conventional liposomes (CLs). The best formula was FL4 which was composedof phospholipid (10%), Tween 80 (1.5%), and ethanol (40%). FL4 showed 143.4 nm vesicle size, 84% EE, and 31-fold ex vivo permeation enhancement through skin compared to CLs. The antiinflammatory activity of FL4 gel showed significant increase compared to control. This study observed the effectiveness of using FLs as carriers for transdermal delivery of MLX.
Received 27 October 2015 Revised 4 January 2016 Accepted 25 January 2016 Published online 24 February 2016
Introduction Meloxicam (MLX) is an effective nonsteroidal antiinflammatory drug (NSAID), with high affinity to inhibit cyclooxygenase-2 (COX-2). MLX was approved by FDA in 2000 (MOBICÕ , Boehringer Ingelheim) for arthritis, osteoarthritis, and degenerative joint disease (Fleischmann et al. 2000; FDA 2000). MLX is available only for oral administration. There is a controversy in the GIT toxicity of MLX. Some authors revealed that the antiinflammatory and analgesic effect of MLX were equivalent to diclofenac, piroxicam, and naproxen (Fleischmann et al. 2000). A number of side effects related to GIT were reported for MLX (Gambero et al. 2005). Accordingly, alternative route of administration, such as transdermal route may help to improve compliance and avoid side effects in subjects who experience these undesirable effects. Considering the fact that most inflammatory diseases occur locally and near the surface of the body, topical application of NSAIDs on the inflamed site can offer the advantage of delivering a drug directly to the site of action producing the desirable effect and avoiding unwanted adverse effects associated with oral route (Prausnitz 2004, Thomas and Finnin 2004). However, some compounds cannot achieve these goals effectively and efficiently, due to their physico-chemical parameters (Southwell and Barry 1983). The major limitation of MLX is the low aqueous solubility, which significantly varies with the pH value of the media (Luger et al. 1996). Its solubility was reported to be 0.012 mg/mL in water, 0.086 10 2 mg/mL in 0.1 M of HCl (Duangjit et al. 2014). MLX has a partition coefficient logP 1.91 and 0.07 at pH 5.0 and 7.4, CONTACT Mohamed M. Badran [email protected] P.O. Box 2457, Riyadh 11451, Saudi Arabia ß 2016 Taylor & Francis
KEYWORDS
Antiinflammatory studies; flexosomes; meloxicam; skin penetration
respectively (Duangjit et al. 2014, Luger et al. 1996). Its molecular weight is 351,4 Da and it has two pKa values: 1.09 and 4.18 (Luger et al. 1996). The pharmacokinetic profile of MLX is characterized by a prolonged and complete absorption with 20-hour elimination half-life. MLX is highly bound to plasma protein (99.5%; Tu¨rck et al. 1996). Because the skin is the route of drug administration for transdermal drug delivery systems, it is important to appreciate skin physiology. The skin acts as an excellent biological barrier which is attributed to the stratum corneum (SC) layer. SC is formed of different layers of corneocytes which are surrounded by the intercellular lipid lamellae (Bouwstra and HoneywellNguyen 2002). This layer hinders permeation of drugs and other substances through the skin (Cevc and Vierl 2010, Menon 2002). Alternative strategies were investigated to overcome the barrier nature of SC include physical (e.g. sonophoresis and electoporation), chemical (e.g. chemical permeation enhancers), vesicular systems (e.g. Liposomes and niosomes), and mechanical (e.g. micro-needles) methods (Barry 2001). Among the applied strategies, vesicular drug delivery systems showed potential and safe approaches for increasing skin penetration of drugs. Despite of their potentials, conventional liposomes (CLs) offer little value as carriers for transdermal drug delivery because they cannot penetrate the skin deep enough. It has been reported that the penetration enhancing of the vesicles through the skin depends on their membrane flexibility. It was shown that liposomes with flexible membrane (i.e. flexosomes;
Department of Pharmaceutics, College of Pharmacy, King Saud University, Building # 23, Office # AA 68
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A. H. ALOMRANI AND M. M. BADRAN
FLs) improved the drug transport across the skin compared with vesicles with rigid membrane (El Maghraby et al. 2001). FLs are vesicular delivery systems that composed of phospholipid and an edge activator (e.g. Tween 80; Dragicevic-Curic et al. 2010). These systems underwent several adaptations and modifications to enhance their drug delivery capability. These modifications comprise adding ethanol which is known as ‘‘ethosomes.’’ Single alkyl chain surfactants such as Tweens are examples of edge activaters that act to destabilize lipid bilayers of the vesicles and increase deformability of these bilayers. Ethosomes are considered as multilayer vesicles containing much ethanol (20–45%; Touitou et al. 2000). Ethosomes have ability to deliver drugs (hydrophilic and lipophilic) through SC into the different strata of the skin efficiently than classical liposomes (Zhu et al. 2013). Thus, ethosomes become an excellent transdermal formulation for skin diseases (Zhang et al. 2014). Alternative trandsderma drug delivery systems were examined to deliver MLX topically. These system comprise niosomes (El-Menshawe and Hussein 2013), liposomes (Duangjit et al. 2014), and nanoemulsion (Khurana et al. 2013). Concerning the characteristics of MLX include small oral dosage (7.5–15 mg/day), low molecular weight (354.1), lipid solubility, and excellent tissue tolerability (Parfitt 1999). MLX is a good candidate for transdermal delivery. Therefore, the main purpose of this work is to investigate the impact of the presence and absence of an edge activator and/or different concentration of ethanol on the MLX-loaded FLs. The prepared systems will be characterized in terms of particle size, zetapotential, entrapment efficiency (EE%), penetration, permeation, and antiinflammatory activity of MLX.
Materials and methods Materials MLX was kindly supplied by Riyadh Pharma Medical & Cosmetic Products Co. Ltd. (Riyadh, Saudi Arabia). Phospholipids, Lipoid S100, was purchased from Lipoid KG, Ludwigshafen, Germany. Sodium deoxycholate and Tween 80 were obtained from BDH, Organics, Poole, England. Cholesterol was obtained from SantaCruz Biotechnology, Santa Cruz, CA. Methanol (HPLC grade) was acquired from BDH Chemicals, Poole, UK. Chloroform (HPLC grade) purchased from Acros Organics (Morris, NJ). All other chemicals were of analytical grade.
Table 1. Composition of the different types of FLs containing MLX. Ingredients (w/v) Lipoid S100 Cholesterol Tween 80 Ethanol MLX
CL 10 5 0.05
FL 10 1.5 0.05
FL1 10 1.5 10 0.05
FL2 10 1.5 20 0.05
FL3 10 1.5 30 0.05
FL4 10 1.5 40 0.05
CL: conventional liposomes; FL: flexible liposomes containing Tween 80; FL1–FL4: flexible liposomes with different concentrations of ethanol; MLX: meloxicam.
liposomes in nano-size range. The formed liposomes were light centrifuged at 1000 rpm for 5 min to remove the undissolved MLX and aggregated particles. FLs containing ethanol (F1–F4) were prepared using the previously described procedure, but the dried film was hydrated with a hydroalcoholic solution (10– 40% v/v; ethanolic solution). All formulations were kept at 4 C for further investigation.
Physicochemical characterization Particle size and zeta potential measurement The particle size, polydispersity index (PDI), and zeta potential of the investigated liposomal dispersions were measured by Zetasizer NS (Malvern Instruments Ltd, Worcestershire, UK). The measurements of the investigated formulations were taken at 25 C after proper dilution with distilled water. All experiments were performed in triplicate and the mean value is represented.
Entrapment efficiency The entrapment efficiency of the prepared vesicles was evaluated by indirect method. An aliquot of preparation was centrifuged using Eppendorf tube at 50,000 rpm and 4 C for 30 minutes using OptimaTM Max-E, Ultra Centrifuge (Beckman Coulter, Pasadena, CA). The supernatant was then analyzed by HPLC for MLX concentration. The entrapment efficiency (EE%) was calculated according to the following equation: EE% ¼
MLXt MLXf 100 MLXt
ð1Þ
where MLXt and MLXf are the amounts of total and free MLX, respectively.
Preparation of MLX-loaded flexosomes in gel form Preparation of liposomes The composition of the different liposomes is presented in Table 1. CLs and FLs containing MLX were prepared using film hydration method (Xiang et al. 2015). Briefly, phospholipids, Tween 80, and MLX were dissolved in a mixture of chloroform: methanol (2:1, v/v) in a round-bottom flask. The dry lipid film was obtained by removing the organic solvents under vacuum at 55–60 C, using a rotary evaporator (Buchi Rotavapor R-200, Flawil, Switzerland). The obtained film was then flushed with nitrogen gas to remove any possible traces of organic solvent. The dry lipid film was then hydrated with an aqueous solution by gently mixing for 30 minutes. Probe sonicator at 40% power for 2 minutes (Badnelin, Berlin, Germany) was used to develop
Optimized FLs formulation was selected based on the criteria of attaining the maximum values of transdermal flux, EE%. And for further investigation utilizing in vivo antiinflammatory activity. The FLs were incorporated with a gel to maintain contact of formuation on the skin of rat. Briefly, CarbopolÕ 934 (1% (w/w) was added into water and kept overnight for complete humectation of polymer chains. The FLs was added slowly to a hydrated carbopolÕ solution with stirring (Chaudhary et al. 2013). Other ingredients like 15% w/v polyethylene glycol-400 and triethanolamine (0.5% [w/v]) were added to get a homogeneous dispersion of gel, and this flexosomal gel formulation is employed for in vivo antiinflammatory study.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
Ex vivo penetration studies Skin preparation The impact of the presence and absence of an edge activator and ethanol in the composition of liposomes on the skin penetration of MLX was invetigated. The abdominal skin of rat was used, which is comparable to human skin (Walters and Roberts 1993), for in vitro skin penetration study of MLX-loaded liposomes and FLs. The skin samples of preparation were then wrapped in aluminum foil and stored at 20 C until use within 4 weeks.
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sampling system (WatersTM 717 Plus Autosampler, USA) were used. The HPLC system was monitored by ‘‘Empower (Water)’’ software (Waters, Inc., Milford, MA). MLX was analyzed using mobile phase consisted of methanol: 0.1 M potassium dihydrogen phosphate (40:60 v/v), the pH was adjusted to 6 by addition of 1-M potassium hydroxide. The mobile phase flowed over a reversed-phase C18 column (mBondapakTM, 4.6 150 mm, 10 mm particle size, Waters) at a rate of 1.2 mL/ min. The injection volume of each MLX sample was 20 mL and detected by UV detector at 256 nm. All the operations were carried out at room temperature.
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Ex vivo skin penetration studies The skin samples were thawed at room temperature for about 30 minutes. Skin samples were mounted onto vertical Franz diffusion cells (Logan Instrument Corp., Somerset, NJ) with the effective diffusion area of 1.76 cm2. The epidermal side of the skin was exposed to ambient conditions while the dermal side was bathed with PBS, pH 7.4 (receptor compartment; 12 mL) containing 5% Tween 80 to keep sink condition (Zheng et al. 2012). The receptor compartment was adjusted at 37 C to keep temperature of the skin surface at 32 C, and continuous stirring was maintained to simulate in vivo condition. After equilibration for 30 minutes, 1 mL of the formulation was applied to the skin surface under occlusive condition, covering doner compartment with parafilm. One-milliliter sample from each cell was withdrawn from the receptor medium at different time intervals: 1, 2, 3, 4, 5, and 6 hours, and replaced by an equal volume of freshly prepared receptor fluid. After 6 hours, the skin samples were carefully wiped off to discard the rest of formulations. The skin samples were fixed onto cork plates and stretched using small pins. The SC was then subsequently removed by tape stripping (3M TransporeTM tape, St. Paul, MN). The tapes with a surface area of approximately 4 cm2 were applied on the SC surface of the skin. The applied tape was firmly pressed on the skin surface and pulled off immediately. Each skin sample was stripped with 10 tapes to confirm the removal of the SC (Hiruta et al. 2006). The amount of MLX in the stripped skin was determined by cutting skin into small pieces. The tapes and stripped skin pieces were placed separately in a solvent mixture of PBS pH ¼ 7.4: ethanol (1:2) overnight followed by 5-minute vortexing and 5-minute sonication for complete extraction of MLX. The amount of MLX in extracts of the tapes and stripped skin as well as the receptor fluids were assayed after proper filtration by HPLC. The validity of the extraction method was evaluated by spiking known amounts of MLX on the tapes and stripped skin samples. More than 90% of MLX was recovered from the spiked samples of the tapes and stripped skin. All experiments were performed in triplicate.
HPLC analysis MLX was determined using a reverse-HPLC method with modification (Badran et al. 2014). The HPLC system (WatersTM 600 controller, USA) equipped with wavelength detector (WatersTM 2487 a Dual Absorbance detector, USA), pump (WatersTM 1252 a Binary pump, USA), and an automating
In vivo antiinflammatory activity The optimum formula of the investigated formulations was selected for in vivo study to verify the antiinflammatory activity of MLX. The optimum formula was incorporated into carbopol gel to maintain its contact with the rat-paw skin during application. Carrageenan-induced paw edema in rats was used to evaluate the antiinflammatory effect of MLX. This test is based on the ability of the investigated sample to inhibit the volume of the hind paw edema that induced by carrageenan (Khurana et al. 2013). In brief, adult Wistar Albino rats of either sex weighing 180–200 g (obtained from the Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia) were divided into six groups; each group contained six rats. The volume of the paw of each rat was measured before the test and 3 hours after carrageenan injection. The first group (Group A) was considered as a negative control and its rats were given normal saline (5 mL/kg). The rats of the second group (Group B) were given an aqueous solution of oxyphenylbutazone (100 mg/ kg) orally as an antiinflammatory reference (positive control). The rats of the third group were given MLX suspension (suspended in normal saline) orally (27 mg/kg). Plain gel (contains no MLX), MLX-loaded gel, and MLX-loaded FLs gel (FL4-gel) were applied on the right paw of the rats of the fourth, fifth, and sixth groups, respectively. One hour later, 0.05 mL of 1% carrageenan sodium salt solution was injected subcutaneously into plantar aponeurosis region of the right hind paw of each rat. After 3 hours of carrageenan injection, the rats were generally anesthetized by urethane, and the volume of the paw of each rat was measured using plethysmometer (Apelex, Massy, France). The inhibitory activity was calculated using the following formula: Anti-inflammatory activity ð%Þ ¼
CT 100 C
ð2Þ
where, C ¼ the mean value of the differences between the volume of the right paw after carrageenan injection and volume of the right paw before oral administration of normal saline (negative control). T ¼ the mean value of the differences between the volume of right paw after carrageenan injection and volume of the right paw before topical applied of the tested samples.
Statistical data analysis Data analysis was carried out with the software package, Microsoft Excel, Version 2010. Results are
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A. H. ALOMRANI AND M. M. BADRAN
expressed as mean ± standard error (n ¼ 3 independent samples).
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Results and discussion Particle size and zeta potential of vesicles The prepared MLX-loaded liposomes and FLs (their composition is presented in Table 1) were characterized with respect to their partical size, EE%, and skin penetration profiles. The vesicular systems and their particle size distribution and zeta potential are reported in Table 2. The impact of the liposomes composition on the size was assessed because vesicular size has ability to influence the penetration of drugs through the skin to the deeper tissues. Verma and his group (Verma et al. 2003) studied the influence of liposomal size on the skin penetration utilizing two fluorescently labeled substances. They found that the penetration of these fluorescent substances was inversely related to the size of the liposomes. They concluded that vesicles with size of 600 nm failed to deliver their loaded molecules into deeper layers of the skin, whereas, those with size of 300 nm are able to deliver their loaded molecules into the deeper layers of the skin. The particle size, PDI, and zeta potential results of the prepared liposomes (CL) and FL (FL1 FL4) are presented in Table 2. The particle size of the CL and FL recorded 155.2 and 129.3 nm (n ¼ 3), respectively. FL showed smaller size than CL which is in agreement with others (Alomrani et al. 2015, Gillet et al. 2011). Such effect could be attributed to the ability of Tween 80 (FL) to decrease the surface tension of the medium and facilitate the packing of the phospholipids in small vesicles (Gillet et al. 2011). In case of FLs containing different concentrations of ethanol, the size of the vesicles was small relative to that not containing ethanol with the exception of liposomes containing 40% (w/v) ethanol. However, the size of these liposomes decreased as the concentration of ethanol increased from 10 to 30% (w/v), which is in agreement with other studies (Verma and Pathak 2010). It was observed the vesicular sizes are 95.7, 66.2, and 62.1 (n ¼ 3) for F1, F2, and F3, respectively, while, the particle size of F4 with 40% ethanol was 143.4 nm (n ¼ 3). The effect of ethanol on the size of the vesicle was attributed to formation of a phase within the hydrocarbon chain, which reduce the membrane thickness (Dubey et al. 2007). Moreover, the presence of the ethanol in lipid vesicles bears a high negative charge to the systems providing some degree of steric stabilization leading to decrease in vesicle size (Sanjay et al. 2008). It was indicated that the addition of ethanol (40%) caused an increase in the viscosity of the system, which leads to the formation of a softsolid-like system (Castangia et al. 2013). This effect is due to the nature and concentration of ethanol, which has a dielectric constant lower than that of water. At high concentration (40%, ethanol), the water has ability to tilt some phospholipid chains to the intervesicle medium, leading to the formation of new and strong vesicle interconnections (Manca et al. 2014). The size of the investigated vesicular systems (CL, FL, FL1, F2, F3, and FL4) was less than 300 nm which means that the investigated systems have potential to deliver MLX through the skin.
Table 2. Physical characteristics of the different types of FLs containing MLX. Codes CL FL FL1 FL2 FL3 FL4
Particle size (nm) 155.2 ± 5.2 129.3 ± 4.4 95.7 ± 2.9 66.2 ± 2.2 62.1 ± 3.3 143.4 ± 1.9
0.329 0.281 0.214 0.121 0.153 0.228
PDI ± 0.0056 ± 0.003 ± 0.005 ± 0.008 ± 0.004 ± 0.005
Zeta potential (mv) 5.11 ± 0.64 3.02 ± 0.12 17.04 ± 0.71 20.09 ± 2.02 14.23 ± 0.35 15.83 ± 1.91
Regarding PDI, the investigated liposomes showed PDI values between 0.153 and 0.329 (Table 2) which lay within the acceptable homogeneity range of dispersions (Haidar et al. 2008, Verma et al. 2003). The surface charge of the vesicular systems play a significant role in the stability and skin interaction of the vesicles (Chen et al. 2010). CL showed a low positive charge, 5.1; however, FLs exhibited a negative charge on their vesicular surfaces. The magnitude of negativity of FLs was increased by increasing the concentrations of ethanol. The reported values of FL, FL1, FL2, FL3, and FL4 were –3, –17, –20, –14, and –16, respectively. Regarding Tween 80, despite it is a nonionic surfactant, the oxyethylene part of Tween 80 could be a source of the negative charges, that is, shown with FL (Marzio et al. 2013). Furthermore, it was reported that ethanol provides a more negative charge on the surface of ethosomes (Verma et al. 2003). But in the case of ethanol concentration (40%), the negative values were decreased. This might be attributed to decrease of the dielectric constant of the media, which leads to a reduction of ionized group on the vesicle surface (Castangia et al. 2015). This study suggested that ethanol has a predominant role in vesicular systems that causes a modification of the net charge and vesicle size.
The entrapment efficiency The EE% of CL reported 32.6% (Figure 1). This low value of EE% was in agreement with other reports (Lo´pez-Pinto et al. 2005). The presence of Tween 80, an edge activator, in the composition of liposomes (FL) enhanced the EE% to 52.6%. Tween 80 is among edge activators that increase the flexibility of the vesicular bilayer and hence improves the EE%. It was reported that edge activators can immerse perpendicularly within the bilayer of the vesicular membrane. This mode of packing causes perturbation of the acyl chains of the lipid matrix, increases the flexibility of the membrane, and consequently enhances the solubility of the lipophilic molecules within the bilayer of the vesicle membrane (Santosh et al. 2012). The presence of ethanol causes an increase in the EE%. The magnitude of the enhancement was in direct relation to the concentration of ethanol. The EE% were 61.1, 70.6, 75.9, and 83.8% for FL1, FL2, FL3, and FL4, respectively (Figure 1). Alternative studies showed that the presence of ethanol in the composition of liposomes resulted in an increase in the EE% of minoxidil (Lo´pez-Pinto et al. 2005), melatonin (Dubey et al. 2007), and tacrolimus (Li et al. 2012). The increase of EE% of vesicular systems by ethanol was attributed to the presence of ethanol in the core of the FLs, which acts to enhance solubility of the drug in the vesicular core (Dubey et al. 2007).
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
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Table 4. Results of skin deposition of MLX in the different layers of rat skin after 6 hours of occlusive incubation. MLX delivered (mg/cm2) h Control CL FL FL1 FL2 FL3 FL4
SC 4.7 ± 5.6 ± 6.9 ± 13.5 ± 17.8 ± 23.1 ± 34.9 ±
0.1 0.3 0.6 1.2 2.5 3.2 3.7
Stripped skin 1.6 ± 0.2 3.5 ± 0.2 4.3 ± 0.1 9.2 ± 2.1 12.4 ± 1.7 15.6 ± 2.4 19.2 ± 1.3
Receptor ND 2.5 ± 0.1 3.8 ± 0.4 7.3 ± 0.9 9.0 ± 2.4 12.3 ± 1.6 15.5 ± 1.9
Figure 1. The entrapment efficiency of MLX-loaded different types of liposomes.
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Table 3. Percutaneous permeability parameters of MLX FLs through rat skin. Formulations Control CL FL FL1 FL2 FL3 FL4
Enhancement Ratio (ER) 3.199 ± 7.278 ± 14.772 ± 20.578 ± 24.962 ± 31.489 ±
0.160 0.998 1.743 2.662 3.282 1.853
Flux (mg cm2 h1)
Permeability coefficient (cm h1) 105
0.028 0.108 0.322 0.671 1.012 1.309 1.545
3.122 8.827 29.180 64.282 87.001 110.622 143.296
± ± ± ± ± ± ±
0.003 0.003 0.0144 0.084 0.146 0.146 0.119
± ± ± ± ± ± ±
0.332 0.257 1.438 1.077 5.256 12.162 3.350
In vitro penetration study
Figure 2. Cumulative permeation of MLX via the rat abdominal skin versus time profile of MLX loaded different types of liposomes.
In vitro skin penetration and permeation of MLX-loaded FLs were performed to evaluate the impact of edge activator and ethanol on MLX skin disposition. The experiments were carried out for 6 hours under occlusive condition. Tables 3 and 4 present the permeation and penetration results of MLX through the skin, respectively. As illustrated in Table 4, the in vitro skin deposition of MLX delivered from MLX solution was 4.7 mg/cm2 in the SC, 1.6 mg/cm2 in the stripped skin, and no MLX was detected in the receptor fluid. These data revealed that MLX has low permeability value through skin, which mainly attributed to its lipophilicity and to the barrier nature of the SC layer (Kapoor and Subbarao 2011, Peeters et al. 2002). For such drug, enough concentration should permeate the skin to the deeper layers to do its desired effect. However, the disposition profile of MLX in different skin compartments was significantly enhanced when CL and FLs (FL and FL1–FL4) systems were applied. For CL, the disposition of MLX in the SC and stripped skin was increased by 1.2- and 2.2-fold, respectively, compared with MLX solution, and detectable amount of MLX was observed in the receptor fluid (see Figure 3 and Table 4). The flux and permeability coefficient of MLX were increased by CL reporting enhancement ratio (ER) 3.2 times of that reporting with MLX solution (see Figure 2 and Table 3). These results revealed that CL facilitates the permeation of MLX to the deeper tissues of the skin. The positive impact of CL on MLX permeation through skin was in correlation with others (Pabyton et al. 2013). The mechanisms were suggested to explain the enhancement of drug permeation through skin by liposomes. First, the similarity between the compositions of the
liposomes and biological membranes may facilitate the interaction and/or fusion between them. This fusion allows liposomes to liberate the loaded drug into SC and other skin layers (Chen et al. 2010, El Maghraby et al. 2001). Second, Phospholipids itself may interact with the cell membrane and perturb the integrity of the SC. Such interaction causes disruption in the integrity of the barrier nature of the SC (Pabyton et al. 2013). Further enhancement in drug permeation through skin could be achieved by altering the composition of liposomes. The permeation of MLX was further improved by using FLs (FL and FL1–FL4; Tables 3 and 4; Figures 2 and 3). FL showed improvement in MLX skin disposition, reporting 1.5-, 2.7-fold of MLX in SC and stripped skin, respectively, compared with MLX solution. MLX flux and ER were 11.5 and 7.3 times of that recorded with MLX solution, respectively (Table 3). This positive impact of FL on MLX disposition and permeation through skin is in agreement with other works using liposomes containing surfactants (El Zaafarany et al. 2010, Hiruta et al. 2006). Such improvement was attributed the influence of Tween 80 on the flexibility of liposomes. The flexibility of FL facilitates MLX migration through paracellular hydrophilic pores within the skin. FL can penetrate SC easily and subsequently change the intercellular lipids; this change alters integrity of the SC and hence increases the disposition of the drug into different skin layers (Monteiro et al. 2012). Further improvement in MLX permeation through skin was observed when ethanol being added to FLs (see Tables 3 and 4). It was reported that the
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A. H. ALOMRANI AND M. M. BADRAN
Figure 3. The amount of MLX delivered into the SC, stripped skin, and the receptor fluid after 6 hours of incubation with rat skin after applying a different type of liposomes.
Figure 4. % Inhibition in edema produced by optimized MLX loaded FLs gel formulation (FL4), and oral administration in carrageenan-induced rat paw.
presence of ethanol promotes MLX permeation and disposition through skin. Linear increases in MLX disposition in the skin layers and MLX permeation were observed with increasing ethanol concentration (see Tables 3 and 4; Figures 2 and 3). The ER of FL1, FL2, FL3, and FL4 was 14.8, 20.6, 25, and 31.5, respectively. F4 gave the highest flux (1.5 mg cm 2 h1) and permeation coefficient 143.3 10 5 cm h1. The improvement of MLX permeation through skin was in direct correlation with ethanol concentration. More specifically, F1, F2, F3, and F4 showed 2.9-, 3.8-, 4.9-, and 7.4-fold increase in MLX in SC compared with that obtained using MLX solution and 2.4-, 3.2-, 4.1-, and 6.2-fold increase compared with that obtained with CL. The amounts of MLX accumulation in the stripped skin were 2.6-, 3.5-, 4.4-, and 5.5-fold for F1, F2, F3, and F4, respectively, compared with CL. The positive impact of FL1–FL4 on the skin
disposition of MLX is in agreement with others (Dubey et al. 2007, Li et al. 2012, Lo´pez-Pinto et al. 2005). According to previous studies, several suggestions were proposed to explain the mechanism of permeation enhancement of FLs in presence of ethanol. One mechanism attributed the enhancement effect of FLs to the presence of ethanol, which may cause the following effects: (1) ‘‘pull effect’’ as a result of skin lipid bilayer disruption by ethanol and (2) ‘‘push effect’’ as a result of increasing of thermodynamic activity of MLX due to ethanol evaporation (Dubey et al. 2007). A synergistic effect of phospholipids and ethanol was also suggested (Chen et al. 2010). The scenario of this synergism starts by interaction of ethanol with polar head groups of lipid molecules of SC causing reduction in the transition temperature of these lipids and consequently increasing their fluidity and as a result, the structure of SC becomes loose and disordered. This action is followed by fusion of vesicles with skin lipids resulting in releasing of the vesicles content into the deep layers of the skin easily via the loosed SC (Dragicevic-Curic et al. 2010). Moreover, the higher EE% and accumulation of MLX in skin layers by FLs were observed.
In vivo antiinflammatory activity Because of its good skin penetration and permeation profiles, FL4 was selected as an optimized formula for in vivo antiinflammatory effect. Because FL4 is present as liquid form, it was prepared as gel, using carbopol as a gelling agent, to maintain its contact with the rat-paw skin during the application. The in vivo antiinflammatory study was performed in rats using carrageenan-induced rat-paw edema model (Winter et al. 1962). Figure 4 presents the antiinflammatory activity of MLXloaded FL4 gel and different control samples.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
According to Figure 4, the antiinflammatory effect of MLX gel reported 34% inhibition of edema, whereas, the oral form of MLX was almost equal to the conventional gel form of MLX. A significant antiinflammatory effect was shown by F4-loaded gel. This system reported antiinflammatory effect as 64% inhibition of edema, which is almost 2-fold of conventional MLX gel. These data are correlated with the skin permeation and penetration studies (see Figures 2 and 3). According to these findings, it could be concluded that MLX-loaded FLs can penetrate SC smoothly and improve MLX antiinflammatory activity which will significantly help in the treatment of the inflammation and revealed the positive impact of FLs on the skin delivery of drug.
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Conclusion In this study, various types of liposomes containing MLX (CL and FLs) were prepared and evaluated. In vitro skin penetration studies revealed that FL composed of phospholipids and Tween 80 delivered high amount of MLX to the SC and deeper skin layers compared with CL and solution. Further enhancement in MLX skin penetration was achieved when ethanol was added to the composition of the liposomes. Such improvement may be attributed to the ability of Tween 80 and ethanol to reduce the size and increase the flexibility of the vesicles in addition to the increase in EE%. The highest penetration and EE% were found in case of the optimized formulation FL4, prepared by 10% (w/v) of lipoid S100, 1.5% (w/v) of Tween 80%, and 40% (v/v) of ethanol. Moreover, F4-loaded carbopol gel revealed comparative higher inhibition of rat paw edema compared with that of oral administration. It can be concluded that FLs containing ethanol could be a promising carrier for cutaneous delivery of MLX.
Acknowledgements The authors also would like to thank Mr. Malik Saud Ahmad for his technical assessment in performing antiinflammatory test, and Riyadh Pharma Medical & Cosmetic Products Co. Ltd. (Riyadh, Saudi Arabia) for providing the free sample of MLX.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding information The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the research group project no RGP-VPP-287.
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ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: http://www.tandfonline.com/loi/ianb20
Flexosomes for transdermal delivery of meloxicam: characterization and antiinflammatory activity Abdullah H. Alomrani & Mohamed M. Badran To cite this article: Abdullah H. Alomrani & Mohamed M. Badran (2016): Flexosomes for transdermal delivery of meloxicam: characterization and antiinflammatory activity, Artificial Cells, Nanomedicine, and Biotechnology, DOI: 10.3109/21691401.2016.1147452 To link to this article: http://dx.doi.org/10.3109/21691401.2016.1147452
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Date: 02 March 2016, At: 11:04
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016 http://dx.doi.org/10.3109/21691401.2016.1147452
Flexosomes for transdermal delivery of meloxicam: characterization and antiinflammatory activity Abdullah H. Alomrania,b and Mohamed M. Badrana,c Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; bNanomedicine Unit (NMU-KSU), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; cDepartment of Pharmaceutics, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
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a
ABSTRACT
ARTICLE HISTORY
The study aims to investigate the impact of flexosomes (FLs) on transdermal delivery of meloxicam (MLX). FLs are composed of phospholipid, Tween 80, and ethanol which were prepared by film hydration method. The prepared FLs were characterized for particle size, zeta potential, and entrapment efficiency (EE). Ex vivo skin penetration studies were perfomed, and the best formulation was further evaluated using in vivo antiinflammatory activity test. FLs were in nano-size scale carring negative charge and observed high EE% and enhanced skin penetration of MLX compared to conventional liposomes (CLs). The best formula was FL4 which was composedof phospholipid (10%), Tween 80 (1.5%), and ethanol (40%). FL4 showed 143.4 nm vesicle size, 84% EE, and 31-fold ex vivo permeation enhancement through skin compared to CLs. The antiinflammatory activity of FL4 gel showed significant increase compared to control. This study observed the effectiveness of using FLs as carriers for transdermal delivery of MLX.
Received 27 October 2015 Revised 4 January 2016 Accepted 25 January 2016 Published online 24 February 2016
Introduction Meloxicam (MLX) is an effective nonsteroidal antiinflammatory drug (NSAID), with high affinity to inhibit cyclooxygenase-2 (COX-2). MLX was approved by FDA in 2000 (MOBICÕ , Boehringer Ingelheim) for arthritis, osteoarthritis, and degenerative joint disease (Fleischmann et al. 2000; FDA 2000). MLX is available only for oral administration. There is a controversy in the GIT toxicity of MLX. Some authors revealed that the antiinflammatory and analgesic effect of MLX were equivalent to diclofenac, piroxicam, and naproxen (Fleischmann et al. 2000). A number of side effects related to GIT were reported for MLX (Gambero et al. 2005). Accordingly, alternative route of administration, such as transdermal route may help to improve compliance and avoid side effects in subjects who experience these undesirable effects. Considering the fact that most inflammatory diseases occur locally and near the surface of the body, topical application of NSAIDs on the inflamed site can offer the advantage of delivering a drug directly to the site of action producing the desirable effect and avoiding unwanted adverse effects associated with oral route (Prausnitz 2004, Thomas and Finnin 2004). However, some compounds cannot achieve these goals effectively and efficiently, due to their physico-chemical parameters (Southwell and Barry 1983). The major limitation of MLX is the low aqueous solubility, which significantly varies with the pH value of the media (Luger et al. 1996). Its solubility was reported to be 0.012 mg/mL in water, 0.086 10 2 mg/mL in 0.1 M of HCl (Duangjit et al. 2014). MLX has a partition coefficient logP 1.91 and 0.07 at pH 5.0 and 7.4, CONTACT Mohamed M. Badran [email protected] P.O. Box 2457, Riyadh 11451, Saudi Arabia ß 2016 Taylor & Francis
KEYWORDS
Antiinflammatory studies; flexosomes; meloxicam; skin penetration
respectively (Duangjit et al. 2014, Luger et al. 1996). Its molecular weight is 351,4 Da and it has two pKa values: 1.09 and 4.18 (Luger et al. 1996). The pharmacokinetic profile of MLX is characterized by a prolonged and complete absorption with 20-hour elimination half-life. MLX is highly bound to plasma protein (99.5%; Tu¨rck et al. 1996). Because the skin is the route of drug administration for transdermal drug delivery systems, it is important to appreciate skin physiology. The skin acts as an excellent biological barrier which is attributed to the stratum corneum (SC) layer. SC is formed of different layers of corneocytes which are surrounded by the intercellular lipid lamellae (Bouwstra and HoneywellNguyen 2002). This layer hinders permeation of drugs and other substances through the skin (Cevc and Vierl 2010, Menon 2002). Alternative strategies were investigated to overcome the barrier nature of SC include physical (e.g. sonophoresis and electoporation), chemical (e.g. chemical permeation enhancers), vesicular systems (e.g. Liposomes and niosomes), and mechanical (e.g. micro-needles) methods (Barry 2001). Among the applied strategies, vesicular drug delivery systems showed potential and safe approaches for increasing skin penetration of drugs. Despite of their potentials, conventional liposomes (CLs) offer little value as carriers for transdermal drug delivery because they cannot penetrate the skin deep enough. It has been reported that the penetration enhancing of the vesicles through the skin depends on their membrane flexibility. It was shown that liposomes with flexible membrane (i.e. flexosomes;
Department of Pharmaceutics, College of Pharmacy, King Saud University, Building # 23, Office # AA 68
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A. H. ALOMRANI AND M. M. BADRAN
FLs) improved the drug transport across the skin compared with vesicles with rigid membrane (El Maghraby et al. 2001). FLs are vesicular delivery systems that composed of phospholipid and an edge activator (e.g. Tween 80; Dragicevic-Curic et al. 2010). These systems underwent several adaptations and modifications to enhance their drug delivery capability. These modifications comprise adding ethanol which is known as ‘‘ethosomes.’’ Single alkyl chain surfactants such as Tweens are examples of edge activaters that act to destabilize lipid bilayers of the vesicles and increase deformability of these bilayers. Ethosomes are considered as multilayer vesicles containing much ethanol (20–45%; Touitou et al. 2000). Ethosomes have ability to deliver drugs (hydrophilic and lipophilic) through SC into the different strata of the skin efficiently than classical liposomes (Zhu et al. 2013). Thus, ethosomes become an excellent transdermal formulation for skin diseases (Zhang et al. 2014). Alternative trandsderma drug delivery systems were examined to deliver MLX topically. These system comprise niosomes (El-Menshawe and Hussein 2013), liposomes (Duangjit et al. 2014), and nanoemulsion (Khurana et al. 2013). Concerning the characteristics of MLX include small oral dosage (7.5–15 mg/day), low molecular weight (354.1), lipid solubility, and excellent tissue tolerability (Parfitt 1999). MLX is a good candidate for transdermal delivery. Therefore, the main purpose of this work is to investigate the impact of the presence and absence of an edge activator and/or different concentration of ethanol on the MLX-loaded FLs. The prepared systems will be characterized in terms of particle size, zetapotential, entrapment efficiency (EE%), penetration, permeation, and antiinflammatory activity of MLX.
Materials and methods Materials MLX was kindly supplied by Riyadh Pharma Medical & Cosmetic Products Co. Ltd. (Riyadh, Saudi Arabia). Phospholipids, Lipoid S100, was purchased from Lipoid KG, Ludwigshafen, Germany. Sodium deoxycholate and Tween 80 were obtained from BDH, Organics, Poole, England. Cholesterol was obtained from SantaCruz Biotechnology, Santa Cruz, CA. Methanol (HPLC grade) was acquired from BDH Chemicals, Poole, UK. Chloroform (HPLC grade) purchased from Acros Organics (Morris, NJ). All other chemicals were of analytical grade.
Table 1. Composition of the different types of FLs containing MLX. Ingredients (w/v) Lipoid S100 Cholesterol Tween 80 Ethanol MLX
CL 10 5 0.05
FL 10 1.5 0.05
FL1 10 1.5 10 0.05
FL2 10 1.5 20 0.05
FL3 10 1.5 30 0.05
FL4 10 1.5 40 0.05
CL: conventional liposomes; FL: flexible liposomes containing Tween 80; FL1–FL4: flexible liposomes with different concentrations of ethanol; MLX: meloxicam.
liposomes in nano-size range. The formed liposomes were light centrifuged at 1000 rpm for 5 min to remove the undissolved MLX and aggregated particles. FLs containing ethanol (F1–F4) were prepared using the previously described procedure, but the dried film was hydrated with a hydroalcoholic solution (10– 40% v/v; ethanolic solution). All formulations were kept at 4 C for further investigation.
Physicochemical characterization Particle size and zeta potential measurement The particle size, polydispersity index (PDI), and zeta potential of the investigated liposomal dispersions were measured by Zetasizer NS (Malvern Instruments Ltd, Worcestershire, UK). The measurements of the investigated formulations were taken at 25 C after proper dilution with distilled water. All experiments were performed in triplicate and the mean value is represented.
Entrapment efficiency The entrapment efficiency of the prepared vesicles was evaluated by indirect method. An aliquot of preparation was centrifuged using Eppendorf tube at 50,000 rpm and 4 C for 30 minutes using OptimaTM Max-E, Ultra Centrifuge (Beckman Coulter, Pasadena, CA). The supernatant was then analyzed by HPLC for MLX concentration. The entrapment efficiency (EE%) was calculated according to the following equation: EE% ¼
MLXt MLXf 100 MLXt
ð1Þ
where MLXt and MLXf are the amounts of total and free MLX, respectively.
Preparation of MLX-loaded flexosomes in gel form Preparation of liposomes The composition of the different liposomes is presented in Table 1. CLs and FLs containing MLX were prepared using film hydration method (Xiang et al. 2015). Briefly, phospholipids, Tween 80, and MLX were dissolved in a mixture of chloroform: methanol (2:1, v/v) in a round-bottom flask. The dry lipid film was obtained by removing the organic solvents under vacuum at 55–60 C, using a rotary evaporator (Buchi Rotavapor R-200, Flawil, Switzerland). The obtained film was then flushed with nitrogen gas to remove any possible traces of organic solvent. The dry lipid film was then hydrated with an aqueous solution by gently mixing for 30 minutes. Probe sonicator at 40% power for 2 minutes (Badnelin, Berlin, Germany) was used to develop
Optimized FLs formulation was selected based on the criteria of attaining the maximum values of transdermal flux, EE%. And for further investigation utilizing in vivo antiinflammatory activity. The FLs were incorporated with a gel to maintain contact of formuation on the skin of rat. Briefly, CarbopolÕ 934 (1% (w/w) was added into water and kept overnight for complete humectation of polymer chains. The FLs was added slowly to a hydrated carbopolÕ solution with stirring (Chaudhary et al. 2013). Other ingredients like 15% w/v polyethylene glycol-400 and triethanolamine (0.5% [w/v]) were added to get a homogeneous dispersion of gel, and this flexosomal gel formulation is employed for in vivo antiinflammatory study.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
Ex vivo penetration studies Skin preparation The impact of the presence and absence of an edge activator and ethanol in the composition of liposomes on the skin penetration of MLX was invetigated. The abdominal skin of rat was used, which is comparable to human skin (Walters and Roberts 1993), for in vitro skin penetration study of MLX-loaded liposomes and FLs. The skin samples of preparation were then wrapped in aluminum foil and stored at 20 C until use within 4 weeks.
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sampling system (WatersTM 717 Plus Autosampler, USA) were used. The HPLC system was monitored by ‘‘Empower (Water)’’ software (Waters, Inc., Milford, MA). MLX was analyzed using mobile phase consisted of methanol: 0.1 M potassium dihydrogen phosphate (40:60 v/v), the pH was adjusted to 6 by addition of 1-M potassium hydroxide. The mobile phase flowed over a reversed-phase C18 column (mBondapakTM, 4.6 150 mm, 10 mm particle size, Waters) at a rate of 1.2 mL/ min. The injection volume of each MLX sample was 20 mL and detected by UV detector at 256 nm. All the operations were carried out at room temperature.
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Ex vivo skin penetration studies The skin samples were thawed at room temperature for about 30 minutes. Skin samples were mounted onto vertical Franz diffusion cells (Logan Instrument Corp., Somerset, NJ) with the effective diffusion area of 1.76 cm2. The epidermal side of the skin was exposed to ambient conditions while the dermal side was bathed with PBS, pH 7.4 (receptor compartment; 12 mL) containing 5% Tween 80 to keep sink condition (Zheng et al. 2012). The receptor compartment was adjusted at 37 C to keep temperature of the skin surface at 32 C, and continuous stirring was maintained to simulate in vivo condition. After equilibration for 30 minutes, 1 mL of the formulation was applied to the skin surface under occlusive condition, covering doner compartment with parafilm. One-milliliter sample from each cell was withdrawn from the receptor medium at different time intervals: 1, 2, 3, 4, 5, and 6 hours, and replaced by an equal volume of freshly prepared receptor fluid. After 6 hours, the skin samples were carefully wiped off to discard the rest of formulations. The skin samples were fixed onto cork plates and stretched using small pins. The SC was then subsequently removed by tape stripping (3M TransporeTM tape, St. Paul, MN). The tapes with a surface area of approximately 4 cm2 were applied on the SC surface of the skin. The applied tape was firmly pressed on the skin surface and pulled off immediately. Each skin sample was stripped with 10 tapes to confirm the removal of the SC (Hiruta et al. 2006). The amount of MLX in the stripped skin was determined by cutting skin into small pieces. The tapes and stripped skin pieces were placed separately in a solvent mixture of PBS pH ¼ 7.4: ethanol (1:2) overnight followed by 5-minute vortexing and 5-minute sonication for complete extraction of MLX. The amount of MLX in extracts of the tapes and stripped skin as well as the receptor fluids were assayed after proper filtration by HPLC. The validity of the extraction method was evaluated by spiking known amounts of MLX on the tapes and stripped skin samples. More than 90% of MLX was recovered from the spiked samples of the tapes and stripped skin. All experiments were performed in triplicate.
HPLC analysis MLX was determined using a reverse-HPLC method with modification (Badran et al. 2014). The HPLC system (WatersTM 600 controller, USA) equipped with wavelength detector (WatersTM 2487 a Dual Absorbance detector, USA), pump (WatersTM 1252 a Binary pump, USA), and an automating
In vivo antiinflammatory activity The optimum formula of the investigated formulations was selected for in vivo study to verify the antiinflammatory activity of MLX. The optimum formula was incorporated into carbopol gel to maintain its contact with the rat-paw skin during application. Carrageenan-induced paw edema in rats was used to evaluate the antiinflammatory effect of MLX. This test is based on the ability of the investigated sample to inhibit the volume of the hind paw edema that induced by carrageenan (Khurana et al. 2013). In brief, adult Wistar Albino rats of either sex weighing 180–200 g (obtained from the Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia) were divided into six groups; each group contained six rats. The volume of the paw of each rat was measured before the test and 3 hours after carrageenan injection. The first group (Group A) was considered as a negative control and its rats were given normal saline (5 mL/kg). The rats of the second group (Group B) were given an aqueous solution of oxyphenylbutazone (100 mg/ kg) orally as an antiinflammatory reference (positive control). The rats of the third group were given MLX suspension (suspended in normal saline) orally (27 mg/kg). Plain gel (contains no MLX), MLX-loaded gel, and MLX-loaded FLs gel (FL4-gel) were applied on the right paw of the rats of the fourth, fifth, and sixth groups, respectively. One hour later, 0.05 mL of 1% carrageenan sodium salt solution was injected subcutaneously into plantar aponeurosis region of the right hind paw of each rat. After 3 hours of carrageenan injection, the rats were generally anesthetized by urethane, and the volume of the paw of each rat was measured using plethysmometer (Apelex, Massy, France). The inhibitory activity was calculated using the following formula: Anti-inflammatory activity ð%Þ ¼
CT 100 C
ð2Þ
where, C ¼ the mean value of the differences between the volume of the right paw after carrageenan injection and volume of the right paw before oral administration of normal saline (negative control). T ¼ the mean value of the differences between the volume of right paw after carrageenan injection and volume of the right paw before topical applied of the tested samples.
Statistical data analysis Data analysis was carried out with the software package, Microsoft Excel, Version 2010. Results are
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A. H. ALOMRANI AND M. M. BADRAN
expressed as mean ± standard error (n ¼ 3 independent samples).
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Results and discussion Particle size and zeta potential of vesicles The prepared MLX-loaded liposomes and FLs (their composition is presented in Table 1) were characterized with respect to their partical size, EE%, and skin penetration profiles. The vesicular systems and their particle size distribution and zeta potential are reported in Table 2. The impact of the liposomes composition on the size was assessed because vesicular size has ability to influence the penetration of drugs through the skin to the deeper tissues. Verma and his group (Verma et al. 2003) studied the influence of liposomal size on the skin penetration utilizing two fluorescently labeled substances. They found that the penetration of these fluorescent substances was inversely related to the size of the liposomes. They concluded that vesicles with size of 600 nm failed to deliver their loaded molecules into deeper layers of the skin, whereas, those with size of 300 nm are able to deliver their loaded molecules into the deeper layers of the skin. The particle size, PDI, and zeta potential results of the prepared liposomes (CL) and FL (FL1 FL4) are presented in Table 2. The particle size of the CL and FL recorded 155.2 and 129.3 nm (n ¼ 3), respectively. FL showed smaller size than CL which is in agreement with others (Alomrani et al. 2015, Gillet et al. 2011). Such effect could be attributed to the ability of Tween 80 (FL) to decrease the surface tension of the medium and facilitate the packing of the phospholipids in small vesicles (Gillet et al. 2011). In case of FLs containing different concentrations of ethanol, the size of the vesicles was small relative to that not containing ethanol with the exception of liposomes containing 40% (w/v) ethanol. However, the size of these liposomes decreased as the concentration of ethanol increased from 10 to 30% (w/v), which is in agreement with other studies (Verma and Pathak 2010). It was observed the vesicular sizes are 95.7, 66.2, and 62.1 (n ¼ 3) for F1, F2, and F3, respectively, while, the particle size of F4 with 40% ethanol was 143.4 nm (n ¼ 3). The effect of ethanol on the size of the vesicle was attributed to formation of a phase within the hydrocarbon chain, which reduce the membrane thickness (Dubey et al. 2007). Moreover, the presence of the ethanol in lipid vesicles bears a high negative charge to the systems providing some degree of steric stabilization leading to decrease in vesicle size (Sanjay et al. 2008). It was indicated that the addition of ethanol (40%) caused an increase in the viscosity of the system, which leads to the formation of a softsolid-like system (Castangia et al. 2013). This effect is due to the nature and concentration of ethanol, which has a dielectric constant lower than that of water. At high concentration (40%, ethanol), the water has ability to tilt some phospholipid chains to the intervesicle medium, leading to the formation of new and strong vesicle interconnections (Manca et al. 2014). The size of the investigated vesicular systems (CL, FL, FL1, F2, F3, and FL4) was less than 300 nm which means that the investigated systems have potential to deliver MLX through the skin.
Table 2. Physical characteristics of the different types of FLs containing MLX. Codes CL FL FL1 FL2 FL3 FL4
Particle size (nm) 155.2 ± 5.2 129.3 ± 4.4 95.7 ± 2.9 66.2 ± 2.2 62.1 ± 3.3 143.4 ± 1.9
0.329 0.281 0.214 0.121 0.153 0.228
PDI ± 0.0056 ± 0.003 ± 0.005 ± 0.008 ± 0.004 ± 0.005
Zeta potential (mv) 5.11 ± 0.64 3.02 ± 0.12 17.04 ± 0.71 20.09 ± 2.02 14.23 ± 0.35 15.83 ± 1.91
Regarding PDI, the investigated liposomes showed PDI values between 0.153 and 0.329 (Table 2) which lay within the acceptable homogeneity range of dispersions (Haidar et al. 2008, Verma et al. 2003). The surface charge of the vesicular systems play a significant role in the stability and skin interaction of the vesicles (Chen et al. 2010). CL showed a low positive charge, 5.1; however, FLs exhibited a negative charge on their vesicular surfaces. The magnitude of negativity of FLs was increased by increasing the concentrations of ethanol. The reported values of FL, FL1, FL2, FL3, and FL4 were –3, –17, –20, –14, and –16, respectively. Regarding Tween 80, despite it is a nonionic surfactant, the oxyethylene part of Tween 80 could be a source of the negative charges, that is, shown with FL (Marzio et al. 2013). Furthermore, it was reported that ethanol provides a more negative charge on the surface of ethosomes (Verma et al. 2003). But in the case of ethanol concentration (40%), the negative values were decreased. This might be attributed to decrease of the dielectric constant of the media, which leads to a reduction of ionized group on the vesicle surface (Castangia et al. 2015). This study suggested that ethanol has a predominant role in vesicular systems that causes a modification of the net charge and vesicle size.
The entrapment efficiency The EE% of CL reported 32.6% (Figure 1). This low value of EE% was in agreement with other reports (Lo´pez-Pinto et al. 2005). The presence of Tween 80, an edge activator, in the composition of liposomes (FL) enhanced the EE% to 52.6%. Tween 80 is among edge activators that increase the flexibility of the vesicular bilayer and hence improves the EE%. It was reported that edge activators can immerse perpendicularly within the bilayer of the vesicular membrane. This mode of packing causes perturbation of the acyl chains of the lipid matrix, increases the flexibility of the membrane, and consequently enhances the solubility of the lipophilic molecules within the bilayer of the vesicle membrane (Santosh et al. 2012). The presence of ethanol causes an increase in the EE%. The magnitude of the enhancement was in direct relation to the concentration of ethanol. The EE% were 61.1, 70.6, 75.9, and 83.8% for FL1, FL2, FL3, and FL4, respectively (Figure 1). Alternative studies showed that the presence of ethanol in the composition of liposomes resulted in an increase in the EE% of minoxidil (Lo´pez-Pinto et al. 2005), melatonin (Dubey et al. 2007), and tacrolimus (Li et al. 2012). The increase of EE% of vesicular systems by ethanol was attributed to the presence of ethanol in the core of the FLs, which acts to enhance solubility of the drug in the vesicular core (Dubey et al. 2007).
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Table 4. Results of skin deposition of MLX in the different layers of rat skin after 6 hours of occlusive incubation. MLX delivered (mg/cm2) h Control CL FL FL1 FL2 FL3 FL4
SC 4.7 ± 5.6 ± 6.9 ± 13.5 ± 17.8 ± 23.1 ± 34.9 ±
0.1 0.3 0.6 1.2 2.5 3.2 3.7
Stripped skin 1.6 ± 0.2 3.5 ± 0.2 4.3 ± 0.1 9.2 ± 2.1 12.4 ± 1.7 15.6 ± 2.4 19.2 ± 1.3
Receptor ND 2.5 ± 0.1 3.8 ± 0.4 7.3 ± 0.9 9.0 ± 2.4 12.3 ± 1.6 15.5 ± 1.9
Figure 1. The entrapment efficiency of MLX-loaded different types of liposomes.
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Table 3. Percutaneous permeability parameters of MLX FLs through rat skin. Formulations Control CL FL FL1 FL2 FL3 FL4
Enhancement Ratio (ER) 3.199 ± 7.278 ± 14.772 ± 20.578 ± 24.962 ± 31.489 ±
0.160 0.998 1.743 2.662 3.282 1.853
Flux (mg cm2 h1)
Permeability coefficient (cm h1) 105
0.028 0.108 0.322 0.671 1.012 1.309 1.545
3.122 8.827 29.180 64.282 87.001 110.622 143.296
± ± ± ± ± ± ±
0.003 0.003 0.0144 0.084 0.146 0.146 0.119
± ± ± ± ± ± ±
0.332 0.257 1.438 1.077 5.256 12.162 3.350
In vitro penetration study
Figure 2. Cumulative permeation of MLX via the rat abdominal skin versus time profile of MLX loaded different types of liposomes.
In vitro skin penetration and permeation of MLX-loaded FLs were performed to evaluate the impact of edge activator and ethanol on MLX skin disposition. The experiments were carried out for 6 hours under occlusive condition. Tables 3 and 4 present the permeation and penetration results of MLX through the skin, respectively. As illustrated in Table 4, the in vitro skin deposition of MLX delivered from MLX solution was 4.7 mg/cm2 in the SC, 1.6 mg/cm2 in the stripped skin, and no MLX was detected in the receptor fluid. These data revealed that MLX has low permeability value through skin, which mainly attributed to its lipophilicity and to the barrier nature of the SC layer (Kapoor and Subbarao 2011, Peeters et al. 2002). For such drug, enough concentration should permeate the skin to the deeper layers to do its desired effect. However, the disposition profile of MLX in different skin compartments was significantly enhanced when CL and FLs (FL and FL1–FL4) systems were applied. For CL, the disposition of MLX in the SC and stripped skin was increased by 1.2- and 2.2-fold, respectively, compared with MLX solution, and detectable amount of MLX was observed in the receptor fluid (see Figure 3 and Table 4). The flux and permeability coefficient of MLX were increased by CL reporting enhancement ratio (ER) 3.2 times of that reporting with MLX solution (see Figure 2 and Table 3). These results revealed that CL facilitates the permeation of MLX to the deeper tissues of the skin. The positive impact of CL on MLX permeation through skin was in correlation with others (Pabyton et al. 2013). The mechanisms were suggested to explain the enhancement of drug permeation through skin by liposomes. First, the similarity between the compositions of the
liposomes and biological membranes may facilitate the interaction and/or fusion between them. This fusion allows liposomes to liberate the loaded drug into SC and other skin layers (Chen et al. 2010, El Maghraby et al. 2001). Second, Phospholipids itself may interact with the cell membrane and perturb the integrity of the SC. Such interaction causes disruption in the integrity of the barrier nature of the SC (Pabyton et al. 2013). Further enhancement in drug permeation through skin could be achieved by altering the composition of liposomes. The permeation of MLX was further improved by using FLs (FL and FL1–FL4; Tables 3 and 4; Figures 2 and 3). FL showed improvement in MLX skin disposition, reporting 1.5-, 2.7-fold of MLX in SC and stripped skin, respectively, compared with MLX solution. MLX flux and ER were 11.5 and 7.3 times of that recorded with MLX solution, respectively (Table 3). This positive impact of FL on MLX disposition and permeation through skin is in agreement with other works using liposomes containing surfactants (El Zaafarany et al. 2010, Hiruta et al. 2006). Such improvement was attributed the influence of Tween 80 on the flexibility of liposomes. The flexibility of FL facilitates MLX migration through paracellular hydrophilic pores within the skin. FL can penetrate SC easily and subsequently change the intercellular lipids; this change alters integrity of the SC and hence increases the disposition of the drug into different skin layers (Monteiro et al. 2012). Further improvement in MLX permeation through skin was observed when ethanol being added to FLs (see Tables 3 and 4). It was reported that the
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A. H. ALOMRANI AND M. M. BADRAN
Figure 3. The amount of MLX delivered into the SC, stripped skin, and the receptor fluid after 6 hours of incubation with rat skin after applying a different type of liposomes.
Figure 4. % Inhibition in edema produced by optimized MLX loaded FLs gel formulation (FL4), and oral administration in carrageenan-induced rat paw.
presence of ethanol promotes MLX permeation and disposition through skin. Linear increases in MLX disposition in the skin layers and MLX permeation were observed with increasing ethanol concentration (see Tables 3 and 4; Figures 2 and 3). The ER of FL1, FL2, FL3, and FL4 was 14.8, 20.6, 25, and 31.5, respectively. F4 gave the highest flux (1.5 mg cm 2 h1) and permeation coefficient 143.3 10 5 cm h1. The improvement of MLX permeation through skin was in direct correlation with ethanol concentration. More specifically, F1, F2, F3, and F4 showed 2.9-, 3.8-, 4.9-, and 7.4-fold increase in MLX in SC compared with that obtained using MLX solution and 2.4-, 3.2-, 4.1-, and 6.2-fold increase compared with that obtained with CL. The amounts of MLX accumulation in the stripped skin were 2.6-, 3.5-, 4.4-, and 5.5-fold for F1, F2, F3, and F4, respectively, compared with CL. The positive impact of FL1–FL4 on the skin
disposition of MLX is in agreement with others (Dubey et al. 2007, Li et al. 2012, Lo´pez-Pinto et al. 2005). According to previous studies, several suggestions were proposed to explain the mechanism of permeation enhancement of FLs in presence of ethanol. One mechanism attributed the enhancement effect of FLs to the presence of ethanol, which may cause the following effects: (1) ‘‘pull effect’’ as a result of skin lipid bilayer disruption by ethanol and (2) ‘‘push effect’’ as a result of increasing of thermodynamic activity of MLX due to ethanol evaporation (Dubey et al. 2007). A synergistic effect of phospholipids and ethanol was also suggested (Chen et al. 2010). The scenario of this synergism starts by interaction of ethanol with polar head groups of lipid molecules of SC causing reduction in the transition temperature of these lipids and consequently increasing their fluidity and as a result, the structure of SC becomes loose and disordered. This action is followed by fusion of vesicles with skin lipids resulting in releasing of the vesicles content into the deep layers of the skin easily via the loosed SC (Dragicevic-Curic et al. 2010). Moreover, the higher EE% and accumulation of MLX in skin layers by FLs were observed.
In vivo antiinflammatory activity Because of its good skin penetration and permeation profiles, FL4 was selected as an optimized formula for in vivo antiinflammatory effect. Because FL4 is present as liquid form, it was prepared as gel, using carbopol as a gelling agent, to maintain its contact with the rat-paw skin during the application. The in vivo antiinflammatory study was performed in rats using carrageenan-induced rat-paw edema model (Winter et al. 1962). Figure 4 presents the antiinflammatory activity of MLXloaded FL4 gel and different control samples.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
According to Figure 4, the antiinflammatory effect of MLX gel reported 34% inhibition of edema, whereas, the oral form of MLX was almost equal to the conventional gel form of MLX. A significant antiinflammatory effect was shown by F4-loaded gel. This system reported antiinflammatory effect as 64% inhibition of edema, which is almost 2-fold of conventional MLX gel. These data are correlated with the skin permeation and penetration studies (see Figures 2 and 3). According to these findings, it could be concluded that MLX-loaded FLs can penetrate SC smoothly and improve MLX antiinflammatory activity which will significantly help in the treatment of the inflammation and revealed the positive impact of FLs on the skin delivery of drug.
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Conclusion In this study, various types of liposomes containing MLX (CL and FLs) were prepared and evaluated. In vitro skin penetration studies revealed that FL composed of phospholipids and Tween 80 delivered high amount of MLX to the SC and deeper skin layers compared with CL and solution. Further enhancement in MLX skin penetration was achieved when ethanol was added to the composition of the liposomes. Such improvement may be attributed to the ability of Tween 80 and ethanol to reduce the size and increase the flexibility of the vesicles in addition to the increase in EE%. The highest penetration and EE% were found in case of the optimized formulation FL4, prepared by 10% (w/v) of lipoid S100, 1.5% (w/v) of Tween 80%, and 40% (v/v) of ethanol. Moreover, F4-loaded carbopol gel revealed comparative higher inhibition of rat paw edema compared with that of oral administration. It can be concluded that FLs containing ethanol could be a promising carrier for cutaneous delivery of MLX.
Acknowledgements The authors also would like to thank Mr. Malik Saud Ahmad for his technical assessment in performing antiinflammatory test, and Riyadh Pharma Medical & Cosmetic Products Co. Ltd. (Riyadh, Saudi Arabia) for providing the free sample of MLX.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding information The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the research group project no RGP-VPP-287.
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