Dermatopharmacokinetic and pharmacodynamic evaluation of ethosomes of griseofulvin designed for...

RESEARCH PAPER

Dermatopharmacokinetic and pharmacodynamic evaluationof ethosomes of griseofulvin designed for dermal delivery

Nidhi Aggarwal • Shishu Goindi

Received: 10 April 2013 / Accepted: 28 August 2013 / Published online: 15 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The present study is aimed at evaluation of

the dermal delivery potential of griseofulvin-loaded

ethosomes. Griseofulvin-loaded ethosomes were pre-

pared using ‘‘Cold technique’’ (Indian Patent Appli-

cation 208/DEL/2009). The optimized formulation

was characterized for vesicular shape and size, drug

entrapment efficiency, drug content, pH, stability, and

spreadability. Ex vivo skin permeation, dermatophar-

macokinetics, and skin sensitivity studies were carried

out using male Laca mice. In vivo antifungal activity

was assessed against Microsporum canis using guinea

pig model for dermatophytosis. The optimized for-

mulation E7 possessing 2 % phospholipid (PL) and

30 % ethanol exhibited the highest drug entrapment

(72.94 ± 0.80 %) and optimum vesicle size

(148.5 ± 0.48 nm). E7 illustrated remarkably higher

drug permeation and skin retention when compared

with liposomes. Pharmacodynamic studies in guinea

pigs induced with M. canis revealed that the dermal

fungal infection was completely cured in 8 days upon

twice daily topical application of griseofulvin-loaded

ethosomes whereas liposomes led to complete cure in

14 days. The formulation was observed to be non-

sensitizing, histopathologically safe, and stable at

5 ± 3, 25 ± 2, and 40 ± 2 �C for a period of 1 year.

Results indicated that dermal delivery of griseofulvin

employing ethosomes could be a commendable alter-

native to reduce the bio-burden associated with

conventional oral formulations.

Keywords Antifungal � Dermal �Dermatophytes � Pharmacodynamic

Introduction

The vehicle components of a dermatological formula-

tion appreciably affect the penetration of drugs into and

through the skin. Ethanol has been used since times

immemorial in the pharmaceutical and cosmetic

industry. It is a well-known skin penetration enhancer

thought to work by exerting a ‘‘push’’ or a ‘‘pull’’ on the

intercellular region of the stratum corneum. Touitou

(2000) first reported the incorporation of high concen-

trations of ethanol into liposomes which resulted in

formation of novel elastic vesicles known as etho-

somes. Ethosomes have provided an ample opportu-

nity to transport active substances more efficaciously

through the stratum corneum into the deeper layers of

the skin than the conventional vesicles i.e., liposomes

(Dubey et al. 2010). These soft malleable vesicles can

be effectively tailored for encasing both hydrophilic

and lipophilic drug molecules. These elastic vesicular

carriers ushered a new era in the field of topical skin

applications and ever since these colloidal nanocarriers

N. Aggarwal � S. Goindi (&)

University Institute of Pharmaceutical Sciences,

Panjab University, Chandigarh 160014, India

e-mail: [email protected]

123

J Nanopart Res (2013) 15:1983

DOI 10.1007/s11051-013-1983-9

have been successfully investigated to enhance the

clinical efficacy of a number of drugs viz., minoxidil,

melatonin, tamoxifen, testosterone, zidovudine, lam-

ivudine, methotrexate, indinavir, cyclosporine A, and

fluconazole (Gangwar et al. 2010). Also, a number of

dermal formulations based upon ethosomes are com-

mercially available in the market like Nanominox�,

Skin Genuity, LipoductionTM, SupraVir cream, Dec-

ocrin� cream, Cellutight EF, and NoicellexTM.

Extensive literature survey revealed that although

griseofulvin is a very effective fungistatic antibiotic its

oral route is not favored due to its poor oral

bioavailability as a consequence of its low water

solubility. Moreover, it also exhibits numerous side

effects upon oral administration. Further in case of

dermatophytosis, usually upper layers of skin are

infected; therefore, it would be beneficial to use

griseofulvin topically. Moreover, it has also been

reported that the skin concentration resulting from

single topical application is much higher than those

obtained after prolonged oral administration and

persists there in measurable amounts for 4 or more

days (Nimni et al. 1990). Although, oral itraconazole

and ketoconazole are acceptable alternatives to gris-

eofulvin, these are not considered to be the first-line

therapy for Tinea owing to their cardiotoxic and

hepatotoxic nature, respectively (Koumantaki–Math-

ioudaki et al. 2005). Due to strong hydrophobic

character and insolubility in polar solvents it is

difficult to deliver griseofulvin in therapeutically

effective concentration using conventional topical

dosage forms such as solutions, ointments, creams,

gels, and spray. In order to overcome the dermal

permeability issues the incorporation of a penetration

enhancer N-methyl-2-pyrrolidone in emulsion and

suspension dosage forms of griseofulvin has been

suggested by Fujii et al. (2000). In a recent study, the

thermogelling microemulsions of griseofulvin based

on poloxamers revealed that there existed a lag time of

2.5–3 h in the ex vivo permeation studies (Peira et al.

2011). A lag phase in permeation may not hold

practical value for dermal delivery system. In fact a

formulation exhibiting instantaneous penetration in

the stratum corneum with drug reservoir forming

properties would be more appreciable.

The present work encompasses the design of

griseofulvin-loaded ethosomes for effective dermal

delivery of this antifungal agent. These vesicles can

deform and pass through skin pores without any

considerable loss of entrapped entities. The optimized

ethosomes were investigated to assess the levels of

dermal deposition of drug in mice skin and antifungal

efficacy against Microsporum canis using guinea pig

model for dermatophytosis.

Materials and methods

Chemicals and reagents

Griseofulvin (Wallace Pharmaceuticals Ltd., Mumbai,

India), Phospholipon� 90G (Phospholipid GmbH,

Germany), and Carbopol� 980 NF (Lubrizol

Advanced Materials India Pvt. Ltd., Mumbai, India)

were obtained as gift samples. Polycarbonate mem-

branes (M/s Whatman, Kent, UK); Cholesterol, extra

pure (Loba Chemie, Mumbai, India); Rhodamine 123,

Sephadex G-50 (medium) and Roswell Park Memorial

Institute (RPMI) 1640 medium (Sigma-Aldrich Inc.,

MO, USA); HPLC-grade acetonitrile, acetic acid, and

methanol (Merck KGaA, Darmstadt, Germany) were

also used in the study. Triple-distilled water (TDW)

was used throughout the study. All other chemicals

and reagents were of analytical grade and were used

without further purification.

Dermatophyte strains

The standard strains of Microsporum gypseum

(MTCC no. 2830), M. canis (MTCC no. 2820),

Trichophyton mentagrophytes (MTCC no. 7250),

and Trichophyton rubrum (MTCC no. 296) were

procured from Microbial Type Culture Collection

(MTCC), Institute of Microbial Technology (IM-

TECH), Chandigarh, India.

Animals

Male Laca mice 8–9 weeks old weighing 30–35 g

were obtained from Central Animal House, Panjab

University, Chandigarh, India. These were housed in

polypropylene cages and employed for performing

ex vivo permeation, histopathology, and dermatophar-

macokinetic studies. Male albino guinea pigs (Duncan

Hartley strain) 8–9 weeks old weighing between

350–400 g were obtained from disease-free small

animal house of College of Veterinary Sciences, Lala

of 15 J Nanopart Res (2013) 15:1983

123

Lajpat Rai University of Veterinary and Animal

Sciences, Hisar, Haryana, India. Guinea pigs were

housed in stainless steel metabolic cages and allowed

to acclimatize for a minimum of 15 days before

initiating the experiment. All the animals were kept at

ambient temperature with a 12-h night/day cycle, and

supplied with a standard pellet diet and water ad libi-

tum. The protocols for animal use and care were

approved by the Institutional Animal Ethics Commit-

tee (IAEC), Panjab University, Chandigarh, India

(IAEC/97 dated 24.03.2011).

Formulation of ethosomes of griseofulvin

Ethosomes were prepared by cold method (Dubey

et al. 2007, 2010). Accurately weighed quantity of

Phospholipon� 90G (PL 90G) and griseofulvin was

dissolved in ethanol at 30 �C in a 25 mL in-house built

closed vessel (Table 1). TDW maintained at 30 �C

was added to the ethanolic phase slowly drop by drop,

in a fine stream with constant stirring (Mechanical

stirrer, Remi equipment, Mumbai) at 1500–2000 rpm

in a closed vessel, to avoid ethanol evaporation.

Mixing was continued for additional 5 min. The

prepared formulations were stored at refrigerated

(4–8 �C) conditions for overnight before conducting

any characterization and evaluation studies.

Conventional liposomes of griseofulvin were pre-

pared using thin-film hydration (TFH) method using

previously optimized formula (Aggarwal and Goindi

2012).

Preparation of vesicular gels

The optimized vesicular dispersions were incorpo-

rated into the secondary vehicle i.e., gel base to make

them suitable for topical application. Carbopol

(500 mg) was dispersed in 50 mL of tepid TDW and

stirred for 2 h at 600 rpm and was neutralized with

triethanolamine solution (1.0 g in 50 mL TDW) with

continuous stirring to obtain a transparent gel. Briefly,

the accurately weighed amount of drug-loaded opti-

mized ethosome and liposome dispersions were

incorporated in pre-gelled Carbopol� 980 NF (0.5 %

by weight) by gentle levigation.

Preparation of Rhodamine 123-loaded ethosomes

Ethosomes loaded with Rhodamine 123 (0.03 %, w/v)

were also prepared for performing vesicle skin pen-

etration study. Unentrapped Rhodamine 123 was

removed from the system using mini-column centri-

fugation technique (Dubey et al. 2010).

Preparation of conventional formulation bases

containing griseofulvin

The conventional systems of griseofulvin were pre-

pared and used for ex vivo permeation comparisons

against the optimized colloidal formulations of gris-

eofulvin, containing the drug in amounts equivalent to

those present in final optimized vesicular dispersions.

Aqueous suspension was prepared by suspending the

Table 1 Influence of varying proportions of PL and ethanol on drug entrapment efficiency of ethosomes

Formulation

codeaPL 90 G

(mg)

Ethanol

(% w/w)

PL drug

weight ratio

Drug

payload

Percent drug

entrapment (n = 3)

%

Transmittance

No. of vesicles/

mm3 9 103

E1 100 20 10:1 2.20 22.00 ± 0.99 77.3 41.0

E2 125 20 12.5:1 2.87 35.86 ± 0.49 73.2 46.5

E3 150 20 15:1 2.81 42.14 ± 1.10 68.4 56.0

E4 175 20 17.5:1 3.09 54.12 ± 0.45 65.8 68.0

E5 200 20 20:1 3.04 60.91 ± 0.43 60.9 86.5

E6 200 25 20:1 3.27 65.40 ± 0.72 55.7 118.0

E7b 200 30 20:1 3.64 72.94 ± 0.80 49.3 139.0

E8 200 35 20:1 3.11 62.12 ± 0.94 59.6 95.0

E9 200 40 20:1 2.84 56.79 ± 1.06 68.3 71.5

Liposomes 150/30 (PL/CHOL) – 15:1 3.04 54.78 ± 0.56 54.5 51.0

a All the formulations contained constant amount of drug (10 mg)b Selected as optimized formulation for further characterization and evaluation

J Nanopart Res (2013) 15:1983 of 15

123

drug in 0.5 % w/v Carbopol suspension in water.

Hydroethanolic gel of griseofulvin was prepared by

dissolving the drug in 40 % v/v ethanol solution which

was subsequently gelled with Carbopol� 980 NF

[(0.5 % by weight) neutralized with triethanolamine].

Oil-in-water (o/w) conventional cream was formu-

lated employing 6 % w/w sorbitan mono–oleate, 3 %

w/w white bees wax, 36 % w/w white soft paraffin,

15 % w/w liquid paraffin, and quantity sufficient to

100 % w/w with distilled water (Raza et al. 2011). The

oil phase as well as the aqueous phase were heated

separately at 65 �C, the drug was incorporated in oil

phase followed by addition of aqueous phase to the oil

phase under continuous stirring. The resulting emul-

sion was allowed to cool down gradually under

constant stirring to obtain a cream.

Pre-formulation studies

Different concentrations of PL were investigated for

their suitability to formulate ethosomes with desired

quality attributes (Table 1). The selection was based

upon various parameters such as the microscopic

observations, vesicle count, transmittance, drug

entrapment, and drug payload. As ethanol is the main

component responsible for elasticity of vesicles its

amount was also optimized by using varying amount

of ethanol keeping PL and drug amount constant

(Table 1). The vesicle dispersions were suitably

diluted with TDW and the vesicles were counted

microscopically employing a hemocytometer; their

number density was calculated using Eq. 1 (Jain et al.

2005):

The vesicular dispersions (0.2 mL) were suitably

diluted with TDW and the transmittance was observed

spectrophotometrically at a wavelength of 500 nm

using TDW as the blank (Jain et al. 2008). The drug

entrapment efficiency of drug-loaded ethosomes and

liposomes were determined using SephadexG-50

(medium) by employing mini-column centrifugation

technique (Aggarwal and Goindi 2012). The empty

ethosomes and liposomes were also treated in the

analogous way to serve as blank during the studies.

Drug entrapment efficiency was calculated using

Eq. 2 and drug payload (drug entrapped/100 mg of

lipid) was calculated using Eq. 3:

Drug entrapment efficiency

¼ Entrapped drug ðmgÞTotal drug added ðmgÞ � 100 ð2Þ

Drug payload ¼ Amount of drug entrapped ðmgÞTotal mass of lipids ðmgÞ

� 100 ð3Þ

Characterization and evaluation of ethosomes

The optimized ethosome dispersion was characterized

for morphology (Hitachi H–7000 Tranmission Elec-

tron Microscope), vesicle size, size distribution pro-

file, zeta potential, and percent deformability

(Malvern Zetasizer, Malvern Instruments Ltd.,

Worcestershire, UK). The optimized ethosome dis-

persion as well as its gel was characterized for total

drug content (TDC) and pH (Labindia Pico?, Mum-

bai, India). The optimized ethosome gel was subjected

to texture analysis for assessment of different rheo-

logical properties like work of shear, force of gel

extrusion, stickiness, and firmness. The optimized

ethosome gel was filled in lacquered aluminum

collapsible tubes and stored at three different temper-

atures 5 ± 3, 25 ± 2, and 40 ± 2 �C for a period of

1 year and evaluated for TDC, pH, permeation

characteristics, and other organoleptic features.

Skin sensitivity studies and histopathological

examination

The skin sensitizing and irritant potential of the

developed ethosome formulation was evaluated. The

dorsal region (2 9 3 cm2) of mice was shaved with

electric clipper in the direction of tail to head without

damaging the skin. The control group was treated with

Total number of vesicles per cubic mm ¼ Total number of vesicles counted � dilution factor � 4000

Total number of squares countedð1Þ


123

normal saline and the optimized ethosome gel was

applied to the treatment group three times a day for

3 days consecutively (n = 5). The animals were

observed for any signs of itching or change in skin

such as erythema, papule, flakiness, and dryness. On

the third day animals were sacrificed, the skin was cut

and processed as reported by Azeem et al. (2012).

Briefly, one specimen from control and one from the

test group were fixed in 10 % (v/v) buffered formalin.

Subsequently, each tissue was rinsed thoroughly with

water, dehydrated using a graded series of alcohols,

embedded in paraffin wax, and microtomed. After-

ward, the sections were stained with hematoxylin and

eosin followed by observation under a high-power

light microscope.

Vesicle skin penetration study using fluorescent

microscopy

The skin of mice was prepared as discussed under skin

sensitivity studies. Ethosome dispersion loaded with

Rhodamine 123 was applied on the hair-free dorsal

region. The animals were sacrificed after 4 h by

overdose inhalation of chloroform and the skin was

incised, washed with excess of saline and sliced into

5-lm-thick sections, placed on slides, and observed

under fluorescence microscope.

Ex vivo drug permeation and skin retention studies

The studies were performed using excised dorsal skin

of Laca mice employing vertical Franz diffusion cell

assembly (PermeGear, Inc., PA, USA) as described by

Aggarwal and Goindi (2012). Briefly, phosphate

buffer saline (PBS) pH 6.4 containing 2.0 % w/v

Tween 20 was used as receptor media and the cell

contents were maintained at temperature of

32 ± 1 �C. 1 mL aliquot was periodically withdrawn

at suitable time intervals from the sampling arm of

receptor chamber and was replaced with fresh buffer.

At the end of the permeation studies (24 h), the skin

surface in the donor compartment was rinsed with

ethanol to remove the excess drug. The receptor

medium was then replaced with 50 % (v/v) ethanol to

extract the drug retained in the skin. Similar perme-

ation and skin retention studies were performed using

blank formulations (without drug) and the absorbance

values were subtracted from test formulations to

account for the effect of skin components as well as

formulation excipients. The cumulative percent

permeation, flux (Jss; lg/hr/cm2), and skin retention

(lg/cm2) were calculated. The data of ex vivo perme-

ation studies were statistically analyzed by one-way

analysis of variance (ANOVA) followed by Dunnett’s

method. Results were quoted as significant where

p \ 0.05.

Dermatopharmacokinetics

The dorsal skin of mice (2 9 3 cm2) was prepared as

discussed under skin sensitivity studies. The prepared

mice were divided into six groups for sampling at

different time points: 5, 15, 30 min, 1, 2, and 4 h

(n = 6). Vesicular dispersions (equivalent to 500 lg

of griseofulvin) were applied on the dorsal prepared

region of animals. 500 lL of blood was collected from

each animal at the specified time intervals and then

they were sacrificed to collect the skin samples which

were stored at -20 �C until analysis.

Reverse-phase HPLC (RP-HPLC) conditions

RP-HPLC method was developed according to the

ICH and US FDA validation guidelines reported by

Aggarwal and Goindi (2012). Waters� 2695 Separa-

tion Module equipped with a 2996 photodiode array

(PDA) detector and Waters Empower 2 software, and

Hibar� 250 9 4.6 mm2 HPLC column (M/s Merck

KGaA, Germany) kept at 40 �C were employed for

analysis. The mobile phase consisted of a mixture of

acetonitrile (ACN) and 0.1 M acetic acid (40:60 %; v/v)

and the flow rate was 1.5 mL/min. The stock solution

of griseofulvin (1 mg/mL) was prepared in ACN and

serially diluted with mobile phase in the concentration

range between 0.5 and 20 lg/mL to prepare the

calibration curve. All the samples were filtered

through 0.22-lm nylon membrane filter before ana-

lysis. The injection volume employed for analysis was

20 lL and the wavelength of detection was 293 nm.

The area under the peak was used to calculate the

concentration of griseofulvin.

Preparation of skin homogenate and extraction

of drug

Skin samples were treated with TDW at temperature

of 60 �C to make it free from subcutaneous fat (Fujii

et al. 2000). Skin homogenates (10 % w/v) were


123

prepared in PBS pH 6.4 and methanol (1:1, v/v), using

Teflon tissue homogenizer (Kim et al. 2005). One part

of skin homogenate was then treated with two parts of

ACN containing 0.5 % (v/v) formic acid and the

contents were vortexed for 1 min followed by centri-

fugation for 10 min at 10,000 rpm at 4 �C. The

supernatant was filtered through 0.22-lm nylon

membrane filter and analyzed.

Processing of blood samples and extraction of drug

The blood samples were centrifuged for 10 min

at 10,000 rpm to separate plasma. One part of plasma

was extracted with two parts of ACN containing

0.5 % (v/v) formic acid and analyzed as mentioned

above.

Antifungal studies

The broth microdilution method was used to deter-

mine the minimal inhibitory concentration of griseo-

fulvin against M. gypseum, M. canis, T.

mentagrophytes, and T. rubrum. The test were

performed using RPMI 1640 medium supplemented

with L-glutamine and without sodium bicarbonate

buffered at pH 7.0 with MOPS [3–(N–morpholino)

propanesulfonic acid] buffer. The agar plate diffusion

method was performed to check the efficacy of

griseofulvin-loaded optimized liposome and ethosome

dispersions against the above-mentioned dermato-

phytes. The cultures were revived and inoculums were

prepared as explained by Barros et al. (2007).

Test procedure for broth microdilution method

The tests were performed in sterile, round-bottomed,

96-well microplates following Clinical and Labora-

tory Standards Institute (CLSI) M38–A protocol;

using the drug concentrations between 0.039 and

16 lg/mL (Araujo et al. 2009).

Antifungal assay using agar plate diffusion method

The standard plot of griseofulvin against all the

dermatophyte strains was prepared in the concentra-

tion range 1–10 lg/100 lL using agar plate diffusion

method. 100 lL of the optimized ethosome and

liposome dispersions equivalent to 10 lg of griseo-

fulvin and their corresponding blanks were placed in

the agar plate wells and incubated for 48 h at 30 �C

after pre-diffusion. After the incubation period, the

zone of inhibition was measured and recorded.

Pharmacodynamic studies in guinea pigs

Microsporum canis was selected as the infecting

fungus because this zoophilic fungus can infect the

skin, resulting in skin and hair root invasion.

Inoculum preparation

Stock inoculum suspensions of the fungi containing

1 9 107 fungal conidia of M. canis in 200 lL of sterile

normal saline were prepared (Ghannoum et al. 2004).

Animal inoculation and antifungal therapy

A total of 20 animals were taken; 5 were treated with

liposome blank, 5 with ethosome blank, 5 with griseo-

fulvin-loaded liposome gel, and the remaining 5 with

griseofulvin-loaded ethosome gel. The inoculation of

animals was done under general anesthesia. An area of

3 9 3 cm2, on the guinea pigs back was made hair-free,

marked and abraded with sterile fine grit sandpaper.

Then 200 lL of the prepared inoculum was applied to

abraded skin (Ghannoum et al. 2004). The animals were

observed on daily basis for signs of infection. The

topical treatment was started after 7 days, after the

appearance and confirmation of fungal hyphae on the

skin of animals using potassium hydroxide microscopy

(Ghannoum et al. 2004). Vesicular gels were applied

twice a day using a dose quantity approximately

equivalent to 1 mg of drug. The clinical as well as

mycological parameters were also evaluated after

1 week of initiation of topical treatment and at the end

of treatment. The clinical assessment was scored on a

scale from 0 to 5 as follows: 0—no signs of infection;

1—few slightly erythematous places on skin; 2—well-

defined redness, swelling with few blistering hairs, and

bald patches with scaly areas; 3—large areas of marked

redness, incrustation, bald patches, and ulcerations; 4—

partial damage to the integument and loss of hair; and

5—excessive damage to the integument and complete

loss of hair at the site of infection.

The hair root invasion test was used to assess the

mycological cure rate resulting from antifungal treat-

ment (Ghannoum et al. 2004). Briefly, the area of

infection was divided into four quadrants and 10 hairs


123

per quadrant were uprooted and planted on the surface

of PDA which was subsequently incubated at 30 �C

for 48 h. After the incubation, the number of hair

exhibiting fungal filaments at the hair root was

counted.

For histopathological examination, skin biopsy

samples were obtained from one representative animal

per group after completion of the treatment period.

The tissue was fixed in 10 % neutral buffered forma-

lin, embedded in paraffin, and processed for histopa-

thological examination. The fungal elements were

visualized using Periodic acid–Schiff (PAS) staining

(Alkhayat et al. 2009).

Results and discussion

Pre-formulation studies

Various PL to drug ratios in the presence of minimum

fixed amount of ethanol were investigated (Table 1). It

was observed that on increasing the amount of PL, the

drug entrapment increased. The increase in drug

entrapment may be related to the increased availability

of the lipid phase for griseofulvin and its subsequent

accommodation in the lipid bilayers. The maximum

drug payload achieved was 3.09/100 mg PL (E4) with

a drug entrapment efficiency of 54.12 ± 0.45 %.

Although further increase in PL resulted in lesser

drug payload, the drug entrapment was enhanced to

60.91 ± 0.43 %; therefore, higher concentration of

PL (E5) was selected for further optimization studies

(Table 1). The effect of varying proportions of PL on

the number of vesicles per cubic mm, percent trans-

mittance, and its relative abundance was in conso-

nance with the drug entrapment studies (Table 1).

From the results of various characterization param-

eters, it was vividly apparent that increasing the

concentration of ethanol from 20 to 30 % increased

the entrapment efficiency owing to increase in fluidity

of membranes leading to higher drug loading in

the vesicles (Table 1). Formulation E7 containing

30 % ethanol exhibited maximum drug entrapment

(72.94 ± 0.80 %). However, further increase in eth-

anol percentage probably made the vesicle membrane

leaky due to greater disturbance of the lipid–bilayer

organization of vesicles due to increased solubiliza-

tion of PL in ethanol resulting in breakdown of

vesicular structures and thus leading to decreased

entrapment efficiency. The influence of increasing

percentage of ethanol on number of vesicles per cubic

mm, percent transmittance, and its relative abundance

was in coherence with the results of entrapment

efficiency.

E7, the optimized ethosome dispersion exhibited

higher drug entrapment as compared to the optimized

liposome dispersion which could be credited to

formulation attributes of the elastic vesicular systems.

The presence of ethanol in ethosomes not only

enhanced drug encapsulation in the PL bilayer but

also led to drug solubilization and entrapment in the

hydroethanolic core of the vesicles. Whereas, lipo-

somes, due to their rigid bilayer structure and aqua-

filled center, illustrated relatively lower drug entrap-

ment (Table 1).

Characterization studies

TEM micrograph of the optimized ethosomes exhib-

ited the presence of unilamellar assembles (Fig. 1),

possessing a mean vesicle size of 148.5 ± 0.48 nm

with a PDI of 0.203 ratifying the narrow variation in

the size range of vesicles (Fig. 2). The zeta potential of

E7 dispersion was observed to be -27.7 ± 0.48 mV

Fig. 1 Transmission electron micrograph of optimized etho-

somes (150,0009)


123

which could be ascribed to the presence of ethanol

which conferred a negative charge to the surface of

ethosomes.

The membrane elasticity of ethosomes is a crucial

and explicit character which differentiates it from

conventional vesicles. Ethosomes were observed to be

highly elastic with only 12 % deformation as com-

pared to liposomes exhibiting 58 % deformation in

their vesicle size after passing through polycarbonate

membrane. The elasticity of ethosomes could be

attributed to the presence of ethanol which might have

reduced the interfacial tension of the vesicle mem-

brane as well as the absence of cholesterol. Conse-

quently these vesicles easily penetrate through the

minute pores of the skin, undergoing changes in shape,

when deformations are enforced by the surrounding

stress or space confinements, which minimizes the risk

of vesicle rupture upon penetrating the skin pores.

TDC for E7 was observed to be 99.14 ± 0.32 %

and for its vesicular gel it was found to be

98.34 ± 0.73 %. The results revealed that the TDC

of the developed formulations was not significantly

different (p \ 0.001) from the initial amount incor-

porated in the formulations. pH of the optimized

ethosome dispersion and gel was observed to be 6.51

and 6.43, respectively.

Texture analysis revealed that the griseofulvin–

ethosome gel possessed fairly good gel strength, ease

of spreading, and adequate cohesiveness; which are

essential for application and retaining the formulation

on the skin (Fig. 3). Further, uniformity of texture

curve, plotted employing Exponent 32� software,

confirmed the smoothness of ethosome vesicular gel

and the absence of any grittiness or lumps.

The optimized ethosome gel exhibited stability

with respect to total drug content which was found to

vary between 99.40 ± 1.01 % and pH which varied

between 6.42 and 6.34 at 5 ± 3, 25 ± 2, and

40 ± 2 �C for a period of 1 year. The permeation

characteristics did not reveal any significant change

and the organoleptic features like the gel viscosity, gel

firmness, gel strength, and physical appearance were

also observed and no significant change was found in

these characters.

Skin sensitivity and histopathological studies

The mice skin from control group (Fig. 4a) on

comparison with the mice skin treated with griseoful-

vin-loaded ethosome gel (Fig. 4b) established the

safety of the prepared formulation with no perceptible

histopathological changes indicating the safety of the

Fig. 2 Graphical representation of particle size distribution of optimized ethosome dispersion (E7)


123

formulation for topical use. There was no apparent

sign of edema, inflammatory cell infiltration, ery-

thema, papule, flakiness, and dryness on mice skin.

Uniformly layered stratum corneum and loosely

textured collagen in the dermis could be observed.

Vesicle skin penetration study using fluorescent

microscopy

Ethosomes loaded with Rhodamine 123 when visualized

under fluorescent microscope indicated fluorescence

Fig. 3 Texture analysis of the prepared ethosome gel of griseofulvin

Fig. 4 Histological photographs of mice skin a control (no treatment); b after treatment with ethosome gel of griseofulvin (2009)


123

only in the vesicles suggesting removal of unentrapped

fluorescent probe via mini-column centrifuge technique

(Fig. 5a). The skin section 4 h after single topical

application of Rhodamine 123-loaded ethosomes

showed fluorescence with intact vesicles in the stratum

corneumas well as deeper layers ofmouse skin (Fig. 5b),

indicating that the vesicles penetrated the skin barrier and

got deposited in the skin. This attribute is desirable and

beneficial in targeting superficial fungal infections of the

skin. The presence of intact vesicles in the skin section

signifies that these can squeeze, pass through skin pores

and retain their structure.

Ex vivo drug permeation studies

The ex vivo permeation performance of conventional

formulations i.e., aqueous suspension, cream, hydroe-

thanolic gel, and liposomes was compared with

griseofulvin-loaded ethosomes. The ex vivo permeation

studies indicated that drug permeation from optimized

ethosomes (70.77 ± 0.83 %) in 24 h was significantly

higher than the liposomes, hydroethanolic gel, conven-

tional cream, and aqueous suspension of griseofulvin

(Fig. 6; p \ 0.05). The aqueous suspension of the drug

exhibited only 9.69 ± 0.86 % drug permeation, cream

base demonstrated 25.58 ± 0.61 %, hydroethanolic gel

showed 39.37 ± 0.79 %, and liposome dispersion

revealed 52.01 ± 0.73 % drug permeation in 24 h.

The ethosome gel depicted slightly lower drug perme-

ation of 62.26 ± 1.65 %, compared with ethosome

dispersion which may be attributed to slow diffusion of

drug through gel network. Besides providing the

optimum structure and viscosity to the ethosomes for

topical application, Carbopol offers an additional

advantage of excellent adhering and constant releasing

formulation (Bhaskar et al. 2008).

Also, the permeation kinetics of ethosomes was

studied by studying the diffusional release exponent

from the plot of log cumulative drug permeated versus

log time. This plot yielded a straight line from which

diffusional release exponent (n) was calculated and

found to be 0.75 ± 0.07 and 0.88 ± 0.08 for ethsome

dispersion and gel, respectively, illustrating non-

Fickian drug permeation pattern.

The enhanced drug permeation from ethosomes

suggests a synergistic mechanism between ethanol,

Fig. 5 a Ethosomes loaded with Rhodamine 123 seen under fluorescent microscope; b fluorescence microscope analysis of mice skin

after 4 h application of Rhodamine 123-loaded ethosomes (1009)

Fig. 6 Comparison of ex vivo permeation profiles of different

compositions of griseofulvin through mice skin (n = 3)


123

vesicles, and skin lipids. The stratum corneum lipid

multi-layers at physiological temperature are densely

packed and possess high conformational order. The

intercalation of ethanol into the polar head group

environment can result in an increase in the membrane

permeability (Berner and Liu 1995). In addition,

ethanol may also provide the vesicles with soft flexible

characteristics which allow their easier penetration

into deeper layers of the skin (Godin and Touitou

2005). The interdigitated, malleable ethosomes thus

can forge paths in the disordered stratum corneum,

justifying their superior delivery properties. However,

in creams and hydroethanolic gels, the diffusion and

partitioning processes occur at much slower rate and to

a lesser extent due to the viscous structure of the

creams and more rigid nature of conventional gels.

In terms of flux (rate of permeation) of griseofulvin,

ethosomes provided a significantly higher flux

(21.32 ± 0.26 lg/h/cm2) as compared to liposomes

(13.41 ± 0.24 lg/h/cm2), hydroethanolic gel (7.27 ±

0.12 lg/h/cm2), cream base (4.35 ± 0.79 lg/h/cm2),

and aqueous suspension (2.375 ± 0.11 lg/h/cm2) as

delivery module (Fig. 7; p \ 0.05). This is due to the

fact that the ethosomes get the most favorable interac-

tion with the skin cells and are able to build the aqua-

filled lipoidal microenvironment to facilitate the drug

transportation. The hydrodynamic/osmotic conditions

result in better drug–skin partitioning which may be

held responsible for the improved transport of drug

molecules within the skin layers. PLs which aid in the

penetration by mixing well with the skin lipid are absent

in conventional systems, leading to poor permeation

flux (Kirajavainen et al. 1999). It was observed that

gelling of ethosomes slightly decreased the flux

(20.13 ± 0.27 lg/h/cm2). This decrease may be due

to slow diffusion and penetration of ethosomes into and

through the skin from gel base.

Skin retention studies revealed that ethosomal

suspension resulted in higher skin retention

(38.75 ± 1.91 lg/cm2) as compared to aqueous sus-

pension (0.74 ± 0.21 lg/cm2), cream base (11.13 ±

0.42 lg/cm2), hydroethanolic gel (12.24 ± 0.51 lg/cm2),

and liposomal suspension (13.41 ± 0.71 lg/cm2). A

significantly greater retention achieved with etho-

somes indicates these to be better delivery systems for

topical use (Fig. 7; p \ 0.05). The retention was

enhanced by 2.89-, 3.16-, 3.48-, and 52.36-folds as

compared to liposomes, hydroethanolic gel, cream,

and aqueous suspension, respectively. Gelling of

ethosomes slightly enhanced the skin retention. As

evident from results of skin retention studies, vesicular

systems have shown better drug retention in skin vis–

a–vis conventional systems i.e., cream, hydroethanolic

gel, and aqueous suspension. This may be attributed to

the depot-forming characteristic of the vesicular

systems. The ethosome dispersion and gel achieved

Fig. 7 Comparison of flux (rate of permeation; lg/cm2/h) and drug retention in skin (lg/cm2) from various formulations of

griseofulvin in mice skin (n = 3)


123

the higher concentrations of drug in skin as compared

to liposome dispersion and gel which could be

ascribed to combined effect of ethanol and PL (Dubey

et al. 2007). Thus, it can be inferred that the prepared

ethosomes could effectively make the drug molecules

accessible within skin layers, retaining them within

close vicinity of the target infection site.

Dermatopharmacokinetics

A well-resolved and sharp peak of griseofulvin was

obtained with a retention time of approximately

8–9 min and a total run time of 15 min using ACN

and 0.1 M acetic acid (40:60 %; v/v) as the mobile

phase, with no interference with other components of

the mobile phase, skin homogenate, and plasma com-

ponents. The method was linear for the standard drug

samples, skin homogenate, and plasma samples, over

the studied concentration range i.e., 0.5–20 lg/mL. The

limit of detection for griseofulvin was 0.05 lg/mL and

the limit of quantification was 0.2 lg/mL in the standard

drug solution, skin homogenate as well as plasma

samples. The drug recovery from skin homogenates was

found to be 98.31–103.49 % showing good accuracy of

the method. The drug recovery from plasma samples

varied from 98.57 to 102.13 % again proving the

accuracy of the method.

The topical application of ethosomes leads to

fluidization of intercellular domains and thus a struc-

tural modification of the stratum corneum, resulting in

enhanced transport of encapsulated drugs in vesicles

(Dubey et al. 2007, 2010). The dermal and transdermal

penetration of vesicles is determined by the vesicle

size (Touitou et al. 2000), surface charge (Gillet et al.

2011), elasticity, and composition of the vesicle

bilayer (Pirvu et al. 2010; Verma and Fahr 2004).

The kinetic studies revealed that the deformable as

well as the conventional vesicles exhibited instanta-

neous penetration in the skin. Liposomes achieved

2.35 % (nearly 12 lg) and ethosomes attained 3.54 %

(almost 18 lg) drug retention in the skin in 5 min

(Fig. 8). Thereafter, in case of liposomes the drug

penetration attained a plateau phase in 30 min with

almost 4.45 lg drug retention until 4 h and in case of

ethosomes nearly 2.06 lg of drug deposition was

observed until 4 h. The malleable nature of ethosomes

leads to significantly higher (p \ 0.05) amount of drug

penetration and deposition as compared to liposomes

in the skin which eventually resulted in a rich drug

reservoir in skin. The plasma samples revealed the

presence of only 0.2 lg of drug after 15 min which

was maintained until 30 min. The plasma samples for

the further time points indicated the absence of drug in

plasma. The increased drug skin permeability with

ethosomes is concordant with the reports published in

literature showing enhanced drug permeation with

lipid vesicles having ethanol as one of their compo-

nents. Ethanol, an indispensible component of etho-

somes fluidizes both the vesicular lipid bilayers as well

as stratum corneum lipids, thus providing a greater

malleability to the vesicles and enhancing permeabil-

ity of the skin. Therefore, ethosomes act as malleable/

elastic carriers for drug and intact vesicles penetrate

the stratum corneum along with the encapsulated drug.

Second, these act as penetration enhancer and interact

with the stratum corneum lipids and alter the perme-

ability, which facilitates penetration of drug molecule

across stratum corneum. Thus, enhanced permeation

of drug with ethosomes could be attributed to com-

bined effect of alcohol and lipid vesicular system.

Antifungal studies

The broth microdilution method revealed complete

inhibition of M. gypseum, M. canis, T. mentagro-

phytes, and T. rubrum at 0.5 lg/mL of the drug

concentration. The blank vesicular dispersions of

liposomes as well as ethosomes did not exhibit any

zone of inhibition indicating the absence of any

antifungal efficacy of the formulation excipients at the

tested count of colony-forming units (cfu/mL) of

1 9 106. The agar plate diffusion protocol revealed

the efficacy of griseofulvin-loaded liposomes and

ethosomes against all the tested dermatophytes

Fig. 8 Comparison of percent drug retention in mice skin at

various time intervals after single topical application of the

griseofulvin-loaded ethosomes and liposomes (n = 6)


123

indicating the retention of antifungal efficacy of drug

encapsulated in the vesicles (Table 2). However, the

zones of inhibition for liposomal dispersion were less

as compared to ethosome dispersion which could be

attributed to the slow rate of drug diffusion from

liposomes. The microbiological studies were observed

to be synchronous with the ex vivo permeation studies

supporting the steady diffusion of drug from the

vesicles which would allow the drug to act on the

fungus for a longer time period.

Pharmacodynamic studies

The infected guinea pigs were observed daily for the

signs of infections. The first signs of infection were

observed on the 3rd day after inoculation in all the

animals manifested in the form of redness and scaling.

These alterations became more evident around the 7th

day with marked hair loss and brittle hair. The lesions

progressively increased in diameter in the animal

groups treated with blank formulations and were found

to be covered with white–yellow crusts strongly

adhered to the epidermis.

Redness and itching at the site of infection in the

treatment groups was allayed in 2–3 days. It was also

observed that there was shedding of the infected skin

scales and appearance of light pink-colored skin with

initiation of very fine hair growth on 5–6 days after the

initiation of treatment. The complete healing of the

infected site was achieved in 8 days in the group treated

with ethosome gel. Subsequently, a fine uniform and

healthy hair growth was observed at the site of infection.

Although treatment with liposome gel showed early

signs of symptomatic relief similar to ethosome gel,

complete mycological cure was achieved in 2 weeks

with a fine, uniform, and healthy hair growth.

The mycological efficacy of topical vesicular gels

of griseofulvin employing hair root invasion test

revealed the absence of fungal growth in hairs

obtained from the guinea pigs treated with drug-

loaded ethosome and liposome gels. However, the test

demonstrated abundant fungal growth in hairs

obtained from animals treated with liposome and

ethosome blank formulations when cultured in PDA

plates.

Skin biopsies were obtained from the test areas,

skin sections were stained with PAS stain, and

histopathological examination of skin sections was

performed to determine whether there was any skin

tissue invasion by M. canis. The histopathogical

results revealed the complete absence of any fungal

element in the skin biopsies of animals treated with

test formulations (Fig. 9a). However, in the animals

receiving ethosome blank treatment fungal elements

in the hair follicles were clearly visible (Fig. 9b).

Liposome gel depicted slower cure rate as compared to

ethosome gel which could be ascribed to the rigid or

less deformable nature of liposomes which might have

led to poor penetration and diffusion of drug in the

skin. The synergistic effect of ethanol and PL in the

vesicles might have played an active role in the fast

recovery from infection. The formulation might have

exhibited drying effect on skin which could be

ascribed to the presence of ethanol and the same

could be responsible for a faster cure rate.

Microsporum canis, the dermatophyte strain

selected for the study commonly results in Tinea

capitis (fungal invasion of scalp) in children (most

prevalent between 3–7 years of age). The infection

results in dry dandruff like scaling, broken hair shaft at

the scalp surface, smooth areas of hair loss, inflamed

mass, and yellow crusts. The conventional treatment

involves application of antifungal shampoo twice a

week for 4 weeks along with oral treatment with

griseofulvin as it is most effective against M. canis. In

lieu of the results of the present research, the ethosome

Table 2 Antifungal efficacy of griseofulvin-loaded vesicular dispersions against different fungal strains (n = 4)

Dermatophytes Liposome dispersion Ethosome dispersion (E7)

Zone of inhibition (cm) Percent drug diffusion Zone of inhibition (cm) Percent drug diffusion

M. gypseum 1.95 ± 0.06 49.10 ± 1.85 2.80 ± 0.08 70.02 ± 2.49

M. canis 1.98 ± 0.10 49.06 ± 1.86 2.78 ± 0.10 70.28 ± 1.36

T. mentagrophytes 1.88 ± 0.05 50.36 ± 1.73 2.65 ± 0.06 69.74 ± 1.16

T. rubrum 1.90 ± 0.08 49.76 ± 3.09 2.68 ± 0.13 69.91 ± 1.90


123

formulation of griseofulvin may be tested clinically

for its therapeutic benefits and clinical compliance in

humans.

Conclusion

The results of the present investigations conclusively

demonstrate the role of ethosomes in efficient dermal

drug delivery of griseofulvin. The ex vivo and the

pharmacodynamic studies of the developed ethosomes

of griseofulvin unambiguously assure their utility in

dermal delivery of griseofulvin. The developed system

may provide better remission from the disease due to

site-specific drug delivery with minimal side effects.

Acknowledgments Gift samples of griseofulvin supplied by

Wallace Pharmaceuticals Ltd., Mumbai, India; Phospholipon�

90G, provided by Phospholipid GmbH, Germany; and

Carbopol� 980 NF from Lubrizol Advanced Materials India

Private Limited, Mumbai, India are gratefully acknowledged.

Conflict of interest The authors report no conflict of interest.

The financial assistance was provided by CSIR, New Delhi for

carrying out the research studies.

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