Pharmaceutical Development and Technology, 12:591–601, 2007 Copyright © Informa Healthcare USA, Inc.ISSN: 1083-7450 print / 1097-9867 onlineDOI: 10.1080/10837450701481181

591

LPDT

Development of Liposomal Systems of Finasteride for Topical Applications: Design, Characterization, and In Vitro Evaluation

Development of Liposomal Systems of Finasteride for Topical ApplicationsRajiv Kumar, Bhupinder Singh, Gautam Bakshi, and Om Prakash KatareUniversity Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

Finasteride (FNS) is a “drug of choice” for benign prostatehypertrophy and prostate cancer. The drug has also been reportedto be useful orally in the treatment of some difficult-to-treatandrogen-dependent skin disorders, such as seborrhea, acne,hirsutism, and androgenetic alopecia. However, the ideal routefor its administration (i.e., topical) remains unexplored. This haslogically suggested the search for strategic formulationapproaches to make the drug effective on topical applications,hitherto unexplored. The present study targets the encasement ofdrug molecules in the interiors of vesicular compartments (lipo-somes) made up of hydrogenated phospholipids, as an attempttoward the development of a trans-epidermal therapeutic systemof FNS. Multilamellar drug-loaded liposomes were prepared bythin-film hydration with sonication method and optimized withrespect to drug payload, entrapment efficiency, and size by for-mulating different vesicular compositions under different processconditions. The vesicular systems consisting of saturated phos-pholipid (100 mg), cholesterol (50 mg), and FNS (5 mg) showedhighest drug payload (2.9 mg/100 mg of total lipids), and drugentrapment efficiency (88.6%). Mean (± SD) vesicle size of theprepared liposomes was found to be 3.66 ± 1.6 μm. Significantlyhigher skin permeation of FNS through excised abdominal miceskin of FNS was achieved from the liposomal formulations vis-à-vis corresponding solution and conventional gels. LiposomalFNS formulations also showed more than fivefold higher deposi-tion of drug in skin than the corresponding plain drug solutionand conventional gel. Stability studies indicated that the liposo-mal formulations were quite stable in the refrigerated conditionsfor 2 months with negligible drug leakage or vesicle size alter-ation during the storage period. Results of the current studieswith FNS-loaded vesicular systems project the high plausibility

of a topical liposomal formulation for effective localized deliveryof Finasteride.

Keywords phospholipids, drug delivery, transepidermal, skin,permeation, vesicles, dermatological disorders

INTRODUCTION

Finasteride (FNS), a synthetic 4-azasteroid com-pound[1] (4-azaandrost-1-ene-17-carboxamide), is an anti-androgen used for benign prostate hyperplasia (BPH) inlow doses (1 mg/day), and for prostate cancer in higherdoses (5 mg/day).[2–7] Recently, oral administration of FNShas also been found to be useful in the treatment of variousdermatological and follicular disorders, such as acne, seb-orrhea, and male pattern baldness (i.e., androgenetic alope-cia).[8–13] It is well documented that some skin or hairfollicle disorders involve alteration in the rate of proteinsynthesis in the nuclei of skin or hair follicle cells.[1,14]

Dihydrotestosterone (DHT), a derivative hormone (metab-olite) of testosterone, has been shown to be critical in thesedisorders, because alteration in the rate of protein synthesisis caused by internalization of DHT-androgen receptorcomplex in the nuclei of skin or of hair follicle cells.[15]

FNS blocks the production of DHT from testosterone bycompetitively and specifically inhibiting type II 5-α reductaseisozyme, thus decreasing many of its effects.[16] Hence,FNS tends to plays a vital role in the treatment of clinicalproblems related with skin and hair follicles.[8,17,18]

Although the importance and potential of FNS in thetreatment of dermatological problems has found a tacitplace, its oral route of administration has been a criticalissue.[19] The treatment of local skin problems involvingdermal layers and follicles would be more logical onlythrough the topical route, because oral administration ofFNS tends to cause several untoward effects in a vastmajority of patients. Common adverse effects following

Received 22 November 2006, Accepted 25 April 2007.Address correspondence to Professor Om Prakash Katare,

University Institute of Pharmaceutical Sciences, Panjab Univer-sity, Chandigarh-160 014, India; E-mail: drkatare@yahoo.com

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

592 R. Kumar et al.

oral intake include impotence, impaired reproductivefunction, erectile dysfunction, and gynecomastia.[20–22]

Nevertheless, the oral route has hitherto been preferred onaccount of unavailability of an effective topical formulationto deliver FNS onto the desired site of action.

Lately, different drug delivery approaches have beensuccessfully explored to achieve effective drug transportinto and across the skin.[23–25] Among these, phospholipid-based self-assembled supramolecular structures likeliposomes have shown promising results.[26,27] By virtue ofthe unique physicochemical character and diversity in theirconstructs, liposomes have proved to be a very effectivemeans of delivery, especially for topical administration.[28–32]

Besides the characteristic merits of liposomes, their choicefor delivery of FNS to the mesodermal site of action(including hair follicles) is also supported by the physico-chemical nature of the drug, which is highly lipophillic (logP = 4.277). It is supposed to get a more conducive milieuwithin the unique amphiphillic interiors of vesicular com-partments, favoring its partitioning and transport across theskin barrier. Furthermore, the carrier-mediated deliverywould significantly augment the interaction of FNS withandrogen receptors of skin and follicles leading to theenhanced drug action.[33] The latter has been recentlyreported by Tabbakhian et al.[34] by preparing negativelycharged liquid state liposomes and niosomes of FNS andevaluating them for follicular deposition of FNS in thepilosebacious units of hamster flank skin and ears.

Thus liposomes were chosen in the current study toexploit the potential of FNS in dermatological problemsthrough topical delivery. The current work relates to thesystematic development of multilamellar FNS-loaded vesi-cles considering various vesicle-specific as well as prepara-tory process-related parameters. Herein, the influence ofliposomal composition and process conditions was investi-gated on entrapment efficiency, drug payload, and vesiclesize. The effect of different lipid compositions, surfacecharge, and vesicle size on the skin permeation behavior ofprepared FNS liposomes was also studied in vitro by usingexcised abdominal mice skin, and the results obtained werecompared with the corresponding conventional FNS formu-lations. Finally, the stability studies on the optimized liposo-mal formulation were carried out to assess the influence ofstorage conditions and time on drug retention characteris-tics, physical stability, and size of the prepared vesicles.

EXPERIMENTAL

Materials

FNS (Cipla Limited, India) and hydrogenated soylecithin (PC; Phosphatidylcholine content 92–97%;

Phospholipon® 90H; Phospholipids GmbH, Germany)used in the current study were generous gifts from therespective sources. Cholesterol (CHOL), Sephadex G-50medium (bead size range 50–150 μm), and Hexadecylphosphate (Dicetyl phosphate; DCP) were procured fromSigma Chemical Co. (St. Louis, MO, USA). Methylcellu-lose (viscosity grade: 3000–5000 mPas, 2%/20°C) wasprocured from LOBA-Chemie (India). All other materialsand solvents obtained from commercial sources were ofanalytical grade. Double-distilled water was used throughoutthe experimental studies.

Preparation of FNS-Loaded Multilamellar Liposomes

Different compositions used for preparation of FNScontaining liposomes are listed as Table 1. Multilamellarvesicles were prepared by using film hydration with soni-cation technique.[35,36] For each formulation, a thin lipidfilm was prepared by dissolving accurately weighedamounts of the drug, PC, and/or CHOL in a minimumvolume of chloroform in a round-bottom flask. The sol-vent was then rotary evaporated at 40°C (Buchi RE 121Rotavapour, Buchi Laboratories, Switzerland) under nitro-gen stream. The thin lipid film formed on the wall of theflask was flushed with a stream of nitrogen for 1 min andsubsequently hydrated with distilled water for 20 min at55 ± 2°C. The liposomal dispersion thus obtained was vor-texed for 5 min and left undisturbed at room temperaturefor 2 hr to allow annealing of the lipid bilayers. Thehomogenous suspension of multilamellar vesicles wassubsequently sonicated (titanium microtip ¼ in.; Probesonicator-Misonix S-3000 USA; 4°C; 36 Watts; 10 min-5cycles of 2 min each) to reduce the size of MLVs. Theresulting liposomal suspensions were stored in nitrogen-flushed vials at 4°C until further studies. Liposomes, con-taining varied amounts of DCP, a surface charge inducer,were also prepared analogously. The content of FNS withrespect to total lipids ranged between 20 and 50 ng per mgof total lipids, and lipid concentrations used in differentliposomal suspensions ranged between and 10 and 17.5mg mL−1.

Estimation of Percent Drug Entrapment and Drug Payload in Liposomes

Unentrapped drug from the prepared liposomes wasseparated by mini-column centrifugation method.[37]

Liposomal suspension (0.2 mL) was placed in SephadexG-50 column, presaturated with empty liposomes, andcentrifuged at 2000 rpm (626 × g) for 3 min. Elutescontaining drug-loaded liposomes were collected and

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

Tab

le 1

E

ffec

t of

com

posi

tion

of v

ario

us F

NS

lipos

omal

for

mul

atio

ns o

n en

trap

men

t eff

icie

ncy,

dru

g pa

yloa

d, a

nd v

esic

le s

ize

Form

ulat

ion

Cod

e

Com

posi

tion

Am

ount

of F

NS

entr

appe

d*(m

g)PD

EFN

S pa

yloa

d (μ

g of

dru

g:m

g of

tota

l lip

ids)

Mea

n ve

sicl

e di

amet

er*

(μm

)FN

S (m

g)PL

90H

(m

g)C

HO

L (

mg)

DC

P (w

t % o

f to

tal l

ipid

s)

FNS

L1

510

0-

−3.

17 ±

0.1

363

.431

.7:1

15.4

± 2

.2FN

S L

25

100

10−

3.49

± 0

.23

69.8

31.7

:118

.1 ±

1.2

FNS

L3

510

025

−3.

93 ±

0.0

878

.631

.4:1

20.3

± 1

.1F

NS

L4

510

050

-4.

43 ±

0.1

888

.629

.5:1

21.7

± 2

.1FN

S L

55

100

75−

4.24

± 0

.12

84.8

24.2

:124

.1 ±

1.7

FNS

L6

590

60−

4.11

± 0

.19

82.2

27.4

:122

.4 ±

2.1

FNS

L7

575

75−

3.60

± 0

.12

72.0

24:1

20.8

± 1

.5FN

S L

85

6090

−3.

50 ±

0.0

370

.023

.3:1

21.3

± 0

.8F

NS

L9

510

050

14.

37 ±

0.1

287

.429

.1:1

14.3

± 0

.9FN

S L

105

100

502

3.90

± 0

.09

78.0

26:1

12.4

± 1

.1FN

S L

115

100

505

2.50

± 0

.12

5016

.6:1

10.8

. ± 0

.8

FNS:

Fin

aste

ride

; PL

90H

: Pho

spho

lipon

90H

; CH

OL

: Cho

lest

erol

; DC

P: D

icet

ylph

osph

ate;

PD

E: P

erce

nt d

rug

entr

apm

ent.

*Eac

h va

lue

repr

esen

ts M

ean

± SD

(n

= 3

).H

ighl

ight

ed r

ows

indi

cate

the

chos

en f

orm

ulat

ions

for

var

ious

stu

dies

.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

594 R. Kumar et al.

observed under optical microscope to confirm theabsence of unentrapped drug particles. Appropriateamount of elute was digested with Triton-X (1% w/v),and the clear solution was analyzed spectrophotometri-cally (UV-visible spectrophotometer, Shimadzu-1601,Japan) for estimation of FNS content at a λmax of 212 nm

. Liposomes prepared without drug, in asimilar manner, served as blank for the above studiesconducted in triplicate. Drug payload (DPL) in theprepared liposomal formulations was calculated asmicrograms of FNS per mg of total lipids. Percent drugentrapment (PDE) for the prepared liposomes was calcu-lated as in Eq. (1).

Vesicular Morphology and Size Analysis

Prepared liposomal dispersions were analyzed fortheir morphological attributes viz. shape, surface charac-teristics, lamellarity, and size by using optical microscopefitted with a CCD camera (Carton; Japan). Mean vesiclesize and size distribution profile of FNS-loaded liposomeswas determined by light-scattering particle size analyzer(Malvern Mastersizer; Model-S, version 2.15, Malvern,UK). The study was conducted in triplicate to assess thebatch to batch uniformity of vesicle size.

Transmission Electron Microscopy

Negative stain micrographs were prepared on coppergrids covered with a formvar/carbon film. The vesicledispersions were pipetted onto the grids and stained with1% phosphotungstic acid. Subsequently, the staineddispersions were viewed and photographed with a PhilipsCM 10 transmission electron microscope at an acceleratingvoltage of 80 kV.

Preparation of Methylcellulose Hydrogel as a Secondary Vehicle

Methyl cellulose hydrogel (2% w/w) was preparedas a secondary vehicle to make the prepared FNS lipo-somal formulations rheologically favorable for topicalapplication. Briefly, 2 g of methyl cellulose was dis-persed in 18 mL of distilled water maintained at 50°C

under continuous gentle stirring. Subsequently, thisdispersion was gradually added to 80 mL of ice-colddistilled water and stirred gently until complete gel-ling. Thereafter, appropriate amounts of FNS liposo-mal suspensions were gently levigated in the preparedmethylcellulose hydrogel to obtain corresponding lipo-somal gel formulations. For comparative studies, con-ventional hydrogel formulation of FNS was alsoprepared analogously by replacing distilled water asused in the above gel formulation with FNS-aqueoussolution (FNS solubilized in distilled water containing10% v/v methanol).

In Vitro Skin Permeation Studies

The experiments using animals were approved by theuniversity ethics committee. Trans-epidermal permeationstudies with FNS-containing liposomal (FNS-liposomesuspension and FNS-liposomes incorporated in methylcellulose gel 2% w/w) and nonliposomal formulations(10% methanolic solution of FNS and FNS dispersed inmethylcellulose gel) were carried out by using excisedabdominal mice skin.

Briefly, following removal of the subcutaneous fatfrom the full thickness abdominal mice skin, hair on thedorsal side of the animal were removed with the help of a0.1-mm animal hair clipper, in the direction of tail tohead. The dermis of the skin was wiped three to fourtimes with a lint-free adsorbent wipes soaked in isopro-panol to remove any adhering fat material. The tissuesamples were stored frozen at a temperature of −20°C fora maximum of 2 weeks before use. Prior to permeationexperiments, skin tissue was thawed and clamped intodonor and the receptor compartment of the jacketed ver-tical Franz diffusion cell (cross-sectional area of 9.61 cm2;capacity 30 mL; fabricated in-house). The receivermedium consisting of phosphate buffer pH 6.4 USP con-taining 10% v/v methanol was thermostatted at 32 ± 2°Cunder constant magnetic stirring up to 24 hr. Solubility ofFNS in the receptor medium was estimated prior to thepermeation experiments to ensure pseudo-sink condi-tions. Liposomal or non-liposomal FNS formulations(equivalent to 5 mg of drug) were applied uniformly onthe dorsal side of mice skin. The donor chamber and thesampling port were covered by parafilm to prevent evap-oration during the study. Aliquots of 3 mL were with-drawn periodically and replaced with an identical volumeof receptor media (i.e., phosphate buffer pH 6.4 USPcontaining 10% v/v methanol) to maintain the receptorphase volume at a constant level. Samples were suitablydiluted and quantified spectrophotometerically at a λmaxof 210 nm ( ).

( )%E cm11 311=

PDEEntrapped drug (mg)

Total drug added (mg)= ×100 (1)

E cm11 310% =

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

Development of Liposomal Systems of Finasteride for Topical Applications 595

Determination of FNS Deposition in Skin

Following permeation studies, the skin tissuemounted on the diffusion cell was removed and washedthrice with saline solution, followed by blotting betweentissue paper to remove any adhering formulation from thesurface. Subsequently, the skin tissue was cut into smallpieces and homogenized with 10 mL of chloroform:meth-anol mixture (2:1 v/v) for extracting FNS. Homogenatesuspension thus obtained was centrifuged for 5 min at5000 rpm (3913 × g). The supernatant was filtered byusing a 0.45-μ, membrane filter (nylon; Millipore, MA,USA) and quantified spectrophotometrically for drug con-tent at a λmax of 245 nm ( ). Fresh skin tissuetreated in the similar manner was taken as blank for theabove study. Each experiment was conducted in triplicate.

Stability Studies

Stability of the FNS-vesicles was studied for 2months. FNS-loaded liposomal suspensions (5 mL each)were transferred into sealed ampoules of 10 mL capacityafter flushing with nitrogen prior to their storage in refrig-erated condition (RF; 4–8°C) and at room temperature(RT; 25 ± 2°C). Ability of vesicles to retain the entrappeddrug (i.e., drug-retentive behavior) was assessed at thefixed time intervals (i.e., once a week during the firstmonth, and every 15 days afterwards). Samples were with-drawn and analyzed for FNS content in the mannerdescribed previously under drug entrapment studies.

The dispersions were transferred into clear test tubes,at different time points, for visual and microscopic (opticaland TEM) observations such as vesicle fusion, aggrega-tion disruption, and sedimentation. Physical stability usingthese visual and microscopic characteristics was assessedon an ordinal scale ranging between 0 and ++++ as below:

Vesicle size was measured at all the time points with0.1 mL of dispersion diluted 1000-fold with distilled wateron a Malvern Mastersizer. The pH of various liposomalsuspensions at different time points was measured byusing a high-sensitivity pH meter (Model: Metrohm 654,Metrohm, Switzerland).

RESULTS AND DISCUSSION

Preparation of FNS-Loaded Liposomes

Multilamellar liposomes (MLVs) containing FNSwere prepared by thin film hydration technique, owing toits stellar advantages, such as higher encapsulation effi-ciency for lipophillic drugs and ease of preparation.[38]

Preparation of FNS-loaded liposomes was affected bystudying the influence of drug-total lipid ratio on the drugencapsulation efficiency of vesicles. Besides the significantinfluence of process conditions, the drug-bearing capacity(i.e., drug payload), and liposome size were found to beinvariably dependent on drug-lipid ratio and other vesicularcomponents viz. CHOL and DCP too.

Table 1 summarizes the influence of various formula-tion variables, such as drug-lipid ratio, CHOL and DCP onpercent drug entrapment (PDE), drug payload (DPL), andmean size of prepared vesicles. As is evident from theresults, the cholesterol-free liposomes (FNS L1) couldentrap up to 63.4% of drug with DPL of 31.7 μg/mg oftotal lipids. Thereafter, considerable enhancement in PDEwas observed with the addition of cholesterol from 10 to50 mg (formulation FNS L2 to FNS L4), at a fixed druglevel of 5 mg. This observation is in consonance with theliterature reports that incorporation of CHOL in liposomesprofoundly affects the membrane properties of the lipidbilayers by reducing the rotational freedom of hydrocar-bon chains.[39] In addition, the incorporation of cholesterolalso eliminates the gel-to-liquid phase transition of vesiclebilayers and induces permanent transition of gel-statebilayer to an ordered liquid crystalline state.[40] Both thesemechanisms make the lipid bilayers more stable and lesspermeable to the encapsulated drug, leading to augmenta-tion in the entrapment efficiency of vesicles. Although thePDE of vesicles increased with CHOL addition in formu-lations FNS L1 to FNS L4, yet the effective drug payloaddecreased due to net increase in the total amount of lipids,with reference to the fixed amount (5 mg) of FNS. Maxi-mum PDE was observed in formulation FNS L4, and thesame tended to decrease with further increase in theamount of cholesterol or varying phospholipid-cholesterolratio (FNS L5 to FNS L8). Addition of cholesterol showeda direct bearing on the vesicle size too as the mean vesiclesize increased from 15.4 μm (FNS L1) to 21.3 μm (FNSL8) with increasing level of CHOL.

Based on the above results, the formulation FNS L4with maximum PDE of 88.6% (drug payload 29.53 μg ofdrug:1 mg of total lipids) was selected for further study.Addition of surface charge-imparting agent (i.e., DCP), upto 1% of total lipids (FNS L9) did not influence the FNSpayload of liposomes. This observation is quite contrary tothe earlier studies[35,41] in which the improvement in drug

Score Physical stability

0 : Unstable+ : Poor++ : Average+++ : Good++++ : Excellent

E cm11 332% =

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

596 R. Kumar et al.

payload in the vesicles containing surface charge inducerwas reported and ascribed to the improved structural integ-rity of the vesicles. However, the vesicle size in the currentstudy reduced further to a considerable extent (FNS L 9 toFNS L11) vis-à-vis neutral FNS-liposomes, attributable tothe electrostatic repulsions in the lipid bilayers during thevesiculation process.[38,41]

Regarding the process conditions, 5-min vortexingwas found to be adequate to obtain liposomal suspensions,free from aggregates. The former did not affect drugentrapment, as was confirmed by PDE determinationsboth before and after vortexing. Because in liposomesmeant for topical applications, size also plays a key role inthe drug transport across the skin, sonication of formula-tion FNS L 9 was carried out in an attempt to obtainsmaller MLVs. The experimental conditions of the sonica-tion process, especially the sonication time, was optimizedby looking into its considerable impact on drug encapsula-tion too.[38,42,43] As construed from Table 2, substantialsize reduction could be attained with 10 min of sonication,as it yielded vesicles with mean size of 3.66 μm (FNSL13) with quite negligible impact on drug entrapment.Figure 1 corroborates the same graphically. Furtherincrease in sonication affected PDE profoundly, probablybecause of the disruption of drug-loaded vesicles leadingto fragmentation of lipid bilayers. Nevertheless, thesebilayers reassemble later to yield the aggregated liposomaldispersions [41,42] as is evident from the increased vesiclesize (16.6 μm) in formulation FNS L14. Based on theresults of this study, FNS L13 liposomes were furthercharacterized and subsequently investigated for skinpermeation behaviour in vitro.

Morphological and Micromeritic Characterization

Results of the optical and TEM characterization of FNSL13 liposomes showed that the liposomes were homogenousin shape and of lamellar structure. The optical photomicro-graph (Figure 2A) of FNS L13 liposomes ratifies the multil-amellear nature of the vesicles (magnification of 1500×).

Further confirmation of vesicle formation and integrity ofbilayers was achieved by using TEM studies (Figure 2 B).

The vesicle size of all the liposomal formulationstended to follow Gaussian (i.e., normal) size distribution. Asvividly apparent from Figure 3A and 3B, portraying vesiclesize distribution of unsonicated and sonicated liposomes,respectively, the sonication reduced vesicle size and poly-dispersity significantly. Mean vesicle size in unsonicatedliposomes (FNS L4) was found to be 21.7 μm with 90% ofthe liposomal population equal to or below 54.17 μm.Smaller MLVs were, however, obtained with optimal soni-cation of FNS L4 liposomal dispersion with a mean vesiclediameter of 3.66 μm, having 90% of the population equal toor below 5.77 μm. Reproducibility of vesicle size of all theFNS formulations was ratified from the low values of stan-dard deviation observed for mean vesicle size (n = 3) of allthe liposomal-FNS formulations (Tables 1 and 2).

In Vitro Permeation and Skin Deposition Studies

The equilibrium solubility of FNS in phosphate bufferpH 6.4 USP containing 10% v/v methanol was found to be0.497 mg mL−1. Table 3 reports the results obtained withdifferent formulations of FNS. Trans-epidermal perme-ation profile of FNS-containing liposomal formulations[liposomal suspension (FNS L13) and liposomes incorpo-rated in methyl cellulose gel (FNS L13-gel)] were studied.The results obtained were compared with that of the non-liposomal formulations of FNS (i.e., 10% v/v methanolicsolution) and methylcellulose gel containing FNS inequivalent amounts. To assess the effect of surface chargeand vesicle size on drug permeability, the other two lipo-somal formulations (i.e., FNS L4 and FNS L9) were alsoincluded in the study.

After 24 hr the liposomal dispersion (FNS L13) andliposomal gel (FNS L13-gel) showed higher FNS perme-ation across skin than that of the hydroalcohlic solution(Control) and the conventional gel containing FNS (Figure 4).As indicated in Table 3, the amount of FNS permeated in24 hr was found to be 54% and 52.4% from the liposomal

Table 2 Optimization of sonication time: effect on PDE and vesicle size

Formulation Code

Liposome compositionSonication time (min) PDE

Mean vesicle diameter (μm)#FNS (mg) PL 90H (mg) CHOL (mg) DCP (wt %)

FNS L9 5 100 50 1 − 87.4 14.3 ± 0.9FNS L12 5 100 50 1 5 88.0 6.1 ± 1.8FNS L13 5 100 50 1 10 85.8 3.66 ± 1.6FNS L14 5 100 50 1 15 71.2 16.6 ± 3.4

#Values represent Mean ± SD (n=3).

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

Development of Liposomal Systems of Finasteride for Topical Applications 597

suspension (FNS L13) and liposomal gel (FNS L13-gel),respectively. Drug permeated in FNS-aqueous solution(Control) and FNS dispersed in methylcellulose gel, how-ever, was quite lower (i.e., 24% and 29%, respectively).Higher flux obtained with FNS L13 (28.369 μg/cm2/hr)and FNS L13-gel (25.715 μg/cm2/hr) than that obtainedwith the aqueous solution (12.069 μg/cm2/hr) and methyl-cellulose gel of FNS (10.377 μg/cm2/hr), unequivocally,substantiate the permeation-enhancing effect of drugvesiculation. Nevertheless, the incorporation of liposomaldispersion into a secondary vehicle (2% w/w methylcellu-lose hydrogel) did not affect the permeation characteristicsof the FNS-liposomal formulation. The observed perme-ation enhancement could be due to the increased fluidityof the skin barrier on account of the interaction of phos-pholipid molecules of the membranous structures with thatof skin cells.[29,30,44,45] Moreover, the enrichment of theskin layers with respect to hydrated lipids[46,47] is also sup-posed to enhanced the drug permeability.

The permeation behavior of FNS-bearing liposomeshas been shown to be influenced by the surface charge aswell as size.[48] Negatively charged smaller MLVs (FNSL9 and FNS L13) showed higher permeation flux, asindicated by the higher values of the enhancement ratio

Figure 1. Effect of sonication on drug payload and size ofFNS-liposomes.

0

25

50

75

100

Sonication time (min)

PD

E

0

6

12

18

Vesicle size (µ

m)

PDE Vesicle size

0 2015105

Figure 2. Photomicrogrpahs of FNS multilamellar liposomesmade up of saturated phospholipids (lipid concentration: 15 mgmL−1). (A) Optical photomicrogrpah. (B) Electronphotomicrograph.

(A)

(B)

Figure 3. Particle-size distribution plots of FNS-liposomes (A)unsonicated and (B) sonicated.

(A)

(B)

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

598 R. Kumar et al.

(ER) [FNS L9: 1.81; FNS L13: 2.13] than that of the neu-tral liposomal formulations [FNS L4: 1.56] with highervesicle size. This observation is in contrast to the conven-tional understanding that the positively charged liposomesare supposed to interact better with the negatively chargedskin cells.[49] However, in this report, the improved perme-ation of FNS with negatively charged liposomes may bedue to the other liposomal characteristics that tend to helpin skin penetration viz. size, skin hydration, and integra-

tion of liposomal phospholipids with the skinlipids.[41,48,49]

The ability of vesicles in retaining drug within theskin milieu (i.e., depot effect) was investigated bydetermining the amount of drug deposited in the skinsamples used in drug permeation studies. The amountof drug retained in the skin was considerably higher(Table 3) in liposomal preparations than in the non-liposomal formulations. This observation may beascribed to the fact that the liposomal phospholipidsmay mix with the intercellular lipids and thereby causethe swelling of intercellular lipids. These swollen lipidssubsequently serve to provide local accumulation of thedrug and the consequent formation of intracutaneousdrug depots.[50–52]

Stability Profile of FNS-Liposomes

Table 4 depicts the stability profile of the optimizedFNS-liposomal formulation at both the storage conditions.The FNS-liposomal preparations showed remarkably bet-ter stability for 2 months when stored at RF than the for-mulation kept at RT. Substantial loss (nearly 13%) of drugwas evident from the samples stored at room temperatures(i.e., 25 ± 2°C). At lower temperature (RF), on the otherhand, the liposomes could retain up to 97% of the incorpo-rated drug. Higher drug leakage observed at elevated tem-perature suggests the need to store liposomal productunder refrigeration conditions. Loss of drug from the

Table 3 Cumulative amount, permeation flux, and skin deposition of Finasteride through abdominal

rat skin with different FNS formulations

Formulation

PermeationPercent skin deposition*Qcum* (μg/cm2) Flux* (μg/cm2h−1) ER$

FNS L13-gel 280.95 ± 5.89 28.369 ± 1.321 2.35 31.56 ± 2.9FNS L13 286.16 ± 8.97 25.715 ± 1.917 2.13 30.87 ± 2.4FNS L9 218.52 ± 5.51 21.954 ± 2.301 1.82 21.76 ± 3.4FNS L4 197.71 ± 6.87 18.862 ± 3.101 1.56 16.75 ± 2.9FNS conventional gel 130.07 ± 7.71 10.377 ± 1.091 0.85 4.99 ± 0.9Control 156.08 ± 4.33 12.069 ± 2.342 − 2.56 ± 1.1

*Values represent Mean ± SD (n=3).$ER: Relative flux enhancement ratio w.r.t. Control; Qcum: Cumulative amount of FNS permeated in 24 hr.FNS L13-gel : Sonicated liposomes incorporated in methylcellulose (2% w/w) gel.FNS L13 : Sonicated liposomes.FNS L9 : Negatively charged liposomes.FNS L4 : Neutral liposomes.FNS Conventional gel : Finasteride in methylcellulose (2.0% w/w) gel.Control : 10% methanolic solution of FNS.

Figure 4. Mean cumulative amount of Finasteride permeatedfrom various liposomal and non-liposomal formulations acrossexcised abdominal skin (n = 3). The error bars represent standarddeviation values.

0

100

200

300

Time (h)

Cum

ulat

ive

drug

am

ount

per

cm

2

FNS 4 L FNS 9 LFNS 13 L FNS 13 L gelConventional gel Control

0 2418126

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

Development of Liposomal Systems of Finasteride for Topical Applications 599

vesicles stored at higher temperature may be assigned tothe effect of temperature on the gel to liquid transition oflipid bilayers together with the possible chemical degrada-tion of the phospholipids, leading eventually to defects inthe membrane packing.[30,31] Drug leakage of less than 3%of the initial load at refrigeration conditions is well withinthe permissible limits. No change in the pH values of vesi-cle suspensions, however, was seen at both the storageconditions.

Microscopic (Optical and TEM) investigations(Figures 5A and 5B) revealed that the liposomes storedat RF were found to be quite stable in terms of aggrega-tion and disruption tendencies over the studied storageperiod. Drug-loaded vesicles also retained their multila-mellar nature and shape uniformity to an appreciableextent. Size determinations were performed, at differenttime periods, as a part of stability protocol. Practically,no change in the vesicle size was observed at RF

vis-à-vis the significant increase observed at RT. Thelatter may again be accredited to vesicle aggregationand, hence, the poor physical stability of the vesiclesstored at higher temperatures. Considerable disruptionand drug leakage were observed from vesicles stored atRT after 6 weeks, and the stability studies were thusdiscontinued. The optimized FNS liposomes incorpo-rated in the secondary vehicle (i.e., methylcellulose gel)also exhibited similar stability profile (in terms ofmicroscopic characters, pH, and FNS content) at RFconditions (data not shown). Moreover, the cellulosicpolymers are also known to stabilize the leakage oflipid soluble drugs from the supersaturated systems,besides improving their physical stability.[42,53] The sta-bilization effect of the polymer can be attributed to theformation of a protective layer on the surface of thevesicles, resulting in inhibition of drug leakage andvesicle aggregation.

Table 4 Effect of storage conditions and time on drug leakage, physical stability, and vesicle size of optimized liposomal

preparation of Finasteride

Time Points

Stability parameters

RF RT

Percent FNS leakage Vesicle size pH

Physical stability

Percent FNSleakage Vesicle size pH

Physical stability

Freshly prepared (0 day)

− 3.65 ± 0.2 6.12 ± 0.1 ++++ − 3.65 ± 0.2 6.12 ± 0.1 ++++

1st week − 3.55 ± 0.1 5.85 ± 0.2 ++++ 3.7 ± 0.2 3.34 ± 0.3 5.91 ± 0.1 +++2 weeks 1.3 ± 0.2 3.75 ± 0.2 6.07 ± 0.4 ++++ 5.5 ± 0.1 4.11 ± 0.2 6.13 ± 0.3 +++3 weeks 1.29 ± 0.3 3.49 ± 0.1 5.98 ± 0.2 ++++ 6.3 ± 0.3 5.89 ± 0.5 5.98 ± 0.2 ++4 weeks 1.5 ± 0.1 3.30 ± 0.2 6.32 ± 0.03 +++ 9.8 ± 0.4 6.4 ± 0.1 6.16 ± 0.3 +6 weeks 2.3 ± 0.3 3.77 ± 0.3 6.11 ± 0.2 +++ 13.3 ± 0.5 8.9 ± 0.2 6.36 ± 0.1 08 weeks 2.8 ± 0.4 3.89 ± 0.2 5.93 ± 0.3 +++ − − − −

Figure 5. Photomicrogrpahs of FNS multilamellar liposomes after storage at RF for 2 months. (A) Optical photomicrogrpah. (B)Electron photomicrograph.

(A) (B)

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

600 R. Kumar et al.

CONCLUSIONS

The present work on the preparation of topical lipo-somes is an attempt to exploit the immense potential ofmultilamellar liposomal carriers to localize the drug ontothe desired target sites in the skin. For this, a steroidalmolecule, Finasteride, was identified owing to its lipophil-lic nature and delivery needs within the mesodermal layers(including follicular sites). Following thorough investiga-tions on its developed liposomal formulations, it can beinferred that Finasteride-loaded liposomal constructs withoptimal characteristics viz. entrapment, drug payload, size,lamellarity, and surface charge, are able to penetrate, parti-tion, and permeate the skin barrier to access its deliverydestinations. Further, the nonchemical modification in thedrug’s behavior by means of close supramolecular associ-ation of phospholipid molecules also promises to prolongthe drug action as revealed by drug deposition studies.Conclusively, the experimental results and the supportivetheoretical analysis unambiguously indicate promisingavenues for Finasteride in dermatological problems whileexploiting the potential of liposomes through topical routeand call for further intensive investigations.

ACKNOWLEDGMENTS

The authors thank Cipla Limited, Ahmedabad, India,and Phospholipids GmbH, Germany, for generously pro-viding Gift samples of Finasteride and Phospholipon® 90 H,respectively. They also thank the University Grants Com-mission, New Delhi, India, for providing financial assis-tance for carrying out this research work.

REFERENCES

1. Stoner, E. The clinical development of a 5 alpha-reductaseinhibitor, finasteride. J. Steroid Biochem. Mol. Biol. 1990,37, 375–378.

2. Xu, Y.; Dalrymple, S.L.; Becker, R.E.; Denmeade, S.R.;Issacs, J.T. Pharmacologic basis for the enhanced efficacy ofdutasteride against prostatic cancers. Clin. Cancer Res. 2006,12, 4072–4079.

3. Canby-Hagino, E.D.; Brand, T.C.; Hernandez, J.; Thompson,I.M. Chemoprevention of prostate cancer with finasteride.Expert Opin. Pharmacother. 2006, 7, 899–905.

4. Teillac, P.; Abrahamsson, P.A. The role of Finasteride in themanagement of BPH and in the prevention of prostate can-cer. Eur. Urol. Suppl. 2006, 5, 627.

5. Thompson, M.; Goodman, P.J.; Tangen, C.M.; Lucia, M.S.;Miller, G.J.; Ford, L.G.; Lieber M.M.; Cespedes, R.D.;Atkins, J.N.; Lippman, S.M.; . Carlin, S.M; Ryan, A.;Szczepanek, C.M.; Crowley, J.J.; Coltman Jr., C.A. The

influence of finasteride on the development of prostate can-cer. N. Engl. J. Med. 2003, 349, 215–224.

6. Geller, J. Five-year follows up of patients with benign pros-tatic hyperplasia treated with finasteride. Eur. Urol. 1995,27, 267–273.

7. Gormley, G.J.; Stoner, E.; Bruskewitz, R.C.; Imperato-McGinley, J.; Walsh, P.C.; McConnell, J.D.; Andriole, G.L.;Geller, J.; Bracken, B.R.; Tenover, J.S. The effect of finas-teride in men with benign prostatic hyperplasia. N. Engl. J.Med. 1992, 327, 1185–1191.

8. Chen, W.; Zouboulis, C.C.; Orfanos, C.E. The 5 alpha-reductase system and its inhibitors. Recent development andits perspective in treating androgen-dependent skin disor-ders. Dermatology 1996, 193, 177–184.

9. Van Neste, D. Natural scalp hair regression in preclinicalstages of male androgenetic alopecia and its reversal by fin-asteride. Skin Pharmacol. Physiol. 2006, 19, 168–176.

10. Moghetti, M.; Castello, R.; Magnani, C.M.; Tosi, F.; Negri, C.;Armanini, D.; Bellotti, G.; Muggeo, M. Clinical and hor-monal effects of 5 alpha-reducatse inhibitor finasteride inidiopathic hirsutism. J. Clin. Endocrinol. Metab. 1994, 79,115–1121.

11. Meidan, V.M.; Touitou E. Treatments for androgeneticalopecia and alopecia areata: current options and future pros-pects. Drugs 2001, 61, 53–69.

12. Wong, L.; Morris, R.S.; Chang, L.; Spahn, M.A.; Stanczy, F.Z.;Lobo, R.A. A prospective randomized trial comparing finas-teride to spironolactone in the treatment of hirsute women.J. Clin. Endocrinol. Metab. 1995, 80, 233–238.

13. Shapiro, J.; Kaufman, K.D. Use of finasteride in the treat-ment of men with androgenetic alopecia (male pattern hairloss). J. Invest. Dermatol. Symp. Proc. 2003, 8, 20–23.

14. Mellin, T.N.; Busch, R.D.; Rasmusson, G.H. Azasteroids asinhibitors of testosterone 5 alpha-reductase in mammalianskin. J. Steroid Biochem. Mol. Biol. 1993, 44, 121–131.

15. Diani, A.R.; Mulholland, M.J.; Shull, K. Hair growth effectsof oral administration of finasteride, a steroid 5-alpha-reductase inhibitor, alone and in combination with topicalminoxidil in the balding stump tail macaque. J. Clin.Endocrinol. Metab. 1992, 74, 345–350.

16. Propecia® (Finasteride): Information for health care profes-sionals. Merck & Co. Inc. Whitehouse Station, NJ 08889,USA. Available at http://www.propecia.com/finasteride/propecia/consumer/index.jsp. Accessed 10 Septemeber 2006

17. Chen, C.; Puy, L.A.; Simrad, J.; Li., X.; Singh, S.M.; Labrie, F.Local and systemic reduction by topical finasteride or fluta-mide of hamster flank organ size and enzymatic activity.J. Invest. Dermatol. 1995, 105, 678–682.

18. Choudhry, R.; Hodgin, M.B.; Vander Kwast, T.H.;Brinkmann, A.O.; Boersma, W.J. Localization of androgenreceptors in human skin by immunohistochemistry: impli-cations for the hormonal regulation of hair growth, seba-ceous gland and sweat glands. J. Endocrinol. 1992, 133,467–475.

19. Price, V.H.; Menefee, E.; Sanchez, M.; Kaufman, K.D.Changes in hair weight in men with androgenetic alopeciaafter treatment with finasteride (1 mg daily): Three- and 4-year results. J. Am. Acad. Dermatol. 2006, 55, 71–74.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

Development of Liposomal Systems of Finasteride for Topical Applications 601

20. Price, V.H.; Roberts, J.L.; Hordinsky M.; Olsen, E.A.; Savin, R.;Bergfeld, W.; Fiedler, V.; Lucky, A.; Whiting, D.A.; Pappas,F.; Culbertson, J.; Kotey, P.; Meehan, A.; Waldstreicher, J.Lack of efficacy of finasteride in postmenopausal womenwith androgenetic alopecia. J. Am. Acad. Dermatol. 2000,43, 768–776.

21. Green, L. Gynacomastia and breast cancer during finasteridetherapy. N. Engl. J. Med. 1996, 335, 823.

22. Steiner, J.F. Clinical phamacokinetics and pharmacodynam-ics of finasteride. Clin. Phamacokinet. 1996, 30, 16–27.

23. Hadgraft, J. Passive enhancement strategies in topical andtransdermal drug delivery. Int. J. Pharm. 1999, 184, 1–6.

24. Cevc, G. Drug delivery across the skin. Expert. Opin. Inves-tig. Drugs 1997, 6, 1887–1937.

25. Hadgraft, J. Recent developments in topical and transdermaldelivery. Eur. J. Drug. Metab. Pharmacokinet. 1996, 21,165–173.

26. Goyal, P.; Goyal, K.; Kumar, V.; Singh, A.; Katare, O.P.;Mishra, D.N. Liposomal drug delivery systems—Clinicalapplications. Acta Pharm. 2005, 55, 1–25.

27. Schmid, M.H.; Korting, H. C. Liposomes: A drug carriersystem for topical treatment in dermatology. Crit. Rev. Ther.Drug Carrier Syst. 1994, 11, 97–118.

28. Fahr, A.; Mueller, R. Cyclosporin invasomes as topical drugdelivery systems for the therapy of immune system relatedskin diseases. European Patent Application 2002, 205420020208.

29. Yarosh, D.B. Liposomes in investigative dermatology. Pho-todermatol. Photoimmunol. Photomed. 2001, 17, 203–212.

30. Singh, B.; Mehta, G.; Kumar, R.; Bhatia, A.; Ahuja, N.;Katare, O.P. Design, development and optimization of nime-sulide-loaded liposomal systems for topical application.Curr. Drug Deliv. 2005, 2, 143–153.

31. Bhatia, A.; Kumar, R.; Katare, O.P. Tamoxifen entrappedtopical liposomes: Development, characterization and in-vitro evaluation. J. Pharm. Pharm. Sci. 2004, 7, 252–259.

32. Agarwal, R.; Katare, O.P.; Vyas, S.P. Preparation and in-vitro evaluation of liposomal/niosomal delivery systems ofantipsoriatic drug dithranol. Int. J. Pharm. 2001, 228, 43–52.

33. Bouwstra, J.A.; Honeywell-Nguyen, P.L.; Gooris, G.S.;Ponec, M. Structure of the skin barrier and its modulation byvesicular formulations. Prog. Lipid Res. 2003, 42, 1–36.

34. Tabbakhian, M.; Tavakoli, N.; Jaafari, M.R.; Daneshamouz, S.Enhancement of follicular delivery of finasteride by lipo-somes and niosomes. 1. In vitro permeation and in vivo dep-osition studies using hamster flank and ear models. Int. J.Pharm. 2006, 323, 1–10.

35. Manosroi, L.K.; Manosroi, J. Stability and transdermalabsorption of topical amphotericin B liposome formulations.Int. J. Pharm. 2004, 270, 279–286.

36. Bangham, D.; Standish, M.M.; Watkins, J.C. Diffusion ofunivalent ions across the lamellae of swollen phospholipids.J. Mol. Biol. 1965, 13, 238–252.

37. Dipali, S.R.; Kulkarni, S.B.; Betagiri, G.V. Comparativestudy of separation of non-encapsulated drug from unilamel-lar liposomes by various methods. J. Pharm. Pharmacol.1996, 48, 1112–1115.

38. New, R.R. C. Liposomes: A Practical Approach; IRL Press:Oxford, 1990.

39. Gupta, P.N.; Mishra, V.; Rawat, A.; Dubey, P.; Mahor, S.;Jain, S.; Chatterji, D. P.; Vyas, S.P. Non-invasive vaccinedelivery in transferosomes, niosomes and liposomes: a com-parative study. Int. J. Pharm. 2005, 293, 73–82.

40. Trotta, M.; Peira, E.; Carlotti, M.E.; Gallarate, M. Deform-able liposomes for dermal administration of methotrexate.Int. J. Pharm. 2004, 270, 119–125.

41. Manosroi, A.; Podjanasoonthon, K.; Manosroi, J. Develop-ment of novel topical tranexamic acid liposome formulation.Int. J. Pharm. 2002, 235, 61–70.

42. Heurtault, B.; Saulnier, P.; Pech, B.; Proust, J.E.; Jean-Pierre, B. Physico-chemical stability of colloidal lipid parti-cles. Biomaterials 2003, 24, 4283–4300.

43. Carafa, M.; Santucci, E.; Lucania, G. Lidocaine-loaded non-ionic surfactant vesicles: Characterization and in vitro per-meation studies. Int. J. Pharm. 2002, 231, 21–32.

44. Valenta C.; Wanka, M.; Heidlas, J. Evaluation of a novelsoya-lecithin formulation for dermal use containing ketopro-fen as a model drug. J. Control. Release 2000, 63, 165–173.

45. Ogiso, T.; Niinaka, N.; Iwaki, M. Mechanism for enhance-ment effect of lipid disperse system on percutaneous absorp-tion. J. Pharm. Sci. 1996, 85, 57–64.

46. Dreher, F.; Walde, P.; Walther, P.; Wehrli, E. Interaction of alecithin microemulsion gel with human stratum corneum andits effect on transdermal transport. J. Control. Release 1997,45, 131–140.

47. Walde, P.; Giuliani, A.M.; Boicelli, C.A.; Luisi, P.L.Phospholipid-based reverse micelles. Chem. Phys. Lipids1990, 53, 265–288.

48. Fahr, A.; Blume, G.; Verma, S.; Verma, D.D. Particle size ofliposomes influences dermal delivery of substances intoskin. Int. J. Pharm. 2003, 258, 141–151.

49. Yu, H.Y.; Liao, H.M. Triamcinolone permeation from differ-ent liposome formulations through rat skin in-vitro. Int. J.Pharm. 1996, 127, 1–7.

50. Abraham, W.; Downing, D.T. Interaction between corneo-cytes and stratum corneum lipid liposomes in vitro. Biochim.Biophys. Acta 1990, 1021, 119–125.

51. Plessis, J. du.; Ramachandran, C.; Weiner, N.; Muller, D.G.The influence of particle size of liposomes on the depositionof drug into skin. 1994, 103, 277–282.

52. Yarosh, D.B. Liposomes in investigative dermatology. Pho-todermatol. Photoimmunol. Photomed. 2001, 17, 203–212.

53. Gabrijelcic, V.; Sentjure, M. Influence of hydrogels onliposome stability and on the transport of liposomeentrapped substances into the skin. Int. J. Pharm. 1995,118, 207–212.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y JH

U J

ohn

Hop

kins

Uni

vers

ity o

n 10

/01/

13Fo

r pe

rson

al u

se o

nly.