POTENTIAL CLINICAL RELEVANCE

Nanomedicine: Nanotechnology, Biology, and Medicine8 (2012) 489–496

Research Article

Nanosized ethanolic vesicles loaded with econazole nitrate for thetreatment of deep fungal infections through topical gel formulation

Poonam Verma, BPharm, MPharm⁎, Kamla Pathak, MPharm, PhDDepartment of Pharmaceutics, Rajiv Academy for Pharmacy, Mathura, India

Received 15 November 2010; accepted 21 July 2011

nanomedjournal.com

Abstract

This project aimed at developing nanovesicles of econazole nitrate (EN) and formulating them as a suitable dermatological gel forimproved therapeutic efficacy, better dispersity, and good storage stability. Ethosomes were prepared by cold method and evaluated for themean diameter, surface charge, and entrapment efficiency. Optimized ethosomes with vesicle size and entrapment efficiency of 202.85 ±5.10 nm and 81.05 ± 0.13%, respectively, were formulated as Carbopol 934 NF gels with varied permeation enhancers (G1–G7), andcompared with liposomal and hydroethanolic gels. The pharmacotechnical evaluation of gels demonstrated G6 with a flux rate of 0.46 ± 0.22μg/cm2 hr1/2 as the best formulation that was able to exhibit controlled release of EN for 12 hours across rat skin, and percent drug diffusedfrom ethosomes was nearly twofold higher than liposomal and hydroethanolic gels. Confocal laser scanning microscopy demonstrated drugpermeation as far as the last layer of epidermis (stratum basale). Stability profile of the prepared system assessed for 180 days revealed verylow aggregation and insignificant growth in vesicular size. The results collectively suggest that because of the controlled drug release, betterantifungal activity, and good storage stability, EN ethosomal gel has tremendous potential to serve as a topical delivery system.

From the Clinical Editor: Ethosomal gel of econazole nitrate was found to have outstanding potential to serve as a topical delivery system,enabling controlled drug release, providing better antifungal activity, and good storage stability.© 2012 Elsevier Inc. All rights reserved.

Key words: Econazole nitrate; Ethosomes; Pharmacotechnical properties; Gels; Epidermal permeation

Econazole nitrate (EN) is a broad-spectrum antifungalmedication used to treat skin infections caused by variousspecies of pathogenic dermatophytes. It is used for the treatmentof candidiasis.1 In some people, usually with weakened immunesystems, Candida invades deeper tissues as well as the blood,causing life-threatening systemic candidiasis.

A number of antifungal creams are now in use and applied fora variety of dermatological and other mycotic infections.However, many types of such fungal infections are proven tobe persistent and defeat any attempts of control or cure them. Inaddition, local reactions, including irritation, burning sensation,erythema, stinging, pruritic rash, and tenderness, may occur inpatients treated topically.2 Other problems associated with thecreams include failed stability test, either chemical stability orphysical separation of emulsion caused by the salting effect ofthe imidazole salt when used in concentration of about 1% ormore. Cream formulations often necessitate the use of emulsi-

No conflict of interest was reported by the authors of this paper.⁎Corresponding author: Department of Pharmaceutics, Rajiv Academy

for Pharmacy, Delhi-Mathura Bye Pass, Near Saiyad, P.O. Chattikara,Mathura-281001, Uttar Pradesh, India.

E-mail address: poonamrajpatel@gmail.com (P. Verma).

1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2011.07.004

Please cite this article as: P. Verma, K. Pathak, Nanosized ethanolic vesiclesthrough topical gel formula.... Nanomedicine: NBM 2012;8:489-496, doi:10.10

fiers or surfactants to maintain their physical stability and the useof antimicrobial preservatives to prevent microbial contamina-tion; these additives tend to generate an undesirable environmentthat can accelerate hydrolysis of drug and physical separationdue to salting out.3 Recently, a number of strategies have beenemployed to address issues related to EN cream, e.g.,microspheres,4 solid lipid nanoparticles,5 nanosponges,6 andsolid lipid microparticles.7

In dermal and transdermal delivery, the skin is used as aportal of entry for drugs. Because of barrier properties of theouter layer of the skin, in many cases, permeation enhancers areneeded to achieve therapeutic concentrations of drug.8 Classicliposomal systems were found to be effective at forming drugreservoirs in the upper layers of the skin, for local skin therapy.In contrast, ethosomal carriers, phospholipid vesicular systemscontaining high concentrations of alcohol, were effective atenhancing dermal and transdermal delivery of both lipophilicand hydrophilic drugs.9

Ethosomes are phospholipid-based elastic nanovesicles con-taining high percentages of ethanol (20–45%). Ethanol is knownas an efficient permeation enhancer and has been added in thevesicular systems to prepare the elastic nanovesicles. It caninteract with the polar head group region of the lipid molecules,

loaded with econazole nitrate for the treatment of deep fungal infections16/j.nano.2011.07.004

Table 1Composition and physical characterization of various ethosomal and liposome formulations of econazole nitrate (EN)

Formulation code EN (mg) Soya PC(w/w)

Ethanol(%v/v)

Cholesterol(mg)

Particle sizerange (nm)

Polydispersityindex

Entrapmentefficiency (%)

Zeta potential(mV)

F1 10 1 30 – 205.52 ± 9.80 0.45 ± 0.05 53.93 ± 0.27 –64.4 ± 1.11F2 10 2 30 – 238.02 ± 12.4 0.56 ± 0.01 72.89 ± 1.14 –72.2 ± 0.96F3 10 3 30 – 250.60 ± 11.63 0.42 ± 0.06 71.81 ± 0.86 –70.8 ± 0.84F4 10 1 40 – 191.67 ± 8.93 0.41 ± 0.02 60.23 ± 0.18 –71.1 ± 1.04F5 10 2 40 – 231.63 ± 8.44 0.80 ± 0.07 73.92 ± 0.26 –72.4 ± 0.62F6 10 3 40 – 202.85 ± 5.10 0.37 ± 0.01 81.05 ± 0.13 –75.1 ± 0.21F7 10 1 45 – 175.92 ± 6.29 0.58 ± 0.01 72.12 ± 1.16 –48.8 ± 1.32F8 10 2 45 – 252.97 ± 7.36 0.47 ± 0.01 74.56 ± 1.04 –71.2 ± 1.64F9 10 3 45 – 268.86 ± 9.11 0.82 ± 0.04 79.06 ± 0.81 –68.8 ± 0.92Liposome 10 1 – 1 412.34 ± 12.32 0.92 ± 0.10 51.08 ± 2.11 –38.3 ± 1.77

490 P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

resulting in the reduction of the melting point of the stratumcorneum lipid, thereby increasing lipid fluidity, and cellmembrane permeability.10 The high flexibility of vesicularmembranes from the added ethanol permits the elastic vesiclesto squeeze themselves through the pores, which are much smallerthan their diameters; thus, ethosomal systems are much moreefficient in delivering substances to the skin in terms of quantityand depth11 than either conventional liposomes or hydroalcoholicsolution. Much research has also indicated that ethosomes alsopossess good storage stability because of the presence of ethanol,which provides a net negative surface charge, thus avoidingaggregation of vesicles due to electrostatic repulsion.12,13

The present work thus focuses on developing a topicalethosomal antifungal gel formulation that can effectively providefast relief of symptoms and eradication of the fungal infectionwhile minimizing the risk of undesirable side effects caused byvarious creams available on the market, and that possesses gooddispersity and good physical and chemical stability withoutrefrigeration and without the need for special additives such asemulsifiers or surfactants or antimicrobial preservatives, and thatenhances delivery of drugs to their respective target sites, therebyensuring a maximum therapeutic advantage.

Methods

Materials

Econazole nitrate was a kind gift from FDC Ltd. (Mumbai,India). Soya phosphatidylcholine (PC; 30% w/w) was purchasedfrom Himedia (Mumbai, India). Other chemicals used in thestudy were of reagent grade.

Preparation of vesicular system

Preparation of ethosomesEthosomal suspension was prepared by cold method14 using

soya lecithin 30% (w/w), ethanol 30% (v/v), active molecules asdescribed, and water to 100% (w/v). Drug was dissolved inethanol, and the mixture of drug and ethanol was added to thephospholipid dispersion in water at 40°C. After mixing for5 minutes, the preparation was sonicated at 4°C for three cyclesof 5 minutes each with a 5-minute rest between cycles using aprobe sonicator (diameter 22 mm, 4000 rpm; Hicon Enterprises,Bangalore, India) to get nanosized ethosomes. Composition ofvarious ethosomal formulations is documented in Table 1.

Preparation of liposomesLiposomes were prepared by solvent evaporation method.9

PC from soya lecithin and cholesterol were dissolved in a smallvolume of diethylether–chloroform (1:1) mixture in a round-bottom flask. The aqueous phase containing drug was added tothe organic phase such that the ratio of organic to aqueous phasewas 5:1 (Table 1). The mixture was then sonicated for 10minutes. A stable white emulsion was produced, from which thewater and the organic solvent mixture were slowly evaporated at55°C using a rotary vacuum evaporator (Buchi type; HiconEnterprises) until a thin film was formed on the flask wall. Thefilm was hydrated with an appropriate amount of an aqueoussolution and left at 55°C in a thermostatically controlled waterbath for 1 hour; the resulting liposomal suspension was leftto stand at room temperature (21–25°C) for 1 hour and thensonicated for 20 minutes.

Purification of ethosomesThe purification of ethosomes was determined by dialysis

membrane method. Cellulose acetate membrane (molecularweight cutoff 50,000; Himedia) was kept in saline solution for2 hours before dialysis to ensure complete wetting of themembrane. One milliliter of the drug-loaded vesicles was placedin a dialysis bag, which was then transferred into 500 mL ofphosphate buffered saline, pH 6.8. The receiver medium wasstirred with a magnetic stirrer at 500 rpm. Ten-milliliter sampleswere withdrawn after 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 hours andreplaced with an equal volume of fresh dissolution medium.15

The samples were diluted appropriately and analyzed for amountof drug by high-pressure liquid chromatography (HPLC).

Evaluation of ethosomes

Vesicle size and zeta potentialThe vesicle size and zeta potential were measured by Malvern

Zetasizer (V. 6.01; Malvern Instruments Ltd., Malvern, UnitedKingdom). The polydispersity index (PDI) was used as aparameter of the size distribution.

Visualization of vesicle by transmission electronmicroscopy (TEM)

Ethosomal vesicles were visualized using a transmissionelectron microscope (Hitachi H-7500 80 kV; Ibaraki, Japan) withan accelerating voltage of 80 kV.

Table 2Formulation design for the preparation of various econazole nitrate (EN)ethosomal gels

Component(% w/w)

Formulation code

G0 G1 G2 G3 G4 G5 G6 G7 LG HE

EN 1 – – – – – – – – 1Carbopol 934 1 1 1 1 1 1 1 1 1 1EN ethosomes – 1 1 1 1 1 1 1 – –EN liposome – – – – – – – – 1 –Ethanol (95% v/v) 1 – – – – – – – – 1Propylene glycol – – 1 – 1 1 2 2 1 –N-methyl-2-

pyrolidone– – – 1 1 2 2 2 1 –

Triethanolamine 1 1 1 1 1 1 1 1 1 1Double-distilled

water100 100 100 100 100 100 100 100 100 100

HE, hydroethanolic gel; LG, liposomal gel.

Table 3Optimization of purification time of ethosomes (using paired sample t-test)

Optimizationtime (min)

tcal ttab at 5% level(dof = 1)

Result Discussion

90–120 10.4 2.92 tcalN ttab SD120–150 2.90 2.92 tcal b ttab No SD, so optimization

time is 120 minutes

dof, degrees of freedom; SD, significant difference; tcal, tcalculated; ttab,tabulated value.

491P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

Determination of EN entrapment efficiency (EE)After purification, 2 mL of suspension were transferred to a

10-mL centrifuge tube. The suspension was diluted with distilledwater up to 5 mL and centrifuged at 20,000 rpm for 30 minutesat 4°C using a cooling centrifuge (C-24 BL; Remi InstrumentsLtd., Vasai, India). Following centrifugation, supernatant andsediment were recovered and their volume measured. Thesediment was lysed using methanol and filtered through a 0.45μm nylon disk filter. The concentration of EN was analyzed byHPLC. The determination of EE was repeated three times persample for three samples at 25°C.

HPLC assay for EN quantificationENwas quantified by the HPLCmethod previously reported.5

The system used was an Adept HPLC Systems Cecil CE 4201(Cecil Instruments Ltd., Cambridge, United Kingdom), equippedwith a Rheodyne injector Hamilton 702 NR (Lab Unlimited,Dublin, Ireland), an Adept 4100 dual-piston pump, and detectorCE 4200 (Cecil Instruments, Ltd. Chorley, Lancashire, UnitedKingdom). The chromatographic separation was performed atroom temperature using a microsorb 5 μm RP-C18 column (250mm×4.6mm)with a flow rate of 1.0mL/min. Twentymicrolitersof sample of calibration standard were injected into the columnand eluted with a solution of methanol 0.05MNH4H2PO4 (85:15,v/v). Detection was carried out monitoring the absorbance signalsat 200 nm. The elution period was 8 minutes, and the retentiontime of EN was about 5.8 minutes.

Preparation of gels

Gel base was prepared by dispersing Carbopol 934 (LubrizolCorp, Wickliffe, Ohio) in distilled water. The polymer wassoaked in water for 2 hours and then dispersed in distilled waterusing a magnetic stirrer so as to obtain a homogeneous gel baseof 1% w/w. For preparation of EN-loaded gel, the ethosomalsuspension(s) was centrifuged at 2000 rpm for 20 minutes, andthe pellets obtained were incorporated into the prepared gel baseto get 1% w/w EN in the gel base. EN reference formulation(control; G0) was prepared by triturating EN with Carbopol 934gel base. Additionally, the liposomal EN gel was also preparedby incorporating liposomes of EN with Carbopol 934 gel base by

trituration. The formulative composition of the gels is docu-mented in Table 2.

Evaluation of gels

pH and rheological measurementsThe pH of various gel formulations was determined by a

digital pH/mV meter (Hicon, Delhi, India). Measurements ofthe rheological behavior of the formulations were performedwith a Brookfield R/S-CPS Rheometer (Brookfield EngineeringLaboratories, Essex, United Kingdom).

Skin permeation of EN from vesiclesTwo albino rats (Wistar strain) 6 to 8 weeks old weighing

120 to 150 g were humanely killed by chloroform inhalation. Allinvestigations were performed after approval by the institutionalethical committee and in accordance with the disciplinaryprinciples and guidelines of the committee for the purpose ofcontrol and supervision of experiments on animals. The hairs oftest animals were carefully trimmed short (b2 mm) with a pairof scissors 24 hours before, to avoid the adsorption of ethosomeson the surface of the hairs, and the abdominal skin was separatedfrom the underlying connective tissue with a scalpel, the excisedskin was placed on aluminum foil, and the dermal side of the skinwas gently teased off for any adhering fat and/or subcutaneoustissue. A preliminary wash of the skin was done with normalsaline, followed by drying between two filter papers. The skinwas used directly in the study without storage. Rat skinmembranes were mounted with the stratum corneum side upand the donor compartment dry and open to the air, and floated onreceiver solution for 12 hours for equilibration and prehydration.This approach was suggested to maintain a transepidermalhydration gradient. A dermatomed 500 μm thickness of skin fromthe abdominal area was obtained from the Postgraduate Instituteof Medical Education and Research (Chandigarh, India). Theprepared skin was mounted on the franz diffusion cell (ElectroLab. Pvt. Ltd, Mumbai, India) with the dermal side facing thereceptor compartment; 0.02 g of the test gel was applied as a thinfilm on the epidermal side with an effective diffusional area of 1cm2 and receptor volume of 10 mL. The receptor compartmentcontained 10 mL of phosphate buffer pH 6.8 stirred at 200 rpmand maintained at 32 ± 1°C. Samples (1 mL) were withdrawnthrough the sampling port of the diffusion cell at 1, 2, 4, 8, 10, and12 hours and analyzed. An equal volume of fresh phosphatebuffer maintained at 32°C ± 1°C was replaced into the receptorcompartment after each sampling. The study was done intriplicate, and average values were calculated. The cumulativeamount of penetrant that permeated the skin per unit surface

Figure 1. Transmission electron micrograph of ethosomal formulation F6.

Table 4Rheological and physical parameters of various EN gels

Formulationcode

SurfacepH

Viscosity (cP)T-spindle S-94

Drugcontent (%)

τ (Yield stress)(pascal)

G0 3.54 4780 ± 0.56 89.95 ± 0.18 1.10 ± 0.87G1 5.25 6720 ± 1.03 79.20 ± 1.21 1.11 ± 1.21G2 5.66 7600 ± 0.67 74.95 ± 0.62 1.21 ± 1.22G3 5.98 7240 ± 0.34 75.70 ± 1.01 1.15 ± 0.73G4 6.08 7220 ± 1.22 72.95 ± 0.29 1.27 ± 0.34G5 5.95 7660 ± 0.96 84.95 ± 0.81 2.71 ± 0.65G6 5.48 7420 ± 0.22 80.67 ± 1.33 2.42 ± 0.21G7 5.67 7620 ± 0.27 81.81 ± 0.43 2.76 ± 0.76LG 4.11 6620 ± 1.32 77.95 ± 1.54 1.07 ± 1.65HE 4.36 6200 ± 1.21 74.87 ± 1.11 1.01 ± 1.34

EN, econazole nitrate; HE, hydroethanolic gel; LG, liposomal gel.

492 P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

area was plotted against time. The linear portion of the plot wastaken as being the steady-state flux, (Js). The permeabilitycoefficient (Kp) was calculated as:

Kp = Js = Cy ð1Þ

where Cv is the concentration of penetrant in the donor solution.Statistically significant difference was determined using theStudent's t-test and the analysis of variance with P b 0.05 as aminimal level of significance.

Confocal laser scanning microscopyDepth and mechanism of skin permeation of rhodamine red

(RR)–loaded ethosomes was investigated using CLSM. RR–loaded ethosomal suspension was prepared with RR, soyalecithin 30% (w/w), 30% (v/v) ethanol, and water to 100% (w/v).Drug and RR were dissolved in ethanol, and the above mixture ofdrug, RR, and ethanol was added to the phospholipid dispersionin water at 40°C. After mixing for 5 minutes, the preparation wassonicated at 4°C for three cycles of 5 minutes each with a 5-minute rest between each cycle using a probe sonicator (diameter22 mm, 4000 rpm; Hicon Enterprises) to get nanosizedfluorescent ethosomes. The formulation (i.e., containing vesiclesloaded with probe) was applied nonocclusively to the dorsal skinof 5- to 6-week-old albino rats (Wistar strain) for 8 hours. The ratwas then sacrificed by heart puncture and dorsal skin was excisedand cleaned with a thin stream of water to remove any residualgel; then, the skin was placed on aluminum foil and adhering fatand/or subcutaneous tissue was removed. The skin sample wassliced in sections of 10–15 μm in thickness through the z-axis andexamined with CLSM (FV fluoview 1000; Olympus, Tokyo,Japan). Optical excitations were carried out with a 488-nm argon

laser beam, and fluorescence emission was detected above 560nm for RR.

Antifungal activityThe antifungal activity was evaluated by cup plate method

with strains of Candida albicans. The fungal strains were spreadover nutrient agar media in a Petri dish, which was thenimpregnated with the solution placed on the surface of the mediainoculated with the fungal strains. The plates were incubated at25°C for 24 hours. After incubation, the zones of growthinhibition around the disk were observed on an antibiotic zonereader (Hicon Enterprises).

Stability studiesStability of the vesicles was determined by storing the

prepared vesicular suspension at 25°C ± 0.5°C. Vesicle size, zetapotential, and EE of the vesicles were measured after 180 daysusing the method described earlier.

Results

Purification of ethosomes

The percent free drug removed increased with time andreached plateau levels by 120 minutes (Table 3). Paired samplet-test was used to check any significant difference between thepercent free drug removed at 120 and at 150 minutes. It wasobserved that although the extent of free drug in variousethosomal formulations was significantly different (P b 0.05),there was no significant difference in percent free drug removedafter purification times of 120 and 150 minutes (P N 0.05). Thus,free EN from ethosomal lipid vesicles could be efficientlyremoved after purification for 120 minutes and hence was usedthroughout the experiment.

Vesicle characterization

Vesicle morphology and size distributionEN-loaded ethosomes of formulation code F6 (see Table 1)

examined by TEM appeared as discrete spherical structures thatwere more or less uniform in size (Figure 1). The particle sizeand zeta potential of ethosomes were measured, and results areshown in Table 1. The vesicle size of the ethosomal system wasconsiderably smaller than that of the liposomal system, with

Table 5Skin permeation parameters of EN calculated from in vitro skin permeation study through rat skin (after 12 hours)

Formulation code % Cumulative drugrelease at 24 hr

Flux (μg/cm2/hr) Release rate(k)(μg/cm2 hr1/2 )

Permeability coefficient(cm/hr) × 10–3

Enhancement ratio Lag time (hr)

G0 50.10 ± 0.24 0.07 ± 2.40 0.020 0.70 1.00 0.5G1 69.88 ± 0.57 0.22 ± 0.72 0.065 2.25 3.21 0.7G2 75.56 ± 0.58 0.34 ± 1.12 0.101 3.40 4.86 0.5G3 79.06 ± 0.50 0.34 ± 0.83 0.104 3.36 4.79 0.5G4 81.34 ± 0.19 0.38 ± 0.72 0.101 3.82 5.45 0.5G5 84.22 ± 0.19 0.42 ± 0.11 0.125 4.17 5.95 0.6G6 91.66 ± 0.42 0.46 ± 0.22 0.133 4.56 6.51 0.5G7 94.46 ± 0.11 0.38 ± 0.03 0.122 3.80 5.43 0.5LG 55.12 ± 0.76 0.03 ± 0.79 0.013 0.38 0.05 0.8HE 53.74 ± 0.63 0.04 ± 0.64 0.011 0.45 0.06 0.7

Table 6Stability test by measuring ethosomal size and zeta potential after 180 days

Formulation Day 0 Day 180

Vesicle size (nm) Entrapmentefficiency (%)

Zeta potential (mV) Vesicle size (nm) Entrapmentefficiency (%)

Zeta potential (mV)

Ethosomal (F6) formulation 202.8 ± 5.10 81.1 ± 0.13 –75.1 ± 0.21 240.9 ± 4.32 72.4 ± 0.01 –49.1 ± 0.36Liposomal formulation 412.3 ± 12.32 51.1 ± 2.11 –38.3 ± 1.77 778.3 ± 18.54 40.9 ± 0.06 –14.3 ± 3.48

493P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

ethosomes size ranging from 268.86 ± 9.11 nm to 175.92 ± 6.29nm; the liposomes measured 412.34 ± 12.32 nm. The zetapotential of all the ethosomal vesicles was of higher magnitudethan liposomal vesicles (measured in millivolts); that the PDIwas found to be minimum in formulation code F6 could indicatethat it is a heterogeneous dispersion.

EE of EN in ethosomal and liposomal vesiclesOf all the ethosomal formulations, F6 (3% w/w soya PC, 40%

ethanol v/v), showed the greatest entrapment of 81.05 ± 0.13%(Table 1), which is around twofold higher in comparison to theliposomal formulation; on the basis of particle size, zetapotential, PDI, and EE, F6 was selected as the optimizedformulation and this formulation was used to prepare differentethosomal gels (G1–G7) with varying concentrations ofpermeation enhancers as shown in Table 3. In the controlformulation of EN ethosomal gel the flux value and permeationrelease is less, hence different concentrations of the penetrationenhancers were used in the formulations G1–G7 so as to attainthe formulation that showed optimum flux and good permeationto the innermost layer of the epidermis.

Characterization of various gels

Physicochemical characterizationIn the evaluation of gels, the pH of all gels was found between

4.26 and 6.54 (Table 4), thus lying in the normal pH range ofskin, 3.0–9.0; hence the preparation will potentially benonirritating. In all the ethosomal gel formulations the drugcontent ranged between 72.95 +/- 0.29 to 84.95% +/- 0.81 andthere was uniformity of drug dispersion in the gels.

The viscosity of all the gels found in the range of 4780 ± 0.56to7660 ± 0.96 centipoise (cP), and the increase of rotation speed

does not significantly change the viscosity of the sample,indicating formation of a stable gel structure. Carbopol 934forms a physically bonded network by formation of the junctionzones, which are responsible for the mechanical strength of thegel. The presence of the drug slightly reduced the gel viscosity,which could be a reason for the decreased viscosity of the controlgel. In the other formulations, however, the difference inviscosity is insignificant, indicating that there are no signs ofinteraction between the vesicles and Carbopol chains.15

The higher the static-yield value, the more readily themedium will maintain vesicles in suspension and minimizesedimentation. Desirable yield stress range for topical drugdelivery dosage form is between 0.1 and 500 pascal (Pa), and themaximum yield value among the formulated gels was found inG7 to be 2.76 ± 0.76 Pa, whereas a minimum of 1.01 ± 1.34 Pawas documented for hydroethanolic gel (HE).

EN permeation from ethosomal and liposomal gelsSkin permeability values from all the systems are summarized

in Table 5. It was observed that the highest in vitro permeationwas found in G7 and G6 (94.46 ± 0.11% and 91.66 ± 0.42%,indicating the enhancing effect of ethanol for drug penetrationacross the skin layers. The presence of ethanol in the ethosomalgel increased the permeation rate around twofold as compared tothe liposomal gel. Similar conclusions could be obtained uponanalysis of drug flux, which was significantly higher inethosomal formulation G6 (0.46 ± 0.22 μg/cm2/hr) than that ofliposomal, HE, and control gels; this could indicate that ethanolenhances drug permeation across the skin layers. All ethosomalgel and liposomal gel permeation profiles fit the zero order,whereas in control and HE gels the permeation profiles fit to thefirst order showing release directly proportional to concentration.

100

90

80

70

60

50

40

30

20

10

00 2 4 6 8 10 12

Time (h)

cum

ula

tive

am

ou

nt

of

dru

g p

erm

eate

d(%

)

liposomal gel

Control

hydroethanolicgel

ethosomal gel

Figure 3. Comparative cumulative amount of econazole nitrate permeatedfrom various formulations in a 12-hour study via rat skin.

110

100

90

80

70

60

50

40

30

20

10

00 1 2 3 4 5 6 7 8 9 10 11 12

G0

G1

G2

G3

G4

G5

G6

G7

Time (h)

Cum

ulat

ive

amou

nt o

f dru

g pe

rmea

ted

(%)

Figure 2. Comparative cumulative amount of drug permeated from ethosomalformulations in a 12-hour study via rat skin.

494 P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

It could be concluded that ethosomal vesicles acted as a reservoirsystem for control delivery of the encapsulated drug.

On comparison of the formulations, the lag time ranged from0.5 to 0.8 hour. The highest lag time of 0.8 hour was found inliposomal gel, which may be attributable to its increased timerequirement to penetrate through the skin and diffuse fromvehicle to the skin, as well as its interaction with skin lipids16;alternately, it may require time to diffuse from the vehicle, reachthe skin surface, permeate through the skin, and modify itsproperty.17 On the other hand, all the other ethosomalformulations displayed shorter lag time than liposomal gels,and G6 displayed the lowest value of 0.5 hour.

The permeability coefficient was deduced in the range of0.38 × 10–3 to 4.56 × 10–3 cm/hr, which lies within thepermeability coefficient limits of human skin of 10–6 to 10–2

cm/hr.18 It was also observed that, as the flux increased,permeability coefficient also increased.

Antifungal studiesIn the in vitro antifungal activity, the decreasing order of zone

of inhibition was G6 b G0 b LG b HE gel (i.e., 18.53, 7.83, 12.4,and 10.73 mm, respectively). The enhanced in vitro antifungalactivity of tested G6 EN ethosomal gel may be attributed toenhanced diffusion of vesicles containing EN through fungal cellwalls to inhibit ergosterol synthesis, because the G6 gel showedmaximum inhibitory activity as compared to other formulations.Statistically significant difference was determined using theStudent's t-test and the analysis of variance with P b 0.05 as aminimal level of significance.

Discussion

Vesicle size influences topical drug delivery; consequently,vesicles smaller than 300 nm are able to deliver their contentsto some extent into deep layers of the skin.19,20 In case ofethosomes and liposomes with average diameters of b200 nm,

there is potential for delivery through the skin. Our study showedthat the vesicle size of ethosomal systems was significantlysmaller than that of the liposomal system (Table 1), and itsuggests that ethanol has a predominant role in ethosomalsystems that causes modification of the net charge of the systemand confers on it some degree of steric stabilization that mayfinally lead to a decrease in mean vesicle size.21 Anotherimportant parameter is the zeta potential of the vesicles, which isa determinant of stability of vesicular systems. A significantdifference was observed between the zeta potential of theethosomal and liposomal vesicles (–75.1 ± 0.21 mV forethosomal formulation and –38.3 ± 1.77 mV for liposomalformulation). Suspensions with a potential above –60 mV showexcellent stability, and particle aggregation slowed to animperceptible rate due to its high zeta potential, whereassuspensions below –30 mV are of limited stability and canundergo pronounced aggregation. Furthermore, high negativecharge is dependent on two factors: (i) ethanol, which providesa net negative surface charge thus avoiding aggregation ofvesicles due to electrostatic repulsion; and (ii) soya PC, whichprovides a greater rigidity to the layers and reduced likelihoodof vesicles fusion, as well as a greater resistance to the highrotational energy exerted by sonication, resulting in a highnegative charge (Table 6).

Theoretically, liposomes and ethosomes can entrap bothhydrophobic and hydrophilic drugs.22 Thus, EN, a hydrophilicdrug, was readily entrapped in the ethanolic vesicles, and the EEwas about two orders of magnitude higher than that of theliposomes. This could be attributed to the greater retentivity ofEN in ethanol present in the ethosomal core.12 EE depends onethanol and lipid concentration, and therefore on increasing theconcentration of ethanol from 30% to 40% the EE increased as aresult of increase in fluidity of the membrane; however, furtherincrease in ethanol concentration (N40%) probably made the

Figure 4. Confocal laser scanning microscopy. (A) CLSM image of control gel; (B) CLSM image of liposomal gel showing less penetration of drug; (C) CLSMimage of ethosomal gel showing penetration of drug as far as the last layer (stratum basale) of epidermis.

495P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

vesicles more leaky, thus leading to a decrease in EE. Theethosomal formulation F6 (3% w/w soya PC, 40% ethanol v/v)showed the greatest entrapment, justifying itself as the optimizedformulation with an EE of 81.05% and optimum size 202.85 nm,thus presenting an ample opportunity to the EN-loadedethosomal system to attain a better skin permeation profile.

The gels are transparent, with a pleasant and smoothappearance. The highest in vitro permeation was found in G7andG6 (94.46 ± 0.11% and 91.66 ± 0.42% (Figure 2), attributableto the presence of ethanol because it provides the vesicles withsoft, flexible characteristics that allow them to more easilypenetrate into the deeper layers of the skin. It was also proposedthat phospholipid vesicles with ethanol may penetrate into theskin and influence the bilayer structure of the stratum corneum,and this may lead to enhancement of drug penetration.23,24

Comparing cumulative drug permeated and other factorsbetween control, ethosomal, liposomal, and HE gel, higherpermeation was observed for all ethosomal gels, followed byliposomal, HE, and control gel G0 (Figure 3), because the controlformulation was a particulate suspension of drug in the gellingagent without penetration enhancer, hence the least permeationwas observed. In the case of liposomes, these vesicular systemsare associated with many problems. For example, liposomes areexternally triggered (e.g., temperature-, pH-, or magnetic-sensitive) carriers that load drugs passively, which may lead tolow drug-loading efficiency and drug leakage in preparation,preservation, and transport in vivo; furthermore, in the case ofliposomes, osmotic force of the vesicles prevented them frompenetrating beyond the level of the lowest level in stratumcorneum,25 because release decreases. So, a potential solution tothese problems is the use of ethosomes. On comparison ofethosomal and HE gel, higher flux and higher release were

observed for the former because of the synergistic mechanismsof ethanol, phospholipid vesicles, and skin lipids interaction,which promoted the passage of EN through rat skin. So,ethosomal systems are much more efficient in deliveringsubstances to the skin in terms of quantity and depth, than eitherliposomes or HE solution of drug.26 In vitro permeation resultswere further confirmed by CLSM.

The confocal photomicrographs corresponding to variousformulations applied onto the rat skin show that the penetrationfrom control gel was confined only to the upper layer of the skin.In the case of liposomes there is little permeation through theskin. In contrast, enhanced delivery of RR in terms of depth andquantity was observed for ethosomal gel (i.e., as far as the fifthlayer of epidermis, the stratum basale); this is due to the ethanoleffect, wherein ethanol interacts with lipid molecules in the polarhead group region, resulting in a reduction in the transitiontemperature of stratum corneum lipids, increasing their fluidityand decreasing the density of the lipid multilayer.27 This isfollowed by the “ethosomes effect,” which includes lipidpenetration and permeation by the opening of new pathwaysdue to the malleability and fusion of ethosomes with skin lipids,resulting in the enhanced delivery of RR in terms of depth andquantity (Figure 4).

One of the major limitations of vesicular systems is theirphysical instability, wherein the vesicles tend to fuse and growinto bigger vesicles. This fusion leads to breakage of vesiclesand consequently leakage of drug. Liposomal systemsfrequently present the said physical instability. The particlesize almost doubles, and reduction in the zeta potential is about62%; this happened because the absence of electrostaticrepulsion is likely to account for the tendency of neutralliposomes to aggregate.28 Ethosomal formulations by contrast

496 P. Verma, K. Pathak / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 489–496

revealed very low aggregation and insignificant growth invesicular size, because of the presence of ethanol, whichprovides a net negative surface charge that avoids aggregationof vesicles due to electrostatic repulsion.15 Thus, the optimizedethosomal formulations upon storage were physically stable for6 months under the tested condition.

An ethosomal system capable of delivering EN at acontrolled rate into the deeper layers through rat epidermiswas successfully developed. The ethosomes elicited an increaseof the percutaneous permeation of EN, thus improving theantifungal activity of the drug. Slow particle aggregation due tohigh zeta potential results in excellent physical stability of thesystem. The enhanced accumulation of EN via ethosomal carrierwithin the skin might help to optimize targeting of this drug tothe epidermal sites, thus creating new opportunities for well-controlled and modern topical application of EN in the treatmentof various fungal diseases.

Acknowledgment

The authors are grateful to Punjab University (Chandigarh,India) for providing the TEM facility; NIPER (Mohali, India) forproviding assistance in CLSM; and CDRI (Lucknow, India) forproviding the sizing facility.

References

1. Dyas AM, Delargy H. Econazole nitrate. In: Florey K, editor. Analyticalprofiles of drug substances, Vol. 23. New York: Academic Press; 1994.p. 125-51.

2. Bhalaria MK, Naik S, Mishra AN. Ethosomes: a novel system forantifungal drugs in the treatment of topical fungal disase. Ind J Exp Biol2009;47:368-75.

3. Wang J, Patel B, Au S, Shah H. Bristol- Myers Squibb Company,assignee. Antifungal gel formulations. United States patent US 1992;5110809.

4. Albertini B, Passerini N, Sabatino M, Vitali B, Brigidi P, Rodriguez L.Polymer-lipid based mucoadhesive microspheres prepared by spray-congealing for the vaginal delivery of econazole nitrate. Eur J Pharm Sci2009;36:591-601.

5. Sanna V, Gavini E, Cossu M, Rassu G, Giunchedi P. Solid lipidnanoparticles (SLN) as carriers for the topical delivery of econazolenitrate: in-vitro characterization, ex-vivo and in-vivo studies. J PharmPharmacol 2007;59:1057-64.

6. Sharma R, Pathak K. Polymeric nanosponges as an alternative carrier forimproved retention of econazole nitrate onto the skin through topicalhydrogel formulation. Pharm Dev Technol 2011;16:367-76.

7. Passerini N, Gavini E, Albertini B, Rassu G, Sabatino M, Sanna V, et al.Evaluation of solid lipid microparticles produced by spray congealing fortopical application of econazole nitrate. J Pharm Pharmacol 2009;61:559-67.

8. Barry BW. Novel mechanisms and devices to enable successfultransdermal drug delivery. Int J Pharm 2001;14:101-14.

9. Bendas ER, Tadros IM. Enhanced transdermal delivery of sulbutamolsulfate via ethosomes. AAPS Pharm Sci Tech 2007;8:1-7.

10. Touitou E, Dayan N, Bergelson L, Godin B, EliazM. Ethosomes—novelvesicular carriers for enhanced delivery: characterization and skinpenetration properties. J Control Release 1999;65:403-18.

11. Touitou E, Godin B, Dayan N. Intracellular delivery mediated byethosomal carrier. J Biomaterials 2001;22:3055-9.

12. Dubey V, Mishra D, Jain NK. Melatonin loaded ethanolic liposomes:Physicochemical characterization and enhanced transdermal delivery.Control Release 2007;67:398-405.

13. Dubey V, Mishra D, Dutta T, Nahar M, Saraf DK, Jain NK. Dermal andtransdermal delivery of an anti-psoriatic agent via ethanolic liposomes.J Control Release 2007;123:148-54.

14. Jain S, Umamaheshwari RB, Bhadra D, Jain NK. Ethosomes: a novelvesicular carriers for enhanced transdermal delivery of an anti HIVagent. Ind J Pharm Sci 2004;66:72-81.

15. Jain S, Tiwary AK, Sapra B, Jain NK. Formulation and evaluation ofethosomes for transdermal delivery of lamivudine. AAPS Pharm SciTech 2007;8:1-9.

16. Attia MA, El-Gillay AI, Dhaltout SE, Feith GN. Transbuccalpermeation, anti-inflammatory activity and clinical efficiency ofpiroxicam formulated in different gels. Int J Pharm 2004;267:11-28.

17. Santoyo S, Ygartua P. Effect of skin pretreatment with fatty acids onpercutaneous absorption and skin retention of piroxicam after its topicalapplication. Eur J Pharm Biopharm 2000;50:245-50.

18. Larrucea E, Arellano A, Santoyos S, Ygartua P. Combined effect ofoleic acid and propylene glycol on the percutaneous penetration oftenoxicam and its retention on the skin. Eur J Pharm Sci 2001;52:113-9.

19. Ghosh TK, Jasti BR. Theory and practical of contemporary pharmaceu-tics. New York: CRC Press; 2005. p. 43.

20. Verma DD, Verma S, Blume G, Fahr A. Ethosomes increases skinpenetration of entrapped and non-entrapped hydrophilic substances intohuman skin: a skin penetration and confocal laser scanning microscopystudies. Eur J Pharm Biopharm 2003;55:271-7.

21. Fang YP, Tsai YH, Wu PC, Huang YB. Comparision of 5-aminolevulinic acid-encapsulated liposomes versus ethosomes for skindelivery for photodynamic therapy. Int J Pharm 2008;356:144-52.

22. Lopez-Pinto JM, Gonzalez-Rodriguez ML, Rabasco AM. Effect ofcholesterol and ethanol on dermal delivery from DPPC liposomes. Int JPharm 2005;298:1-12.

23. Mishra D, Mishra PK, Dubey V, Nahar M, Jain NK. Systemic andmucosal immune response induced by transcutaneous immunizationusing Hepatitis B surface antigen-loaded modified liposomes. J ControlRelease 2007;33:424-33.

24. Thong HY, Zhai H, Maibach HI. Percutaneous enhancers: an overview.J Skin Pharmacol Physiol 2007;20:272-82.

25. Ceve G. Lipid vesicles and other colloids as drug carrier on the skin.J Adv Drug Del 2004;56:675-711.

26. Godin B, Touitou E, Athamna A. A new approach for treatment of deepskin infection by an ethosomal antibiotic preparation: an in vivo study.J Antimicrob Chemother 2005;55:989-94.

27. Elsayed MA, Abdallah YO, Naggar FV, Khalafallah NM. Lipid vesiclesfor skin delivery of drugs: reviewing three decades of research. Int JPharm 2006;332:1-16.

28. Rao LS. Preparation of liposomes on the industrial scale: problems andperspectives. In: Gregoriadis G, editor. Liposome technology, Vol. 1.Boca Raton (FL): CRC Press; 1984.