http://informahealthcare.com/phdISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, Early Online: 1–18! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.882939

RESEARCH ARTICLE

Quality by design approach for formulation, evaluation and statisticaloptimization of diclofenac-loaded ethosomes via transdermal route

Shashank Jain, Niketkumar Patel, Parshotam Madan, and Senshang Lin

College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA

Abstract

The objective of this study was to fabricate and understand ethosomal formulationsof diclofenac (DF) for enhanced anti-inflammatory activity using quality by design approach.DF-loaded ethosomal formulations were prepared using 4� 5 full-factorial design withphosphatidylcholine:cholesterol (PC:CH) ratios ranging between 50:50 and 90:10, and ethanolconcentration ranging between 0% and 30% as formulation variables. These formulations werecharacterized in terms of physicochemical properties and skin permeation kinetics. Theinteraction of formulation variables had a significant effect on both physicochemical propertiesand permeation kinetics. The results of multivariate regression analysis illustrated that vesiclesize and elasticity of ethosomes were the dominating physicochemical properties affectingskin permeation, and could be suitably controlled by manipulation of formulation variables tooptimize the formulation and enhance the skin permeation of DF-loaded ethosomes.The optimized formulation had ethanol concentration of 22.9% and PC:CH ratio of 88.4:11.6,with vesicle size of 144 ± 5 nm, zeta potential of �23.0 ± 3.76 mV, elasticity of 2.48 ± 0.75 andentrapment efficiency of 71 ± 4%. Permeation flux for the optimized formulation was12.9 ± 1.0mg/h cm2, which was significantly higher than the drug-loaded conventionalliposome, ethanolic or aqueous solution. The in vivo study indicated that optimized ethosomalhydrogel exhibited enhanced anti-inflammatory activity compared with liposomal and plaindrug hydrogel formulations.

Keywords

Anti-inflammation, design of experiment,diclofenac, ethosomes, permeation, qualityby design

History

Received 5 November 2013Revised 31 December 2013Accepted 4 January 2014Published online 3 February 2014

Introduction

Diclofenac (DF) is a widely prescribed non-steroidal anti-inflammatory drug (NSAID) that exhibits anti-inflammatory,anti-osteoarthritic and analgesic activities. Its oral administrationis associated with severe gastrointestinal (GI) side effects such asbleeding, ulceration or perforation of the intestinal wall. It isestimated that approximately 75 000 people are hospitalized and7500 people die each year due to the NSAIDs, especiallyDF-related GI side effects1. Furthermore, the short half-life(1–2 h) and extensive first-pass metabolism attenuate the oralbioavailability of DF to merely 50%2. Due to these impediments,international guidelines for osteoarthritis care and AmericanGeriatrics Society strongly recommend transdermal route as analternative to oral DF administration3. Along with minimizingthe first-pass metabolism and GI side effects, transdermal routealso provides a site-specific application, enhanced physiologicaland pharmacological response, and better patient compliance.

The greatest challenge for the researchers, till date, is toovercome the inherent limitations of transdermal drug absorptionimposed by impervious stratum corneum of skin. This is obvious

from the fact that despite commercial success of transdermal DFpreparations, most of these marketed products are consideredtherapeutically ineffective due to the inability of DF to crossstratum corneum4. The best known marketed transdermal formu-lation of DF, Voltaren Emulgel�, is considered only 10%biologically available and needs to be supplemented with anoral Voltaren dosage form to ensure suitable therapeutic effects ofthe drug5. The other marketed transdermal DF formulations arefound to be less effective than Voltaren Emulgel�6.

Many techniques have been utilized to improve transdermaldrug absorption via physical enhancers (e.g. iontophoresis andmicroneedle), chemical enhancers (e.g. sulphoxides, glycols,alkanols and terpenes), and delivery systems (e.g. liposome,transfersome and microemulsion)7,8. Many of these approacheshave also been investigated to enhance skin delivery of DF9–12.However, these approaches are subjected to some seriouslimitations. For instance, physical approaches are mostly painfuland expensive while chemical permeation enhancers are known tocause permanent skin damage. Lipid-based colloidal deliverysystems, especially liposomes, have shown promise to deliverdrugs across stratum corneum, but with limited commercialsuccess13–15. This is due to the fact that the conventionalliposomes are mostly confined in the upper layer of the stratumcorneum with limited penetration to the deeper tissues, owing totheir large vesicle size and lack of elasticity16.

A promising lipid-based recent alternative for transdermaldelivery is ethosomes, which are elastic lipid vesicles composedmainly of lipids and ethanol (10–40%). Although conceptuallynovel and sophisticated, ethosomes are characterized by

Address for correspondence: Senshang Lin, Ph.D., College of Pharmacyand Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens,NY 11439, USA. Tel: (001) (718) 990 5344. Fax: (001) (718) 990 1877.E-mail: linse@stjohns.edu

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simplicity in their preparation, along with their overall safety, andefficacy – a combination that can highly expand their applicationin pharmaceutical and cosmetic industries. The skin permeationenhancement via ethosomes is attributed to the presence ofethanol that increases fluidization of highly ordered lipophilicstructure of stratum corneum and modulates the physicochemicalcharacteristics of ethosomes by lowering the main transitiontemperature (Tm) of the lipids. It has also been reported thatethosomes show no significant irritation to human skin and causeno toxicity to the cells in vitro17,18.

Most research studies on ethosomes have either focused on(i) the effect of formulation variables on physicochemicalproperties of ethosomes18–20, (ii) skin permeation mechanism21,22

and/or (iii) comparison of skin permeation with conventionalliposomes or marketed formulation20–24. These studies have notexplored the interaction of formulation variables and physico-chemical properties on the skin permeation kinetics of ethosomes.The latter information is particularly relevant in order tomanipulate and control the ethosomal delivery system to achievethe desired transdermal permeation, which can also eventuallyprovide a basis for commercialization of these products in themarket. Also, despite the overwhelming success of therapeuticpotential of ethosomes, no research has been reported utilizingthis carrier for DF, which is one of the most prescribed drugfor inflammation worldwide25.

The present investigation was undertaken to utilize quality bydesign (QbD) approach to develop the DF-loaded ethosomaldelivery system and to understand the effect of interaction offormulation variables and physicochemical properties in enhan-cing transdermal DF permeation in a defined design space.The QbD approach emphasizes the understanding of variouscomponents of the system for improved control over desiredoutput. Design of experiments (DoE) and multivariate statisticaldata analysis are essential elements of QbD, recognized by recentInternational Conference of Harmonization Q8 guideline26. Thesetools facilitate varying all the formulation variables simultan-eously, allowing quantification and prioritizing the effectsproduced by these variables, along with any possible interactionbetween them, in the defined design space. Following thefabrication of DF-loaded ethosomal formulations, they werecharacterized for physicochemical properties and skin permeationkinetics. The effect of formulation variables on physicochemicalproperties and skin permeation kinetics of ethosomal formulationswas studied using QbD approach. Since skin permeation ofethosomes is mainly controlled by their physicochemical charac-teristics, the physicochemical properties of ethosomal formula-tions were correlated with permeation flux using multivariateregression analysis to identify dominant properties affectingskin permeation. Based on the knowledge gained by regressionanalysis, the concept of ‘‘desirability function’’ was applied toachieve optimum formulation conditions in the defined designspace. The optimized formulation was incorporated into asuitable hydrogel to improve skin retention during transdermalapplication. Anti-inflammatory activity of optimized DF-loadedethosomal formulation was then evaluated in Sprague-Dawley(SD) rats.

Materials and methods

Materials

Soy phosphatidylcholine (PC) (99% pure) was purchased fromAvanti Polar Lipids (Alabaster, Al). DF sodium, cholesterol (CH),chloroform, ethanol, glacial acetic acid, acetonitrile, carrageenanand rhodamine 123 were purchased from Sigma Chemicals(St. Louis, MO). All chemicals were of analytical grade and usedas received.

Assay of DF

DF assay was performed based on the reported literature withslight modification to avoid any interference by solvents27.Briefly, the method employed a reverse-phase HPLC (HP1100series, Agilent Technologies, Wilmington, DE) with a4.6 mm� 250 mm C-18 column (Macherey-Nagel, Bethlehem,PA). The mobile phase consisted of 60% v/v acetonitrile and 40%v/v of 0.5% v/v acetic acid. A flow rate of 2 ml/min was set andDF content was detected at UV wavelength of 280 nm. Theretention time of DF was 4.5 min. The area under the peak wasused to calculate the concentration of DF and linearity over theconcentrations ranging between 1 and 500mg/ml was evaluated.The peak area was observed to increase linearly with respectto the increase in DF concentrations with correlation coefficient(r2) of 0.9987. All measurements were performed in triplicatefor two days.

Preparation of DF-loaded ethosomes

DF-loaded ethosomes and liposomal control formulations wereprepared by the rotary evaporation-sonication method28. Briefly,accurately weighed amounts of soy PC and CH were dissolved in10 ml of chloroform (5% w/w total lipid concentration) in a clean,dry, round-bottom flask. The organic solvent was removedby rotary evaporation at 90 rpm for 15 min at 45 �C to obtainhomogeneous thin lipid film on the inner surface of the flask andwas kept under vacuum overnight to achieve complete drying.The deposited lipid film was then hydrated with 1% w/v DFsodium at room temperature, either in ethanolic solution (10%,15%, 20% and 30% v/v) for ethosomal formulations or distilledwater for liposomal formulations (0% ethanol), by vigorousvortexing for 15 min. The resulting multilamellar vesicles (MLVs)were kept at 4 �C overnight for swelling. In order to preparevesicles of smaller size (SMLVs), the obtained MLVs weresonicated by a bath sonicator at 4 �C for predetermined timeand extruded 11 times through a sandwich of 400 nm followed by200 nm polycarbonate membrane filter assembly29.

Effect of formulation variables of DF-loaded ethosomes

To study the effect of formulation variables on physicochemicalproperties and skin permeation kinetics of DF-loaded ethosomes,a full-factorial design was applied with four levels of PC:CHratio and five levels of ethanol (Table 1). The full-factorialdesign enables all the formulation variables to be variedsimultaneously, allowing quantification of the effects producedby these variables along with any possible interactionbetween them.

Effect of ethanol concentration

Ethanol in the hydration phase of ethosomes manipulates thephysicochemical properties of the ethosomes to enhance skinpermeation30. However, it has been reported that higher ethanolconcentration can cause leakage of drug from the ethosomes aswell as severe skin irritation. Therefore, based on publishedreports, ethanol concentration of 0%, 10%, 15%, 20% and 30% v/vwas studied20,31.

Effect of PC:CH ratio

The ratio of PC:CH significantly influences the physicochemicalproperties and skin permeation kinetics of ethosomes32,33.Therefore, lipid phase with PC:CH (w/w) ratio of 50:50, 70:30,80:20 and 90:10 was studied to cover a wide concentration rangeof the lipid ratios. The PC:CH ratio at values lower than 50:50 andhigher than 90:10 was not used because they were unable to

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produce uniform thin lipid film in round bottom flask duringpreliminary investigation.

Physicochemical characterization of DF-loadedethosomes

The DF-loaded ethosomal and control liposomal formulationsobtained from 4� 5 full-factorial design were characterized interms of their physicochemcal properties to understand the effectof formulation variables on these physicochemcial properties.Since physicochemical properties subsequently affect skin per-meation kinetics of ethosomes, the information derived from thisstudy would also help in achieving better understanding andcontrol over skin permeation of these lipid vesicles.

Morphology and lamellarity

The surface morphology and lamellarity of vesicles wereconfirmed with a confocal laser-scanning microscope (CLSM)equipped with a 488 nm argon, 568 nm krypton and 647 nmHe–Ne laser (Leica TCS-SP 2, Exton, PA). Briefly, the obtaineddried lipid film (PC:CH composition of 70:30) was hydrated with10 ml of a 20% ethanolic solution of rhodamine 123 (0.015 mg/ml) instead of DF. The un-entrapped rhodamine 123 was removedby ultracentrifugation at 30 000 rpm for 30 min (4 ± 0.5 �C). Asmall portion of the pellet settled at the bottom of the centrifugetube was transferred to a microscope slide, covered with a coverslip and observed directly under CLSM.

Phase transition temperature

The effects of ethanol concentration and PC:CH ratio on phasetransition temperature were determined using differential scan-ning calorimetry (Diamond DSC, Perkin Elmer, Waltham, MA)

calibrated with indium23. Briefly, samples weighing approxi-mately 5 mg were placed in the porous aluminum pan and heatingtemperature was set in the range of �10 to 50 �C at the scanningrate of 10 �C/min using dry nitrogen flow.

Vesicle size

The mean diameter and size distribution of various formulationswere determined by photon correlation spectroscopy (PCS)using particle-sizing systems (PSS) Nicomp 380/ZLS, (PSS,Santa Barbara, CA) and the polydispersity index was usedas a parameter of size distribution. All vesicles were diluted100-fold with de-ionized water before size measurements.The determinations were performed in triplicate at roomtemperature.

Zeta potential

The zeta potential of various formulations was measured usingPSS (Nicomp 380/ZLS). The samples were diluted with de-ionized water to obtain adequate count rate. The determinationswere performed in triplicate. All zeta potential measurementswere conducted at room temperature.

Elasticity

The elasticity of ethosomes and control liposomes was determinedas reported previously with few modifications2,12. The deviceused for this study was built exclusively for this purpose. Thedispersion (0.5 ml) was loaded into a gas tight syringe andextruded through a 50-nm membrane, held in a metal filter holder,at a constant pressure for 45 s. The amount of dispersion extrudedduring 45 s was determined and the vesicle size as well as sizedistribution before and after extrusion was obtained by PCS

Table 1. Vesicle size, zeta potential, elasticity and EE of DF-loaded ethosomal and liposomalformulations fabricated from 4� 5 full-factorial design (n¼ 3).

Formulation codeaVesicle size(nm ± SD)b

Zeta potential(mV ± SD)

Elasticity(mean ± SD)

EE(% ± SD)c

PC:CH¼ 50:50A0 252 ± 13 �5.38 ± 0.15 0.05 ± 0.04 34.6 ± 2.2A10 223 ± 8 �13.61 ± 0.77 0.37 ± 0.06 34.8 ± 2.3A15 208 ± 3 �16.14 ± 1.20 0.62 ± 0.11 42.1 ± 0.9A20 201 ± 3 �20.00 ± 0.80 0.81 ± 0.02 48.2 ± 1.2A30 132 ± 8 �23.05 ± 0.85 1.05 ± 0.14 44.1 ± 0.7PC:CH¼ 70:30B0 237 ± 5 �4.97 ± 0.27 0.11 ± 0.18 45.9 ± 0.9B10 204 ± 7 �12.79 ± 0.92 1.01 ± 0.09 52.4 ± 1.4B15 194 ± 2 �15.30 ± 1.10 1.03 ± 0.03 54.0 ± 0.2B20 191 ± 2 �20.00 ± 0.88 1.09 ± 0.07 55.2 ± 1.0B30 123 ± 3 �21.63 ± 1.00 1.32 ± 0.18 47.4 ± 1.1PC:CH¼ 80:20C0 200 ± 9 �4.31 ± 0.55 0.42 ± 0.03 49.6 ± 1.1C10 193 ± 9 �12.21 ± 0.38 1.39 ± 0.07 59.8 ± 0.6C15 183 ± 3 �13.71 ± 0.43 1.51 ± 0.04 61.9 ± 0.2C20 172 ± 4 �19.80 ± 0.89 1.55 ± 0.05 62.0 ± 0.7C30 120 ± 3 �20.85 ± 0.90 1.83 ± 0.04 57.1 ± 1.0PC:CH¼ 90:10D0 183 ± 12 �4.30 ± 0.28 0.62 ± 0.13 53.6 ± 1.2D10 178 ± 4 �9.25 ± 0.35 1.63 ± 0.04 62.4 ± 1.1D15 170 ± 2 �11.81 ± 0.94 1.64 ± 0.02 65.0 ± 1.4D20 132 ± 6 �23.00 ± 0.76 1.78 ± 0.10 72.6 ± 2.4D30 120 ± 4 �28.08 ± 0.82 3.20 ± 0.13 61.2 ± 1.8

PC, Phosphatidylcholine; CH, cholesterol.aA, B, C and D represent PC:CH ratio of 50:50, 70:30, 80:20 and 90:10 w/w, while 0 (liposomes), 10,

15, 20 and 30 represent % v/v of ethanol concentration, respectively.bVesicle size data represent the size obtained from bath sonication (sonication time of 20 min)

followed by extrusion.cEE represents data from the dialysis method.

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 3

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measurement. The filter was also checked visually for clogging.Elasticity was calculated using the following equation34:

E ¼ J � rv

rp

� �2

, ð1Þ

where E is the elasticity of vesicles, J is the amount of dispersionextruded during 45 s, rv is the vesicles size (after extrusion)and rp is the pore size of the barrier. All determinations wereperformed in triplicate.

Entrapment efficiency

The entrapment efficiency (EE) of drug-loaded ethosomes isgenerally determined using either dialysis or ultracentrifugationtechniques. However, the literature reports regarding the suitabil-ity of dialysis and ultracentrifugation techniques for determiningthe EE of ethosomes are not consistent18,20,35–37. Therefore, EEof DF-loaded ethosomal and control liposomal formulations wasdetermined using both techniques.

Determination of drug entrapment using ultracentrifugationwas performed at 30 000 rpm at 4 �C for 30 min. The obtainedethosomal or liposomal pellet at the bottom of the centrifuge tubeafter ultracentrifugation was then lysed with 1% v/v Triton X100and analyzed for drug content by the HPLC method36.

The Spectra/Por 2 dialysis membrane (molecular weight cutoff 12 000–14 000) was used to determine EE of ethosomal andliposomal formulations by the dialysis technique37. The mem-brane was soaked in distilled water overnight prior to its use.A 2 ml volume of ethosomal or liposomal dispersion was placedinto the dialysis bag, both open sides were clipped and kept in aclean beaker containing 250 ml of corresponding ethanolicsolution or distilled water as receiver compartment. The systemwas kept at a temperature below 4 �C and stirred continuously ata constant rate. Samples were removed at predetermined timeintervals, replaced with the fresh medium and analyzed for drugcontent by the HPLC method, until constant drug concentrationvalues were obtained in subsequent withdrawn samples from thereceiver compartment. At the end of dialysis experiment, thecontent of the dialysis bag was transferred into a clean beaker,lysed with 1% Triton X100 and analyzed for drug content by theHPLC method. Both ultracentrifugation and dialysis techniqueswere performed in triplicates for ethosomal as well as liposomalformulations and the EE% was determined using the followingequation:

EE % ¼

amount of drug present

in ethosomes or liposomes

� �

initial amount of drug� 100: ð2Þ

The difference in EE was determined by subtracting thevalue obtained by ultracentrifugation from the value obtainedby dialysis.

In vitro skin permeation study of DF-loaded ethosomes

After physicochemical characterization of DF-loaded ethosomes,skin permeation parameters, namely permeation flux, cumulativedrug permeated and drug deposition in the skin were determinedby in vitro skin permeation study on the formulations obtainedfrom the 4� 5 full-factorial design.

Franz permeation cells (Crown Glass Co., Inc., Somerville,NJ) having receptor compartment volume of 4.8 ml with effectivesurface area of 0.63 cm2 and synchronous driving assembly wasused to evaluate the in vitro skin permeation of DF-loadedethosomal formulations along with controls [liposomes, aqueousand ethanolic (20% v/v) drug solution]. Male SD rats were

obtained from Charles River Laboratories Inc. (Wilmington,MA). All rats were 5–6 weeks old and weighed approximately200–250 g. The experimental procedure and conditions weresimilar to the published literatures31,37. The protocol for per-forming animal studies was approved by the Animal CareCommittee at St. John’s University (Queens, NY). Briefly, SDrats were sacrificed by carbon dioxide asphyxiation. The abdom-inal hairs were clipped carefully and a section of full-thicknessabdominal skin was excised from the fresh carcasses of animal.The skin was thoroughly washed with pH 7.4 phosphate-bufferedsolution (PBS) and subcutaneous fat was carefully removed.The skin specimens were examined for any furrows or crack andthen sliced in 3.5 by 3.5 cm2 sections. The receptor compartmentof Franz permeation cells was filled with degassed pH 7.4 PBSand continuously stirred with a magnetic bar. The skin tempera-ture of 32 ± 1 �C was maintained in these cells by circulatingwater bath (Fisher Scientific Company, Fair Lawn, NJ). The skinsections obtained were mounted between donor and receptorcompartments of the Franz permeation cells with the stratumcorneum side facing toward the donor compartment.

Ethosomal formulations, liposomal formulations, aqueous orethanolic (20% v/v) drug solutions as the controls, with the totaldrug concentration of 1% w/v (500 ml), were then added into thedonor compartment. The donor compartment was covered withParafilm� in order to achieve occlusive condition. At predeter-mined time (1, 2, 3, 4, 5, 6, 8 and 24 h), samples (300 ml each)were withdrawn from the receptor compartment and DF concen-tration was analyzed by the HPLC method. The withdrawnvolume from the receptor compartment was replaced with freshPBS (maintained at 32 ± 1 �C), in order to maintain constantvolume for sink condition and drug concentrations measured werecorrected for the dilution factor. Care was taken to avoidintroduction of air bubbles beneath the dermis during the entirecourse of the experiments.

Skin deposition study was carried out to estimate the amountof drug deposited in the skin. Briefly, at the end of skinpermeation experiment (24 h), the side of the skin exposed todonor compartment was washed five times with pH 7.4 PBS (2 mleach), to remove excess drug from the skin surface. The washingprotocol was initially verified and was found to remove more than95% of the applied dose at zero time. The skin was then keptin 50% methanol (10 ml) for 24 h to extract the drug into themedium. At the end of 24 h, 1 ml of the medium was analyzedby the HPLC method for DF content. All experiments wereperformed in triplicate.

Formulation optimization of DF-loaded ethosomes

As mentioned earlier, by tailoring formulation variables (i.e. etha-nol concentration and PC:CH ratio) simultaneously, the desiredphysicochemical characterstics of ethosomes can be obtained,which can eventually influence skin permeation. Therefore, theessential information required for comprehesive understandingand control over skin permeation of ethosomes includes thecorrelation of formulation variables with the physicochemicalproperties as well as the correlation of these physicochemicalproperties with skin permeation flux. These data can also providethe basis for the optimization of formulation conditions forethosomal drug delivery via skin.

Correlation of formulation variables and physicochemcialproperties

The correlation between formulation variables and physicochem-ical properties was performed by regresssion analysis using JohnMacintosh Program (JMP, SAS Institute, Cary, NC). The designinvolved two independent variables and four dependent variables.

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The independent variables (Xi) were X1¼ ethanol concentration(0%, 10%, 15%, 20% and 30% v/v) and X2¼PC:CH ratio (50:50,70:30, 80:20 and 90:10 w/w). The four dependent variables(Yi) were Y1¼ vesicle size, Y2¼ zeta potential, Y3¼ elasticity andY4¼EE. The model obtained from regression analysis isexpressed in the form of the following equation:

Yi ¼ b1X1 þ b2X2 þ b3X1X2 þ b4X21 þ b5X2

2 , ð3Þ

where b1–b5 are the standardized beta coefficients for theobserved experimental values of Yi, X1X2 is the interaction term,and X2

1 and X22 represent the quadratic term of the independent

variables. The positive and negative signs of the coefficient valuesin the equations obtained after data analysis represent the agonistand antagonist effect of the independent variables while themagnitude of beta coefficient represents the extent of impact ofthe corresponding independent variable. Quadratic terms wereincluded in the model to account for the curvature effect ofindependent variables, if any. For comparing the effects of variousformulations, standardized beta coefficients instead of estimatedbeta coefficients were considered because the latter are dependenton the units of Xi, rendering them inappropriate to compareindependent variables38. The standardized beta coefficient hasbeen defined as the change in standard deviation of a dependentvariable per unit standard deviation increase in the independentvariable. For prediction of the dependent variable, standardizedbeta coefficient value was replaced by estimated beta coefficientin the above equation38.

Statistically significant F ratio (p50.05) and adjusted coeffi-cients of determination (adjusted R2) between 0.8 and 1.0 wereset as criterion for the adequacy of the model39. To validatethe obtained model, three additional checkpoint formulationswere prepared, characterized and compared with the predictedresponse. These checkpoint formulations were selected in order tovalidate the model at lower, middle and higher range of individualphysicochemical properties, within the studied design space.After validation of the model, the effect of linear and interactionterms of formulation variables were studied simultaneously usingresponse surface graph. For comprehensive understanding, theinteraction term was studied using interaction plots at lowerand higher levels of ethanol concentration (0% and 30% v/v) andPC:CH ratio (50:50 and 90:10 w/w).

Correlation of physicochemical properties and permeation flux

The physicochemical properties were correlated with permeationflux by regression analysis in order to identify the dominantphysicochemical properties contributing to the skin permeation ofethosomes. The identified physiochemical properties can then bealtered by the change in formulation variables, namely ethanolconcentration and PC:CH ratio to enhance the permeation flux.Therefore, regression analysis similar to the one describedpreviously was performed to determine the contribution ofphysicochemical properties, namely vesicle size (X1), zetapotential (X2) and elasticity (X3) on the permeation flux (Y) toobtain a model that can be expressed as Y¼ f(X1, X2, X3).Response surface and interaction plot were further studied formore comprehensive understanding.

Approach of formulation optimization

Based on the information derived from the correlation analysis,the physicochemical properties essential for the suitability ofDF-loaded ethosomes as a transdermal delivery system couldbe identified. Since these properties might have different goals(e.g. maximum, minimum or target permeation flux), therefore,desirability function was applied to simultaneously optimize

formulation variables with regard to these properties. Thisfunction combines the models obtained for various physicochem-ical properties, as a geometric mean, to derive an overall modelfor the delivery system, whose maximum value could then belocated within the design space to provide the optimizedethosomal formulation. To confirm efficiency of the model, theoptimized formulation along with two additional checkpointformulations was prepared, characterized and compared with thepredicted responses.

In vivo evaluation of optimized DF-loaded ethosomes

The anti-inflammatory activity of DF-loaded optimized ethoso-mal hydrogel was studied by a carrageenan-induced rat pawedema model reported in the literature2,12,40. Male SD rats werefasted for 24 h before the experiment with free access to water.The animals were divided into three groups with three animalsin each group. Accurately measured 50 ml of 1% suspension ofcarrageenan in saline was injected into the plantar side of hindpaws of each rat. After 1 h, group 1 received optimized ethosomalhydrogel, group 2 received liposomal hydrogel with similarcomposition corresponding to optimized ethosomal hydrogel(except ethanol), and group 3 received plain drug hydrogel.All hydrogel formulations contained same drug concentrationand were applied on the inflamed paw of rats by gently rubbingwith index finger.

The paw edema thickness was measured by a vernier caliper(Scienceware, Pequannock, NJ) at 0, 2, 4, 8 and 12 h. Thepercentage inhibition of carrageenan-induced paw edema wascalculated for each formulation using the following equation:

Tc � Tt

Tc

� �� 100, ð4Þ

where Tc is the mean edema thickness of rats in control group andTt is the mean edema thickness of rats in test group.

Statistical analysis

The Student’s t-test or one-way analysis of variance wasperformed to determine statistically significant differencebetween the data obtained from vesicle size, zeta potential,elasticity, EE studies, skin permeation study and in vivo studies.A PRISM software (GraphPad, version 5, San Diego, CA) wasused and a value of p50.05 was considered statisticallysignificant.

Results and discussions

Physicochemical characterization of DF-loadedethosomes

Morphology and lamellarity

To evaluate the surface morphology and lamellarity of vesicles byCLSM, rhodamine 123, a flurochrome marker was used because ithas approximately similar molecular weight as that of DF sodiumand it localizes preferentially in the bilayers of the vesicles41.Figure 1 represents the confocal image of rhodamine-loadedethosomes (formulation B20) indicating the smooth and concen-tric lamellar architecture of these vesicles.

Phase transition temperature

Lipid vesicles have a characteristic phase (main) transitiontemperature (Tm), which is the temperature at which the gel statetransitions into the liquid-crystalline state. It is reported that lipidmolecular motions are severely restricted in the gel state while inliquid-crystalline state the conformational disorder predominates

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 5

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rendering the vesicles small and elastic in nature42,43. Therefore,the effects of ethanol concentration and PC:CH ratio on the Tm ofthe vesicles were studied to provide the basis for mechanisticunderstanding of the effect of formulation variables on vesicle sizeand elasticity of DF-loaded ethosomal formulations.

As shown in Figure 2(a), increase in ethanol concentrationfrom 0% to 30% v/v at constant PC:CH ratio (formulations D0–D30), the Tm value decreased from 14.13 �C to 8.66 �C, and theendotherm width also decreased with the addition of ethanol. Thiscould be attributed to the fact that short chain alcohols (e.g.ethanol) interact with the hydrophilic head group region ofPC44,45, and their location and concentration near the head groupregion disturbs the natural microstructure of the lipid membranewhich eventually results in decrease in Tm and reduction in theendothermic width46,47. The reduction in Tm consequently facili-tates transition of lipid vesicles from the gel state to the liquid-crystalline state and renders the vesicle partially fluidized. Thelower values of Tm observed in ethosomes (formulations D10,D20 and D30) in comparison to liposomes (formulation D0) aretherefore an indication of higher malleability or softness of theformer23,48.

Furthermore, as shown in Figure 2(b), at constant ethanolconcentration, decrease in PC:CH ratio (increase in CH amount),increased the Tm value from 8.78 �C (formulation D20) to11.26 �C (formulation A20) and the endotherm width broadened.These results may be explained on the basis of CH amountembedded in lipid bilayer. Being amphiphilic in nature, CHmolecule inserts itself into the bilayer structure of the vesicle,orienting its hydrophilic head toward the aqueous surface andaliphatic portion lines itself parallel to the hydrocarbon chains ofPC. This reorientation of bilayer structure increases the packingand rigidity of the vesicles, and consequently results in theincrease in Tm and broadening of the phase transition of the gel tothe liquid-crystalline phase49.

Vesicle size

Published reports have shown that vesicle size plays a crucialrole in skin permeation of ethosomes50. Lower vesicle size

facilitates ethosomes to pass through the small pores of the skinresulting in enhanced skin permeation. The effect of ethanolconcentration and PC:CH ratio on the vesicle size of formulationsobtained from the full-factorial design (Table 1) show thatincrease in ethanol concentration from 0% to 30% v/v, signifi-cantly decreased the vesicle size at all PC:CH ratios. Ethosomalformulation exhibited smaller vesicle size in comparison toliposomes, at respective PC:CH ratios. Also, at respective ethanolconcentration, increase in PC:CH ratio from 50:50 to 90:10 w/w,the vesicle size of ethosomal and liposomal formulationsdecreased significantly. Based on the results of the DSC studydiscussed earlier, these findings suggest that the addition ofethanol decreased Tm resulting in partial fluidization of thesevesicles, which eventually leads to decrease in vesicle size23. Theeffect of PC:CH ratio on vesicle size can be mainly attributedto the fact that CH, unlike ethanol, increases the Tm and broadensthe transition of bilayers from the gel to the liquid-crystallinephase. It has been proposed that in this liquid-crystalline phase,CH increases the packing of phospholipid, increases the thicknessof the hydrophobic portion of the bilayer and decreases the rateof motion of the lipid tails, resulting in increase in the size ofvesicles49.

Regression analysis was then applied to further understand theeffect of formulation variables simultaneously on the vesicle size.The results are summarized in Table 2. The following equationwas obtained from the results of the analysis:

Vesicle size ¼ �0:82ðAÞ� � 0:59ðBÞ� þ 0:15ðABÞ�

� 0:22ðAÞ2� þ 0:20ðBÞ2�,ð5Þ

where A is the ethanol concentration and B is the PC:CH ratio, thecoefficient in this equation represents the standardized betacoefficient and the asterisk sign indicates significance of thevariable. The obtained regression model was found to bestatistically significant (p50.0001) with a high adjusted R2

value of 0.94. In addition, the observed vesicle size for threeadditional checkpoint formulations was not significantly differentfrom the predicted values with low prediction error, indicating thevalidity of the regression model (data not shown).

Figure 1. Confocal images illustrating (a) surface morphology and (b) three-dimensional projection of rhodamine-loaded ethosomal MLV.

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It can be seen from Table 2 and Equation (5) that both linear(A and B) and interaction (AB) terms, along with the quadraticterms (A2 and B2) of independent variables, were statisticallysignificant (represented by the asterisk sign). The significance

of quadratic terms A2 and B2 on the vesicle size indicates thatthe model has a curvature at higher levels of formulationvariables. The effect of linear and interaction terms on thevesicle size is also represented simultaneously in response surface

Figure 2. Thermograms representing (a) effect of ethanol of formulations D0, D10, D20 and D30 (0%, 10%, 20% and 30% ethanol, respectively)containing PC:CH ratio at 90:10 on main transition temperature (Tm), and (b) effect of PC:CH of formulations A20, B20, C20 and D20 (PC:CH ratioat 50:50, 70:30, 80:20 and 90:10, respectively) containing ethanol concentration at 20% on main transition temperature (Tm).

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 7

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graph (Figure 3a). As indicated in Equation (5) and Figure 3(a),the magnitude of the effect of ethanol concentration or PC:CHratio on vesicle size is different at different concentration levelsof ethanol or PC:CH ratio, indicating possible interactionbetween the two variables. It appears that ethanol concentrationcan affect vesicle size in different magnitudes at lower andhigher levels of PC:CH ratio, with more drastic change (i.e.decrease in vesicle size) observed at lower ratios (Figure 4a).This could be due to fluidization phenomenon of ethanol beingmore dependent on the amount of PC (rather than CH) availablefor ethanol. Therefore, at lower level of PC:CH ratio (less PC),a more effective fluidization of PC bilayer occurs with increasein ethanol concentration from 0% to 30% v/v. Similarly, theeffect of PC:CH ratio on vesicle size was also different at lowerand higher levels of ethanol, with more drastic change in vesiclesize observed at the low level of ethanol concentration.It appears that at lower levels of ethanol, CH (or PC:CH ratio)becomes a dominating factor, which is known to drasticallyaffect the vesicle size35. Therefore, at lower level of ethanol,vesicle size decreased with decrease in CH (increase PC:CHratio). At higher level of ethanol concentration, ethanol becomes

a dominating factors rendering the effect of PC:CH on vesiclesize less important35.

Zeta potential

Zeta potential is an important parameter that can influence bothvesicular stability and vesicle–skin interaction of ethosomes31.Electrostatic repulsion between the charged vesicles can preventaggregation during storage. Also, owing to the negative surfacecharge of skin, these vesicles can affect skin permeation.Zeta potential of ethosomes can be manipulated by the ethanolconcentration and PC:CH ratio20. As illustrated in Table 1, zetapotential of the vesicles decreased with increase in ethanolconcentration from 0% to 30% v/v, at respective PC:CH ratios.This is because ethanol provides a concentration-dependentsurface negative charge to polar head region of PC, avoiding,or at least delaying the formation of vesicle aggregates, due tothe electrostatic repulsions20. Moreover, at respective ethanolconcentration, increase in PC:CH ratio from 50:50 to 90:10 didnot produce a significant effect on the zeta potential, which isin agreement with a previously published report51.

Table 2. Summary of regression analysis of DF-loaded ethosomal and liposomal formulations for correlationsbetween formulation variables and physicochemical properties as well as between physicochemical properties andskin permeation flux.

TermEstimated beta

coefficient p ValueStandardized

beta coefficient

Correlation between formulation variables and physicochemical propertiesVesicle sizeIntercept 257.66829 50.0001* 0Ethanol �3.0815 50.0001* �0.8207PC:CH �7.357568 50.0001* �0.59405(Ethanol �15) (PC:CH �4.0825) 0.1814279 0.0171* 0.146485(Ethanol �15) (ethanol �15) �0.079365 0.0013* �0.21659(PC:CH �4.0825) (PC:CH �4.0825) 0.7948122 0.0296* 0.197053Zeta potentialIntercept �6.034447 50.0001* 0Ethanol �0.647225 50.0001* �0.94715PC:CH 0.2085225 0.3691 0.092509(Ethanol �15) (PC:CH �4.0825) �0.035467 0.0326* �0.15735(Ethanol �15) (ethanol �15) 0.0067821 0.1476 0.101701(PC:CH �4.0825) (PC:CH �4.0825) �0.086632 0.2561 �0.11802ElasticityIntercept �0.100318 0.4011 0Ethanol 0.048575 50.0001* 0.68469PC:CH 0.1882821 50.0001* 0.804559(Ethanol �15) (PC:CH �4.0825) 0.0058026 0.0023* 0.247954(Ethanol �15) (ethanol �15) �0.000606 0.2097 �0.08752(PC:CH �4.0825) (PC:CH �4.0825) �0.020116 0.0194* �0.26395EEIntercept 42.261722 50.0001* 0Ethanol 0.26775 0.0027* 0.274796PC:CH 3.8569242 50.0001* 1.200025(Ethanol �15) (PC:CH �4.0825) 0.0050523 0.8385 0.01572(Ethanol �15) (ethanol �15) �0.031774 0.0006* �0.33415(PC:CH �4.0825) (PC:CH �4.0825) �0.61602 0.0001* �0.58854

Correlation between physicochemical properties and skin permeation fluxIntercept 17.499428 0.0963 0Vesicle size �0.083743 0.0442* �0.59823Zeta potential 0.0845731 0.4601 0.130224Elasticity 4.5699437 0.0259* 0.662998(Vesicle size �195.033) (vesicle size �195.033) �0.004046 0.0718 �1.24626(Vesicle size �195.033) (zeta potential +12.9113) �0.014609 0.0999 �0.76256(Zeta potential +12.9113) (zeta potential +12.9113) 0.0080478 0.6086 0.069381(Vesicle size �195.033) (elasticity �0.97688) �0.601725 0.0327* �2.75778(Zeta potential +12.9113) (elasticity �0.97688) �0.494224 0.151 �0.41984(Elasticity �0.97688) (elasticity �0.97688) �14.67972 0.0918 �1.0048

PC, Phosphatidylcholine; CH, cholesterol.*Statistical significance of independent variables.

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To further understand the effect of formulation variables onzeta potential simultaneously, regression analysis was performedand the results are summarized in Table 2. The following equationfor zeta potential was obtained:

Zeta potential ¼ �0:95ðAÞ� þ 0:09ðBÞ � 0:16ðABÞ�

þ 0:10ðAÞ2 � 0:12ðBÞ2,ð6Þ

where the terms A, B and the coefficients have been definedearlier in Equation (5). The regression model was found to bestatistically significant (p50.0001) with a high adjusted R2 valueof 0.92. In addition, there was no statistical difference between theobserved zeta potential of three additional checkpoint formula-tions from the predicted values indicating validity of theregression model (data not shown).

It can be observed from Table 2 and Equation (6) that the zetapotential of the vesicles is significantly dependent on ethanol

concentration (A) and interaction between ethanol concentrationand PC:CH ratio (AB). Since Equation (6) and the responsesurface graph (Figure 3b) indicate a statistically significantinteraction effect (AB) of ethanol concentration and PC:CH ratioon zeta potential, the interaction plot was studied further. Theeffect of ethanol concentration on zeta potential (i.e. decrease inzeta potential) was similar (Figure 4b) both at lower and higherlevels of PC:CH ratio. This indicates that ethanol is a moredominating factor compared with PC:CH ratio and can effectivelyinteract and impart negative charge regardless of PC:CHcomposition. On the other hand, the effect of PC:CH ratio onzeta potential of the vesicles was different at different levels ofethanol. With increase in PC:CH ratio, the zeta potential of thevesicle increased at lower levels of ethanol, while at higher levels,the zeta potential of the vesicles decreased. The former observa-tion could be due to the less amount of CH available to interact,and therefore ethanol imparted negative charge to the polar head

Figure 3. Response surface graph indicating the effect of ethanol concentration and PC:CH ratio on (a) vesicle size, (b) zeta potential, (c) elasticity and(d) EE of diclofenac-loaded ethosomal and liposomal formulations fabricated from 4� 5 full-factorial design (n¼ 3).

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 9

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region of PC20. However, at higher ethanol level, this phenom-enon was not observed, which could again be attributed to themore dominating effect of ethanol resulting in decrease of zetapotential.

Elasticity

Compared with the conventional liposomes, the most importantfeature of ethosomes is their elasticity20,52. The elasticity of thevesicular membrane enables the vesicles to pass through the skinpores having diameter much smaller than the actual diameterof the vesicles. The effects of ethanol concentration and PC:CHratio on the elasticity of formulations obtained from the full-factorial design are shown in Table 1. The elasticity increasedwith increase in ethanol concentration from 0% to 30% v/v, withethosomes showing a much higher elasticity than that shown byliposomes at respective PC:CH ratios. This is due to concentra-tion-dependent ethanol interaction with the lipid bilayers, result-ing in lowering of Tm of the lipid and subsequently increasing theelasticity of ethosomal formulations. Also, the elasticity decreasedwith increase in the amount of CH (decreased PC:CH ratio)at respective ethanol concentration. This could again be attributedto the improved packing of lipid bilayer by CH at low PC:CH

ratios, resulting in increase in the Tm and rigidity of the lipidvesicles. Therefore, it may be concluded that elasticity of thevesicles is the result of the interaction between CH and ethanol.

The results of the following regression analysis are summar-ized in Table 2. Based on the regression analysis, the followingequation for elasticity was obtained:

Elasticity ¼ 0:68ðAÞ� þ 0:80ðBÞ� þ 0:25ðABÞ�

� 0:09ðAÞ2 � 0:26ðBÞ2�,ð7Þ

where the terms A, B and the coefficients have been definedearlier in Equation (5). The regression model was found to bestatistically significant (p50.0001) with high adjusted R2 valueof 0.92. There was no statistical difference between the observedelasticity of three additional checkpoint formulations fromthe predicted values, indicating validity of the model (data notshown).

Table 2 and Equation (7) show that elasticity is significantlyaffected by linear (A and B) and interaction (AB) terms of ethanolconcentration and PC:CH ratio. The quadratic term of PC:CHratio (B2) also has a significant effect on the elasticity of thevesicles indicating that the model has a curvature at higher PC:CHratio. The effect of linear and interaction terms on elasticity is

(b)(a)

(d)(c)

Figure 4. Effect of interaction of ethanol concentration (0% and 30% v/v) and PC:CH ratio (1 and 9 w/w) on (a) vesicle size, (b) zeta potential,(c) elasticity and (d) EE of DF-loaded ethosomal and liposomal formulations fabricated from 4� 5 full-factorial design (n¼ 3).

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represented simultaneously in response surface graph (Figure 3c).Since Equation (7) and Figure 3(c) suggest a statisticallysignificant interaction effect of ethanol and PC:CH, interactionplot was studied (Figure 4c). It was observed that ethanol affectsthe vesicular elasticity to different magnitudes at lower and higherlevels of PC:CH ratio, with more drastic changes occurring athigher PC:CH ratio. This is because at higher PC:CH ratio, theamount of CH in the lipid vesicle is less which facilitatesinteraction of ethanol with PC, consequently fluidizing thesevesicles to higher extent. Similarly, the effect of PC:CH ratio wasof different magnitudes at different levels of ethanol concentra-tion, with a more pronounced effect at higher ethanol concentra-tion. This is because at higher ethanol concentration, the vesiclesare already partially fluidized and therefore increasing the amountof PC:CH ratio (decreasing CH) further exaggerates the fluidiza-tion effect rendering these vesicles more elastic.

Entrapment efficiency

When the ultracentrifugation technique was used, EE obtainedwas lower than that by the dialysis technique for most of theformulations, irrespective of ethanol concentration and PC:CHratio. In order to further understand the suitability of bothmethods, the difference of EE between dialysis and ultracentri-fugation was calculated and plotted against formulation ratiosshown in Figure 5. Interestingly, the difference obtained, inmost cases, increased with increase in ethanol concentration atrespective PC:CH ratios. Also, as CH is decreased (or PC:CHratio increased) in the vesicles at respective ethanol concentration,the difference in EE increased in most case. As discussed above,increasing ethanol and decreasing the CH will increase theelasticity of the vesicles. It is therefore suggested that theentrapped drug might be lost during ultracentrifugation fromthese elastic vesicles, probably due to the deformation of lipidmembranes at such a high speed35. These findings illustrate that,in the case of elastic liposomes like ethosomes, dialysis is a moresuitable method than ultracentrifugation. Since EE obtained fromultracentrifugation might not represent the true EE due todeformation of lipid, therefore EE values obtained from thedialysis method were used in further studies.

The effects of ethanol concentration and PC:CH ratio on theEE of formulations obtained from the full-factorial design are

shown in Table 1. It was observed that at respective PC:CH ratios,EE increased with the increase in ethanol concentration from 0%to 20% v/v, which could be due to the cosolvent effect of ethanol,facilitating the accommodation of drug in the aqueous coreof the vesicle. However, at 30% v/v ethanol concentration,EE of formulations (formulations A30, B30, C30 and D30)decreased. This could be attributed to partial fluidization oflipid bilayers by ethanol, indicated by decrease in Tm andsubsequently higher elasticity value, resulting in leakage ofentrapped drug from these formulations. At respective ethanolconcentrations, EE of formulations increased with increase inPC:CH ratio (decrease in CH) from 50:50 to 90:10. This could beexplained based on the fact that at higher PC:CH ratio, morevolume of aqueous phase is entrapped around lipid bilayer tohydrate PC. Because DF sodium is in the aqueous phase of thevesicles, increasing the volume of entrapped aqueous phase willresult in increased EE.

To understand the effect of formulation variables on EEsimultaneously, regression analysis was performed. The results ofregression analysis are summarized in Table 2 and the followingequation was obtained for EE:

Entrapment efficiency ¼ 0:27ðAÞ� þ 1:20ðBÞ� þ 0:02ðABÞ� 0:33ðAÞ2� � 0:59ðBÞ2�,

ð8Þ

where the terms A, B and the coefficients have been definedearlier in Equation (5). The regression model was found to bestatistically significant (p50.0001) with high adjusted R2 value of0.90. There was no statistical difference between the observed EEof three additional checkpoint formulations from the predictedvalues, indicating the validity of the model (data not shown).

It can be observed from Table 2 and Equation (8) that EE issignificantly affected by both linear (A and B) and quadratic(A2 and B2) effect of ethanol concentration and PC:CH ratio.The significance of quadratic terms A2 and B2 on the vesicle sizeindicates that the model has a curvature at higher levels offormulation variables. From the response surface graphs of linearformulation variables on the elasticity as well as interaction plotof formulation variables (Figures 3d and 4d), it can be seen thatthe effect of ethanol and PC:CH at lower and higher levels of theircounterparts are almost similar indicating no interaction betweenethanol and PC:CH. Hence, it may be concluded that in order

Figure 5. Difference in mean EE% deter-mined by dialysis and ultracentrifugationmethods (n¼ 3).

A0A10 A15 A20 A30 B0

B10 B15B20 B30 C0C10C15C20C30 D0

D10D15D20D30

-2

0

2

4

6

8

10

Formulation code

Diff

eren

ce in

mea

n E

E%

0% Ethanol10% Ethanol

15% Ethanol

20% Ethanol

30% Ethanol

50:50 80:20 90:1070:30PC:CH

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 11

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to manipulate the EE, ethanol concentration and PC:CH ratio canbe individually modulated.

In vitro skin permeation study of DF-loaded ethosomes

Effect of ethanol concentration

Compared to liposomal formulations (0% v/v ethanol), increase inethanol concentration (10%, 15%, 20% and 30% v/v) of ethosomalformulations exhibited higher values of skin permeation param-eters at respective PC:CH ratios (Figure 6 and Table 3). Forexample, at PC:CH ratio of 90:10 w/w, compared with

formulation D0, permeation flux and cumulative drug permeatedincreased approximately 3 and 1.9 times for formulation D10,3.76 and 2.22 times for formulation D15, 3.8 and 3.3 times forformulation D20, and 3.3 and 2.9 times for formulation D30,respectively. Furthermore, drug deposition in the skin increasedapproximately 1.6 times for formulation D10, 1.7 times forformulation D15, 2 times for formulation D20 and 1.9 timesfor formulation D30, respectively, in comparison to formulationD0. Similar enhancement trend for the skin permeation param-eters was also observed at PC:CH of 50:50, 70:30 and 80:20w/w in comparison to respective liposomal formulations

0 4 8 12 16 20 240

25

50

75

100

125

Formulation A0Formulation A10Formulation A15

Formulation A20Formulation A30

Aqueous drug solutionEthanolic drug solution (20%)

Time (h)

Cum

ulat

ive

drug

perm

eate

d (µ

g/cm

2)

0 4 8 12 16 20 240

50

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Formulation C0Formulation C10Formulation C15Formulation C20Formulation C30Aqueous drug solutionEthanolic drug solution (20%)

Time (h)

Cum

ulat

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drug

per

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ted

(µg/

cm2)

0 4 8 12 16 20 240

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Formulation B0Formulation B10Formulation B15Formulation B20Formulation B30Aqueous drug solutionEthanolic drug solution (20%)

Time (h)C

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(µg/

cm2)

0 4 8 12 16 20 240

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Formulation D0Formulation D10Formulation D15Formulation D20Formulation D30Aqueous drug solutionEthanolic drug solution (20%)

Time (h)

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drug

per

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ted

(µg/

cm2)

(d) PC:CH=90:10

(b) PC:CH=70:30

(c) PC:CH=80:20

(a) PC:CH=50:50

Figure 6. Effect of ethanol concentration and PC:CH ratio on in vitro rat skin permeation profiles of DF-loaded ethosomal and liposomal formulationsoutlined in Table 1 along with aqueous drug solution and 20% ethanolic drug solution as controls (data represented as mean ± SD, n¼ 3).

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(i.e. formulations A0, B0 and C0, respectively). In addition,ethosomal formulations (10%, 15%, 20% or 30% v/v ethanol), atrespective PC:CH ratios, showed a concentration-dependentenhancement in skin permeation parameters as compared withaqueous and ethanolic drug solution (controls), except forformulations with 50:50 w/w PC:CH ratio (Table 3). The latterobservation could be attributed to large and relatively rigidvesicular structure due to high CH content in the formulationscomposed of PC:CH ratio of 50:50 w/w. These data clearly reflectthe advantage of ethosomes as a skin delivery carrier for DFcompared with liposomes, aqueous and ethanolic drug solutions.However, although the skin permeation enhancement of ethoso-mal formulations was higher compared with control groups,within ethosomal formulations at respective PC:CH ratios, thepermeation flux, cumulative drug permeated and drug depositionin the skin increased with the increase in ethanol concentration upto 20% v/v and then decreased at 30% v/v (Table 3 and Figure 6).

The enhanced skin permeation of ethosomes compared withcontrol formulations can be attributed to the solvent effect ofethanol on skin lipids followed by skin permeation of ethosomesvia ‘‘ethosome effect’’20,50. When ethosomal formulation comesinto contact with skin lipids, ethanol present in the formulationspartially fluidizes the intercellular lipids and consequentlyenhances the permeation flux, cumulative drug permeated aswell as drug deposition in the skin53. The solvent effect of ethanolis then followed by the ‘‘ethosome effect’’, which representsthe skin permeation enhancement due to physicochemicalproperties of ethosomes, mainly vesicle size, zeta potential andelasticity30,50,52. As shown in Table 1, at respective PC:CHratios, an ethanol concentration-dependent decrease in vesicle

size and increase in elasticity occurs for ethosomal formulations.Lower vesicle size and higher elasticity of ethosomes facilitatethe permeation of vesicles from the small skin pore. It hasbeen reported that since skin is negatively charged, vesicleswith negative zeta potential, create channels in the skin dueto an electrostatic repulsive force, subsequently facilitatingskin permeation52,54. This indicates that the physicochemicalproperties, namely vesicle size, zeta potential and elasticityare crucial in controlling and enhancing skin permeation ofethosomes.

As shown in Table 3 and Figure 6, at respective PC:CH ratios,the magnitude of skin permeation parameters was significantlyless for formulations containing 30% v/v ethanol concentration(formulations A30, B30, C30 and D30), than correspondingformulations with 20% v/v ethanol concentration (formulationsA20, B20, C20 and D20). This could be attributed to the fact thatethanol may have a dehydrating effect on the skin at higherconcentration (in this case, 30% v/v), resulting in decrease in skinpermeation parameters55.

Effect of PC:CH ratio

At respective ethanol concentrations, the skin permeation flux andcumulative drug permeated at 24 h increased with increase inPC:CH ratio from 50:50 to 90:10 w/w (Table 3 and Figure 6).Although drug deposition in the skin also increased at respectiveethanol concentration, the flux and permeated amount enhance-ment were not significantly different. Except for formulation withPC:CH ratio of 50:50 w/w, formulations with PC:CH ratio of70:30, 80:20 and 90:10 w/w (excluding formulations B0 and C0)exhibited higher skin permeation flux, cumulative drug permeatedas well as drug deposition in the skin compared with controlgroups (aqueous and ethanolic solutions) (Table 3).

The aforementioned observations can be related to theindividual effects of phospholipid and CH. Higher amount ofphospholipids (high PC:CH ratio) in the vesicles has beenreported to disturb the intact structure of skin and facilitatespenetration of vesicles through the lipid phase of stratumcorneum56. However, a more dominant role in skin permeationis played by CH, mainly by affecting the physicochemicalproperties of these lipid vesicles. The addition of CH in thelipid vesicles provides vesicular integrity (or rigidity) to thevesicles during skin permeation, as indicated by DSC study.However, the rigidity imparted by the addition of CH (low PC:CHratio) negatively impacts permeation of these vesicles from thesmall pores of the skin. Besides decreasing elasticity, the additionof CH increased vesicle size, which further attenuates the skinpermeation of vesicles. This was evident from the formulationswith PC:CH ratio of 50:50 w/w (formulations A0–A30), which iscomposed of the highest amount of CH (50%) and consequentlyhave large vesicle size and less elasticity. Owing to theseproperties, at respective ethanol concentration, the skin perme-ation flux and cumulative drug permeated for formulations withPC:CH ratio of 50:50 w/w were significantly lower than those atPC:CH ratio of 70:30, 80:20 and 90:10 w/w along with aqueous(in most cases) and ethanolic drug solutions.

It is therefore concluded that both ethanol concentration andPC:CH ratio modulate the physicochemical properties, mainlyvesicle size, zeta potential and elasticity of the ethosomes, andsubsequently affect the skin permeation kinetics of ethosomes.

Correlation between physicochemical properties andpermeation flux

As discussed above, the physicochemical properties of ethosomesplays a major role in affecting the skin permeation kinetics ofethosomes. For better understanding and control over their skin

Table 3. Effect of formulation variables on skin permeation parametersof DF-loaded ethosomal and liposomal formulations fabricated from4� 5 full-factorial design (n¼ 3).

Formulationcodea

Permeation flux(mg/h cm2 ± SD)

Cumulative drugpermeated at 24 h

(mg/cm2 ± SD)

Drug depositedat 24 h

(mg/cm2 ± SD)

PC:CH¼ 50:50A0 – – 249.3 ± 32.8A10 1.1 ± 0.4 61.3 ± 7.0 490.7 ± 86.6A15 2.1 ± 0.3 79.3 ± 8.5 596.0 ± 11.4A20 3.3 ± 0.5 98.8 ± 10.7 688.4 ± 70.6A30 2.7 ± 0.4 88.2 ± 10.9 556.6 ± 62.9PC:CH¼ 70:30B0 – 31.3 ± 2.2 276.1 ± 41.2B10 4.0 ± 1.6 108.6 ± 7.3 519.1 ± 18.8B15 4.0 ± 0.4 116.6 ± 14.6 574.8 ± 27.9B20 5.0 ± 0.6 170.2 ± 55.2 697.7 ± 59.7B30 3.7 ± 0.5 131.5 ± 15.1 588.1 ± 31.5PC:CH¼ 80:20C0 2.4 ± 0.3 52.0 ± 4.5 355.9 ± 27.6C10 5.1 ± 0.6 117.9 ± 16.6 527.6 ± 40.4C15 6.2 ± 0.4 194.7 ± 30.5 573.6 ± 41.6C20 9.7 ± 0.9 276.4 ± 17.6 739.4 ± 102.4C30 8.4 ± 1.6 236.9 ± 60.7 642.6 ± 37.3PC:CH¼ 90:10D0 2.8 ± 0.6 94.0 ± 11.5 356.2 ± 67.2D10 8.1 ± 2.7 176.7 ± 68.2 558.1 ± 15.6D15 10.4 ± 0.3 208.7 ± 37.1 589.1 ± 48.3D20 10.5 ± 1.2 307.8 ± 24.4 756.1 ± 125.7D30 9.1 ± 1.2 275.0 ± 22.8 686.6 ± 53.2ControlsAqueous 2.7 ± 0.2 64.6 ± 4.0 341.5 ± 32.8Ethanol (20%) 3.5 ± 1.3 103.0 ± 5.5 548.1 ± 29.4

PC, Phosphatidylcholine; CH, cholesterol.aA, B, C and D represent PC:CH ratio of 50:50, 70:30, 80:20 and 90:10

w/w, while 0 (liposomes), 10, 15, 20 and 30 represent % v/v of ethanolconcentration, respectively.

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 13

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permeation, it is essential to investigate the physicochemicalproperties that are predominantly contributing to permeationenhancement of ethosomes. Therefore, to identify such physico-chemical properties, vesicle size, zeta potential and elasticity werecorrelated with the skin permeation parameters. Among theskin permeation parameters evaluated and outlined in Table 3,permeation flux was selected because it is regarded as the mostvital tool to predict the skin permeation kinetics23. Therefore,regression analysis was performed and following equation wasobtained:

Permeation flux ¼ �0:60ðAÞ� þ 0:13ðBÞ þ 0:70ðCÞ�

� 0:76ðABÞ � 2:76ðACÞ�

� 0:42ðBCÞ � 1:25ðAÞ2

þ 0:07ðBÞ2 � 1:01ðCÞ2,

ð9Þ

where A, B and C are vesicle size, zeta potential and elasticity,respectively, the coefficient represents standardized beta coeffi-cients and asterisk sign indicates statistical significance of thevariable. The statistical analysis indicated that the model issignificant (p50.0001) with adjusted R2 value of 0.94. Equation(9) and Table 2 illustrate that permeation flux of ethosomes ismainly dependent of vesicle size and elasticity of the vesiclesand on the interaction between them.

Equation (9) and response surface graph shown in Figure 7(a)indicate that permeation flux increased with decrease in vesiclesize and increase in elasticity, with latter playing a more dominantrole in skin permeation of ethosomes based on the magnitude ofbeta coefficient. This is in accordance to our understanding fromprevious discussion that due to small opening of the skin pores,ethosomes with smaller vesicle size can easily pass through them.Also, higher elasticity of the vesicles permits them to squeezethrough the small opening of skin pores to the deeper skin layers.

Figure 7. (a) Response surface graph indicat-ing the effect of vesicle size and elasticity and(b) the effect of interaction of vesicle size(132.1 and 252.03 nm) and elasticity (1.78and 0.05) on permeation flux of DF-loadedethosomal and liposomal formulations fabri-cated from 4� 5 full-factorial design (n¼ 3).

14 S. Jain et al. Pharm Dev Technol, Early Online: 1–18

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Furthermore, Equation (9) indicates that interaction betweenthe vesicle size and elasticity also plays an important rolein enhancing skin permeation of vesicles. As indicated inFigure 7(b), vesicle size affects permeation flux differently atdifferent levels of elasticity. When elasticity of the vesicles is low,the decrease in vesicle size has little effect on permeationflux while if the elasticity of the vesicles is high, the decrease invesicle size greatly enhances skin permeation. Similar observationwas observed with increase in elasticity at low (132.1 nm)and high levels (252.03 nm) of vesicle size obtained from theformulations evaluated. It is perhaps due to extreme small size ofskin pores compared with vesicle size of ethosomes. Therefore,if the ethosomes were not elastic enough, decrease in vesicle sizewould not impact skin permeation significantly due to its inabilityto pass through the much smaller skin openings. However, if theelasticity is high, decrease in vesicle size augments the skinpermeation of ethosomes. Similar reasoning can be applied tothe effect of elasticity at low and high levels of vesicle size. Theseobservations therefore indicate that the elasticity and vesicle sizeare the predominant physicochemical properties that can affectskin permeation while zeta potential has an insignificant effect.

Formulation optimization of DF-loaded ethosomes

From the above correlation analysis, it is clear that skinpermeation kinetics of DF-loaded ethosomal formulations isdepended on the vesicle size and elasticity of these vesicles.However, apart from skin permeation, stability of ethosomesindicated by zeta potential, needs to be considered for practicalapplicability of this delivery system. In other words, the optimizedformulation should be a compromise between skin permeation

and stability of ethosomes. Hence, physicochemical properties,namely vesicle size, elasticity and zeta potential were selected forformulation optimization. To achieve the optimal ethosomalformulation for transdermal application, based on the datasummarized in Tables 1 and 3 as well as data publishedelsewhere18,23,50,57,58, minimum vesicle size (130–250 nm),target zeta potential (�10 to �30 mV) and maximum elasticity(1–3) were used for the formulation optimization. Based on thisapproach, an optimization model displayed in Figure 8 wasanalyzed and an optimized ethosomal formulation, having ethanolconcentration of 22.9% v/v and PC:CH ratio of 7.62 (88.4:11.6)with desirability of 0.86, was obtained. The optimizedethosomal formulation was fabricated and subjected to thephysicochemical characterization and in vitro skin permeationstudy. Vesicle size of 144 ± 5 nm, zeta potential of�23.0 ± 3.76 mV, elasticity of 2.48 ± 0.75, EE of 71 ± 4% andpermeation flux of 12.9 ± 1.0mg/h cm2 from the optimizedethosomal formulation were observed (Table 4). To furthervalidate the optimized model, two additional checkpoint formu-lations were fabricated and subjected to the same evaluations asdescribed for the optimized ethosomal formulation. The com-parison of vesicle size, zeta potential and elasticity betweenthe respective experimental and theoretical values is shown inTable 4, which indicates the validity of the optimized modelestablished in this investigation. Furthermore, it was observedthat in vitro skin permeation profile of optimized formulationdemonstrated significantly higher permeation flux, cumulativedrug permeated and drug deposition in the skin than thecorresponding drug-loaded conventional liposomes, aqueous andethanolic solutions (Figure 9). Although 22.9% of ethanol wasused for the fabrication of optimized DF-loaded ethosomal

Figure 8. Optimization of formulation com-position in terms of vesicle size, zeta poten-tial and elasticity using desirability function.

DOI: 10.3109/10837450.2014.882939 Transdermal diclofenac-loaded ethosomes 15

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formulations, the actual concentration of ethanol presented in theoptimized DF-loaded ethosomal formulations would be muchlower than in ethanolic solutions (22.9%) evaluated. Therefore,this finding indicates that the enhanced permeation flux ofoptimum ethosomal formulation was mainly dominated by thenature of ethosomes instead of the effect of ethanol content.

In vivo evaluation of optimized DF-loaded ethosomes

The results of in vivo evaluation indicate that there was astatistically significant inhibition of paw edema in SD rats withoptimized ethosomal hydrogel in comparison to liposomalhydrogel and plain drug hydrogel. The percentage inhibitionof paw edema by optimized ethosomal hydrogel (63.2 ± 6%)was approximately three times (21.3 ± 7%) and four times(15.8 ± 12%) higher than liposomal hydrogel and plain drughydrogel formulations, respectively. These results are supple-mented by skin permeation study, which indicates that smallvesicle size and high elasticity of ethosomes along withthe solvent effect of ethanol facilitated DF-loaded ethosomalhydrogel to cross the stratum corneum and provide enhancedanti-inflammatory activity compared with liposome and plaindrug hydrogel.

Conclusion

The present investigation demonstrates that skin permeationof DF-loaded ethosomes was essentially dependent on the

interaction of formulation and physicochemical properties.Based on the regression analysis, it was established that vesiclesize and elasticity are the dominating physicochemical propertiesaffecting skin permeation of ethosomes, which can be suitablymanipulated by formulation variables to achieve their desired skinpermeation. The optimized ethosomal formulation obtained basedon aforementioned understanding showed enhanced in vitropermeation through SD rat skin and anti-inflammatory activityon SD rat as compared with the control formulations studied.

Declaration of interest

The authors declare no conflict of interest (monetary or otherwise) inconducting this research. The authors alone are responsible for the contentand writing of the paper. The authors acknowledge St. John’s Universityfor providing financial assistance and research facilities to carry out thisresearch.

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