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International Journal of Biological Macromolecules 67 (2014) 99–104

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

nhanced anti-inflammatory activity of carbopol loaded meloxicamanoethosomes gel

bdul Ahad ∗, Mohammad Raish, Abdullah M. Al-Mohizea,ahad I. Al-Jenoobi, Mohd Aftab Alamepartment of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 16 February 2014eceived in revised form 5 March 2014ccepted 11 March 2014vailable online 19 March 2014

eywords:nti-inflammatory activitythosomes

a b s t r a c t

The aim of the current investigation is to develop nanoethosomes for transdermal meloxicam delivery.The ethosomes were prepared by varying the variables such as concentrations of phospholipids 90G,ethanol, and sonication time while entrapment efficiency, vesicle size and transdermal flux were thechosen responses. Results indicate that the nanoethosomes of meloxicam provides lesser vesicles size,better entrapment efficiency and improved flux for transdermal delivery as compared to rigid liposomes.The optimized formulation (MCEF-OPT) obtained was further evaluated for an in vivo anti-inflammatoryactivity in rats. Optimized nanoethosomal formulation with vesicles size of 142.3 nm showed 78.25%entrapment efficiency and achieved transdermal flux of 10.42 �g/cm2/h. Nanoethosomes proved to be

nflammationeloxicam

ransdermal

significantly superior in terms of, amount of drug permeated into the skin, with an enhancement ratio of3.77 when compared to rigid liposomes. In vivo pharmacodynamic study of carbopol® loaded nanoethoso-mal gel showed significant higher percent inhibition of rat paw edema compared with oral administrationof meloxicam. Our results suggest that nanoethosomes are an efficient carrier for transdermal deliveryof meloxicam.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The greatest obstacle for transdermal delivery is the barrierroperty of the stratum corneum (SC). Many overtures have beensed to breach the obstacle property of skin, amongst them the usef lipid vesicles is gaining interest to modulate the SC [1–3]. Therst papers to report on the effectiveness of conventional vesiclesliposomes) for skin delivery were published in the early 1980s [4].ater on, most studied concluded that liposomes only enhanced therug disposal in the skin, suggesting that vesicles are only useful foropical dermal delivery [5]. Conventional liposomes have been gen-rally reported to remain confined to the upper layer of the SC ando accumulate in the skin appendages, with minimal penetrationo deeper tissues, owing to their large size and lack of flexibility6–9]. Further intensive research over the past years led to thentroduction and development of a new class of lipid vesicles, the

ltra-elastic liposomes that have been termed as ethosomes. Etho-omes, are novel lipid carriers which are composed of phospholipid,thanol and water. Ethosomes were reported to enhance the skin

∗ Corresponding author. Tel.: +966 557124812.E-mail addresses: abdulahad20@yahoo.com, aahad@ksu.edu.sa (A. Ahad).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.03.011141-8130/© 2014 Elsevier B.V. All rights reserved.

permeation of drugs due to the interdigitation effect of ethanol onthe lipid bilayer of liposomes and increasing fluidity of SC lipids[10,11]. The high flexibility of vesicular membranes from the addedethanol permits the elastic vesicles to squeeze themselves throughthe pores, which are much smaller than their own diameters; thus,ethosomal systems are considerably more efficient in deliveringsubstances to the skin in terms of quantity and depth than eitherconventional liposomes or hydroalcoholic solution [12–14].

In present investigation, we attempted to develop nanoetho-somes loaded with meloxicam (MC) (Fig. 1) for improvedpercutaneous permeation. MC has been previously identified asa promising candidate for transdermal drug delivery [15–21]. MCclassified as a BCS class II drug (high permeability and poor solu-bility) is a potent, nonsteroidal anti-inflammatory drug approvedby FDA in 2000 for the treatment of arthritis, osteoarthritis anddegenerative joint disease [22]. In view of the characteristics of MCincluding small oral dosage (7.5–15 mg/day), low molecular weight(354.1 daltons), good solubility in lipophilic solvents (log P = 1.9,in octanol–water), excellent tissue tolerability and adverse effects

on the gastrointestinal tract such as stomachache and indiges-tion, and patient compliance are weaknesses of oral and injectableMC administrations [23–25], it seems that there is potential forinvestigating the MC loaded nanoethosomal system. In this study,

100 A. Ahad et al. / International Journal of Biological Macromolecules 67 (2014) 99–104

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Table 1Formulation code and composition of MC ethosomal formulation (MCEF).

Runs PL90 (% w/v) Ethanol (% v/v) Sonication time (min)

MCEF1 2 25.0 2MCEF2 2 42.5 5MCEF3 2 60.0 8MCEF4 3 25.0 2MCEF5 3 42.5 5MCEF6 3 60.0 8MCEF7 4 25.0 2MCEF8 4 42.5 5

(T − C)/T × 100,

where T is the amount of total MC and C is the amount of MCdetected only in the supernatant. The MC analyses were carried out

Table 2Formulation code and observed response for MC ethosomal formulation (MCEF).

Formulations Vesicle size (nm) PDI EE (%) Flux (�g/cm2/h) ERa

MCEF1 112.6 0.247 68.82 4.20 1.52MCEF2 102 0.210 75.18 5.85 2.12MCEF3 41.21 0.281 66.65 3.53 1.28MCEF4 153.6 0.156 79.22 5.28 1.91MCEF5 142.3 0.261 78.25 10.42 3.77MCEF6 107 0.263 69.39 6.32 2.28MCEF7 217 0.590 80.65 5.18 1.87MCEF8 207.9 0.404 85.20 7.40 2.67

Fig. 1. The chemical structure of meloxicam.

anoethosomes were used as a novel MC transdermal therapeu-ic system (TTS). The system was developed and evaluated forts physicochemical characteristics, such as vesicle size, shape,ntrapment efficiency (EE%), in vitro skin permeation and in vivonti-inflammatory activity.

. Materials and methods

.1. Materials

MC was purchased from Winlab Laboratory Chemicals Leices-ershire, UK. Phospholipon® 90G (PL90G) was received as a gratisample from Lipoid AG, Sennweidstrasse, CH-6312 Steinhausen,witzerland. Ethanol was purchased from Avonchem, Maccles-eld Cheshire, UK. High Performance Liquid ChromatographyHPLC) grade acetonitrile was purchased from BDH, Poole, England.arbopol® 934 and potassium dihydrogen orthophosphate wereurchased from Alpha Chemika Mumbai, India and Loba Chemievt. Ltd. Mumbai, India respectively. Carrageenan and cholesterolere purchased from Sigma–Aldrich, St. Louis, MO, USA and FlukaG, Buchs, Switzerland respectively. Polyethylene glycol-400 andriethanolamine were purchased from Merck, Germany. Doubleistilled water was used for all experiments.

.2. Animals

Wistar albino rats weighing approximately (180–200 g) werebtained from Experimental Animal Care Center, College of Phar-acy, King Saud University, Riyadh, Saudi Arabia. Animals wereaintained in accordance with the recommendations of the ‘Guide

or the Care and Use of Laboratory Animals approved by the centerNIH publications no. 80-23; 1996). All animals were maintainednder standard laboratory conditions on a 12 h light/dark cycle at5 ◦C ± 2 ◦C. The animals were given a pellet diet with water ad

ibitum. The hairs on the skin of animals were clipped and sub-utaneous tissues were surgically removed, and dermis side wasiped with isopropyl alcohol to remove residual adhering fat [26].

he skin samples were mounted over the diffusion cells in such aay that SC side faced the donor compartment whereas the dermis

aced the receiver compartment [27,28].

.3. Preparation of nanoethosomes

MC ethosomal formulations (MCEF) were prepared by thin layervaporation technique [29,30]. Precisely, PL90G and the drug wereaken in a clean, dry, round bottom flask and dissolved in chlo-oform: methanol, 2:1 v/v. The organic solvent was removed byotary evaporation above the lipid transition temperature (BuchiTM

otavaporTM R-210, Zurich, Switzerland). Final traces of solvent

ere removed under vacuum overnight. The deposited lipid filmas hydrated with different concentration of ethanolic-PBS (pH

.5) mixture (Table 1) by rotation at 60 rpm for 1 h at room tem-erature. The resulting vesicles were swollen for 2 h at room

MCEF9 4 60.0 8

Abbreviations: MC, meloxicam; PL90, Phospholipon® 90G.

temperature to get large multilamellar vesicles. To prepare smallervesicles, large multilamellar vesicles were probe sonicated usingtitanium probe Sonopuls HD 2070 (Bandelin electronic GmbH & Co.KG, Berlin). The sonicated vesicles were extruded through a sand-wich of 200 and 450 nm polycarbonate membranes (Chromafil®

Xtra, Macherey-Nagel GmbH & Co. KG, Germany). The optimiza-tion of formulations was carried out by varying PL90G and alcoholconcentration from 2 to 4% and 25–60%, and sonication time(2–8 min) respectively. The compositions of various formulationsare reported in Table 1. The vesicle size, EE% and transdermal fluxobtained from in vitro skin permeation study of nanoethosomesbearing MC are presented in Table 2.

2.4. Vesicles shape, size and size distribution

Nanoethosomes vesicles were visualized by using a transmis-sion electron microscope (JEM-1011, JEOL, Tokyo, Japan) [31].The vesicles size, and polydispersity index (PDI) were determinedby the dynamic light scattering method, using a computerizedinspection system Zetasizer Nano ZS (Malvern Instruments, UnitedKingdom) at 25 ± 1 ◦C and at a scattering angle of 90◦ [32,33].

2.5. Determination of vesicle EE%

EE% expressed as a percentage of the total amount of MC foundin the studied formulations at the end of the preparation proce-dure. The EE% of MC vesicles was determined by ultracentrifugationat 30,000 rpm and 4 ◦C for 1 h using T-1250 rotor, Sorvall Discov-ery 90SE ultracentrifuge (Hitachi, Tokio, Japan) [34,35]. Followingcentrifugation, the supernatant and vesicles were separated. Thesupernatant was removed and drug quantity was analyzed by HPLC.The EE% was calculated as follows:

MCEF9 166 0.329 71.85 5.63 2.03

Abbreviations: EE (%), entrapment efficiency; ER, enhancement ratio; MC, meloxi-cam; PDI, polydispersity index.

a In comparison to conventional liposomes.

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A. Ahad et al. / International Journal of

ith a liquid chromatography (Shimadzu Class-VPV 5.02 instru-ent), equipped with a UV detector. Separation was achieved using

n Atlantis (250 × 4.6 mm ID, 5.0 �m) column (Water®, Milfordassachusetts, USA). Binary elution was carried out at a flow rate

.0 mL/min with the mobile phase containing acetonitrile: water:% aq. glacial acetic acid (56:34: 10% v/v/v). Mobile phase was pre-ared daily, filtered by passing through a 0.45 �m membrane filternd degassed. All chromatographic separations were performedt room temperature. Detection was carried out at 362 nm withV-detector [36].

.6. In vitro skin permeation studies

The in vitro skin permeation of MCEF was studied using trans-ermal franz diffusion sampling system (Logan Instrument Corp.,J, USA) with an effective permeation area and receptor cell vol-me of 1.7 cm2 and 12 mL, respectively. The temperature of theeceiver vehicle (ethanol: PBS, pH 7.4) (20:80)) was maintainedt 37 ± 1 ◦C and was constantly stirred by a magnetic stirrer at00 rpm [37,38]. The rat skin was subsequently carefully checkedhrough a magnifying glass to ensure that skin samples were freerom any surface irregularity such as tiny holes or crevices in theortion that was used for transdermal permeation studies. The skinas later mounted on a receptor compartment with the SC side

acing toward the donor compartment. 1 mL of nanoethosomes ofC was placed in the donor compartment. Samples of 500 �L wereithdrawn from the receptor compartment via the sampling port atifferent time intervals i.e. 0, 1, 2, 3, 4, 6, 8, and 24 h and analyzed forrug content by HPLC method [36]. The receptor phase was imme-iately replenished with equal volume of fresh diffusion buffer.imilar experiments were performed with conventional liposomalormulations. In order to determine the extent of enhancement, annhancement ratio (ER) was calculated as follows:

R = steady state flux of formulationsteady state flux of control

.7. In vivo anti-inflammatory activity

Carrageenan induced rat paw edema volume model [18,39]as used to assess the anti-inflammatory activity of developedanoethosomal MC carbopol® gel. Left hind paws of each rat werearked just beyond the tibiotarsal junction, so that every time the

aw is dipped up to the fixed mark to ensure constant paw volume.he rats (180–200 g) were randomly divided into four groups ofix rats each. Group A, normal control received normal saline only.dema was induced in the remaining groups (Groups B–D). Group

(toxic control) received carrageenan only without the drug. Theroups C and D received an application of MCEGF-OPT (1 g), andC oral treatment respectively. One hour after the gel application,

ub plantar injection of 0.1 mL of a 1% w/v freshly prepared car-ageenan in normal saline was given into the left hind paw of eachat. Measurements of the paw volume up to the ankle joint wereerformed before and at different time intervals (1, 2, 3, 4, 5, 6, 8,0, 12 h) following the carrageenan injection using plethysmome-er (UGO Basile 7140, Italy). Percentage reduction in edema wasalculated as follows:

Inhibition = % edema (control) − % edema(formulation treated)% edema (control)

.8. Statistical analysis

Data were analyzed using the GraphPad Instat software (Graph-ad Software Inc., CA, USA). Data are expressed as mean ± standardeviation (S.D.) and were assessed by paired t-test throughout the

ical Macromolecules 67 (2014) 99–104 101

comparisons of edema size. p < 0.05 was considered to show signif-icant difference for all comparisons made.

3. Results and discussion

Ethosomes are vesicular system with hydrated bilayers. MCloaded nanoethosomes were prepared by thin layer evaporationtechnique using varying concentration of phospholipid, ethanoland sonication time and were characterized for appropriate phys-icochemical attributes (Tables 1 and 2).

3.1. Effect of variables on vesicles size analysis

The vesicle size of various MCEF is presented in the Table 2. Theleast vesicle size was observed for MC loaded ethosomes formu-lations MCEF3 is 41.21 nm while the maximum vesicle size wasobtained as 217 nm for MCEF7 were found lower vesicle size thanthat of conventional liposomes (249 nm) prepared by the samemethod. Inclusion of ethanol used for formulation of ethosomeswhich interact with lipid bilayers, instead of cholesterol (conven-tional liposomes), could explain this reduction in vesicle size. Thesize distribution of vesicles was determined by dynamic light scat-tering. We observed an initial decrease in the average size of thevesicles with increasing amounts of ethanol. However, a furtherincrease in the ethanol concentration from 42.5% up to 60% leads toa further reduction in the size of vesicles; these results further sup-ported by the Dubey et al. [14]. This indicates that at higher ethanolconcentration, the membrane thickness is reduced considerably,probably due to the formation of a phase with interpenetratinghydrocarbon chain. Further, an interesting possible mechanismgiven by Lasic et al. [48], which state that ethanol causes a modifi-cation of the net charge of the system and confers it some degree ofsteric stabilization that may finally lead to a decrease in the vesiclesize, could also be considered. The effects of variables on vesiclessize is illustrated in Fig. 2.

3.2. Effect of variables on EE%

Primarily, the EE% of MC increased significantly with increas-ing ethanol concentration from 25% to 42.5%, which could be dueto its co-solvent effect. Therefore, the more drug amount could beaccommodated in the aqueous core of the vesicles however, fur-ther increase in ethanol concentration to 60% showed a decrease inEE% because ethanol makes the vesicles leakier and this lead to areduction in EE% of vesicles (Table 2). The EE% was least found with66.65% for the formulation MCEF3 and maximum EE% was found85.20% for MCEF8. The formulation MCEF5 (PL90G (3%): ethanol(42.5%)) showed optimum EE%. It was observed from Table 2 thatEE% has a direct positive relationship with concentration of phos-pholipid. As the concentration of the phospholipid increase, the EE%increases, but it also depends upon other factors such as amount ofethanol. The effects of variables on EE% is presented in Fig. 3.

3.3. Effect of variables on skin permeation studies

In vitro skin permeation studies from nanoethosomes wereperformed. The in vitro permeation profile of MCEF show thatethosomes formulation (MCEF5) presents maximum flux value, i.e.10.42 �g/cm2/h over rigid liposome formulation (2.77 �g/cm2/h)with an enhancement ratio of 3.77 through rat skin. The reason forthis better performance of ethosomes formulations is the flexibil-ity and the ability to retain vesicle integrity, while the aggregates

undergo a dramatic change in shape in comparison with conven-tional liposomes, and all these characteristics allow the ethosomesto pass through the skin pores, which are much smaller than etho-somes diameter [40,41]. The effects of variables such as PL90G,

102 A. Ahad et al. / International Journal of Biological Macromolecules 67 (2014) 99–104

Fig. 2. Effect of PL90G, ethanol and sonication time on vesicular size of MC-loaded nanoethosomal formulations.

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Fig. 3. Effect of PL90G, ethanol and sonication time on ent

thanol and sonication on transdermal flux is shown in Fig. 4 andhe cumulative percentage of drug release via rat abdominal skinersus time profile of various meloxicam loaded ethosomal formu-ations is illustrated in Fig. 5.

Fig. 4. Effect of PL90G, ethanol and sonication time on transd

ent efficiency of MC-loaded nanoethosomal formulations.

In our study, we observed that the transdermal flux firstincreased with increasing ethanol concentration and thendecreased (Table 2). These results suggested that a too low or atoo high concentration of ethanol is not beneficial in vesicular

ermal flux of MC-loaded nanoethosomal formulations.

A. Ahad et al. / International Journal of Biological Macromolecules 67 (2014) 99–104 103

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was 25.23%. These results are similar to those reported by Khuranaet al. [15,18,47], who measured the anti-inflammatory activity ofnano-size formulations based MC gels.

Fig. 5. Cumulative percentage of drug release via rat abdominal skin

elivery through skin. These findings are in agreement with pub-ished report [42]. A possible explanation for lower drug delivery at

high ethanol concentration may be that the ethanol at high con-entration decreased the EE% and disrupted the lipid membrane sohat it becomes leakier to the entrapped drug. This will, in turn,educe the delivery.

The optimized formulation of MC-loaded nanoethosomes sys-ems was selected based on the criteria of attaining the maximumalue of transdermal flux and EE%; minimizing the vesicles size.he formulation composition with PL90G (3% (w/v)), ethanol (42.5%v/v)), and sonication time (5 min) was found to fulfill requisitesf an optimized formulation i.e. MCEF-OPT. The optimized formu-ation has the EE% of 78.25% with vesicles size and transdermalux across rat skin of 142.3 nm and 10.42 �g/cm2/h, respectively.he transmission electron micrograph of MCEF-OPT is shown inig. 6 (A). They show the outline and core of the well identifiedpherical vesicles, displaying sealed nanovesicular structure. Sizeistribution of optimized MCEF-OPT nanoethosomal loaded withC is presented in Fig. 6 (B).Optimized nanoethosomal formulation (MCEF-OPT) was

elected for further in vivo pharmacodynamic activity. The MCEF-PT was converted into gel. Briefly, Carbopol® 934 (1% (w/w)) wasdded into water and kept overnight for complete humectation ofolymer chains [43]. The MCEF-OPT was added slowly to hydratedarbopol® solution with stirring [44,45]. Other ingredients like5% w/v polyethylene glycol-400 and triethanolamine (0.5%w/v)) were added to get homogeneous dispersion of gel and thisptimized MC loaded ethosomal gel formulation (MCEGF-OPT) ismployed for in vivo anti-inflammatory study.

.4. In vivo anti-inflammatory activity

The in vivo anti-inflammatory activity of the prepared MCEGF-PT was evaluated in wistar rats using carrageenan-induced

at-paw edema model [46]. Results revealed that injection ofarrageenan (selected as inflammagen) produced a pronounceddema. The percent inhibition of swelling of rat paw edema forCEGF-OPT and MC-oral administration at various time intervalsere presented in Fig. 7. The prepared MCEGF-OPT showed higher

nhibition of swelling of rat paw edema compared with that of oral

dministration over a period of 12 h, indicating improved perme-tion profile by the newly formulated MCEGF-OPT. Peak activity (%nhibition of edema) for oral administration was observed between

and 5 h as shown in Fig. 7. MCEGF-OPT application resulted in 19%

s time profile of various meloxicam loaded ethosomal formulations.

inhibition at the end of 1 h and it further increase to 65% at 12 h.Inhibition produced by the MC oral administration was found to be22 and 25% after 1 and 12 h respectively (Fig. 7).

Statistical evaluation of the mean percentage inhibition ofedema shows a significant difference between MCEGF-OPT com-pared with oral administration as reference using paired t-test(p < 0.05). Our results revealed that the MCEGF-OPT, showed 65%inhibition of edema after 12 h, while that of the oral administration

Fig. 6. (A) Visualization of optimized meloxicam loaded nanoethosomal formu-lation (MCEF-OPT) by transmission electron microscopy. (B) Size distribution ofMCEF-OPT.

104 A. Ahad et al. / International Journal of Biological Macromolecules 67 (2014) 99–104

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ig. 7. Time-dependent % inhibition in edema produced by optimized meloxicam loanduced rat paw.

. Conclusion

Various MC-loaded nanoethosomal vesicles were preparedased on thin layer evaporation technique. Highest transdermalux (10.42 �g/cm2/h) was found in case of optimized formula-ion MCEF5-OPT, prepared using 3% (w/v) Phospholipon® 90G,2.5% (v/v) ethanol sonicated for 5 min. The entrapment efficiencynd vesicles size of MCEF5-OPT were found to be 78.25% and42.3 nm respectively. The in vivo anti-inflammatory activity in ratssing carrageenan-induced rat-paw edema model demonstratedomparative higher inhibition of swelling of rat paw edema byarbopol® gel containing MC-loaded nanoethosomes comparedith that of oral administration. Overall, these findings concluded

hat nanoethosomes accentuates the transdermal flux of MC andould be used as a carrier for effective transdermal delivery of MC.

onflict of interest

All authors have approved the final manuscript and the authorseclare that they have no conflicts of interest to disclose.

cknowledgement

The authors would like to extend their sincere appreciation tohe Deanship of Scientific Research at King Saud University for itsunding of this research through the Research Group Project no.GP-VPP-268.

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