elastic liposomes mediated transdermal delivery of an anti-hypertensive agent: propranolol...

Elastic Liposomes Mediated Transdermal Deliveryof an Anti-Hypertensive Agent: Propranolol Hydrochloride

DINESH MISHRA,1 MINAKSHI GARG,1 VAIBHAV DUBEY,1 SUBHEET JAIN,2 N.K. JAIN1

1Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences,Dr. Hari Singh Gour University, Sagar (MP), 470003 India

2Department of Pharmaceutical Sciences and Drug Research, Panjabi University, Patiala, Punjab, 147002 India

Received 28 December 2005; accepted 24 June 2006

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20737

ABSTRACT: One major problem encountered in transdermal drug delivery is the lowpermeability of drugs through the skin barrier. In the present investigation ultra-deformable lipid vesicles, that is, elastic liposomes were prepared incorporatingpropranolol hydrochloride for enhanced transdermal delivery. Elastic liposomes bearingpropranolol hydrochloride were prepared by conventional rotary evaporation methodand characterized for various parameters including vesicles shape and surfacemorphology, size and size distribution, entrapment efficiency, elasticity, turbidity, andin vitro drug release. In vitro flux, enhancement ratio (ER), and release pattern ofpropranolol hydrochloride were calculated for transdermal delivery. In vivo studyconducted on male albino rats (Sprague Dawley) was also taken as a measure ofperformance of elastic liposomal, liposomal, and plain drug solution. The betterpermeation through the skin was confirmed by confocal laser scanning microscopy(CLSM). Results indicate that the elastic liposomal formulation for transdermal deliveryof propranolol hydrochloride provides better transdermal flux, higher entrapmentefficiency, ability as a self-penetration enhancer and effectiveness for transdermaldelivery as compared to liposomes. � 2006 Wiley-Liss, Inc. and the American Pharmacists

Association J Pharm Sci 96:145–155, 2007

Keywords: propranolol hydrochloride; transdermal delivery; elastic liposomes

INTRODUCTION

The systemic treatment of disease via transder-mal route is not a recent innovation. But, in thelast two decades, transdermal drug delivery hasgained increasing interest. So, transdermal-controlled drug delivery systems have beeninvestigated or developed in order to either avoidhepatic first-pass effect improving drugs bioavail-ability or to decrease the dosing frequencyrequired for oral treatment. However, at present,marketed transdermal drug delivery patches are

available only for a few drugs. Most investigateddrugs do not cross the skin in adequate amount toproduce the therapeutic effect.

Propranolol hydrochloride (nonselectiveb-adrenoreceptor blocker) is widely used inthe treatment of many cardiovascular diseaseslike hypertension, angina pectoris, cardiac arrhy-thmia, and myocardial infarction. But it is subjectto an extensive and highly variable hepatic first-pass metabolism following oral administrationwith a very low bioavailability of between 15%and 23%. This would explain that many systemscontaining propranolol hydrochloride have beenrecently developed for oral and buccal1,2 andtransdermal administration.3,4 Theweak intrinsicability of propranolol hydrochloride to cross the

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007 145

Correspondence to: Dinesh Mishra (Telephone: 07582-220758; Fax: 07582-220758; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 96, 145–155 (2007)� 2006 Wiley-Liss, Inc. and the American Pharmacists Association

skin has been partially improved using prodrugs,micellar solubilization, and chemical penetrationenhancers.5–7 However, all these approaches arenot successful in improving the transdermal fluxto desired level.

To overcome the limitation of poor skin perme-ability of above-mentioned approaches we haverecently reported the elastic vesicles, transfer-somes or elastic liposomes, and ethosomes fortransdermal delivery of Norgestrel, Dexametha-sone, and Zidovudine.8–10 In the present study wereport the elastic liposomes as a carrier for thetransdermal delivery of propranolol hydrochlor-ide. Elastic liposomes are specially optimized,ultradeformable lipid supramolecular aggregates,which are able to penetrate the intact mammalianskin. Elastic liposomes overcome the skin penetra-tion difficulty, possibly by squeezing themselvesalong the intracellular sealing lipids of the stra-tum corneum. The reason for this is the highvesicle deformability, which permits them torespond to the mechanical stress of the surround-ing in a self-adapting manner.11

Elastic liposomes, the ultradeformable carriersfor potential transdermal application, contain amixture of lipids and biocompatible membranesoftners. The optimalmixture leads to flexibility ofthe elastic liposomes membranes and to thepossibility of penetration through channels of theskin, which are opened by the carriers.

Propranolol hydrochloride is hydrophilic innature and its absorption through skin is verypoor. Therefore, in the present study, to increasepercutaneous penetration, the drug was incorpo-rated into elastic liposomes, which would givegreater bioavailability and a very good skin per-meation capability.

EXPERIMENTAL SECTION

Materials

Propranolol hydrochloride was received as giftsample from M/s Sun Pharma Ltd., Vadodara,India. Soyaphosphatidylcholine (PC), cholesterol,Sephadex-G-50, Triton X-100 were purchasedfrom Sigma chemicals Co. (St. Louis, MO). Span40, 60, and 80 were purchased from M/s LobaChemie, Mumbai, India. Rhodamine red-X1,2 dihexadecanoyl-sn-glycero-3-phosphoethano-lamine trimethylammonium salt (RR) was pur-chased from Molecular Probes (Eugene, OR).Ethanol, chloroform, and xylene were procuredfrom M/s E. Merck, Goa, India. Cellophane

membrane (molecular weight cut off, 12000–14000) was procured fromHimedia Ltd., Mumbai,India. All other reagents were of analyticalreagent grade and double distilled water wasused in all experiments.

Preparation of Vesicular Formulations

The elastic liposomes were prepared by conven-tional rotary evaporation sonication methoddescribed by Cevc et al.12 Precisely, phospholipidand surfactant were taken in a clean, dry, roundbottom flask and the lipid mixture was dissolvedin methanol or chloroform:methanol, 2:1 v/v. Theorganic solvent was removed by rotary evapora-tion above the lipid transition temperature(Rotary Evaporator, Superfit, Ambala, India).Final traces of solvent were removed undervacuum overnight. The deposited lipid film washydrated with drug solution in PBS (pH 6.5) byrotation at 60 rpm for 1 h at room temperature.The resulting vesicles were swollen for 2 h at roomtemperature to get large multilamellar vesicles(LMLVs). To prepare smaller vesicles, LMLVswere probe sonicated at 48C for 20 min at40 W (Probe Ultrasonicator, Imeco Ultrasonics,Mumbai, India). The sonicated vesicles wereextruded through a sandwich of 100 and 200 nmpolycarbonate membranes (Millipore; Sigmachemicals Co., St. Louis, MO). The final lipidand drug concentrations in all vesicular formula-tions were 5% w/v and 0.4% w/v, respectively.Similarly Rhodamine Red (0.03% w/v) loadedelastic liposomes were prepared. The liposomes(Phosphatidylcholine:Cholesterol; 7:3) that serv-ed as control in the present study were alsoprepared by the same method as described above.The final lipid and drug concentrations were 5%w/v and 0.4% w/v, respectively.

Incorporation of Propranolol into VesicleDispersion at Saturated Concentration

Propranolol hydrochloride was incorporated intoall vesicle formulations at saturated concentra-tion to obtain equal thermodynamic activities. Todetermine the maximum amount of drug thatcould be added, increasing amounts of propranololwere added during preparation of elastic liposo-mal formulation. It was assumed that the pre-sence of propranolol crystals would indicate thatthe formulation was saturated with propranolol.Therefore, all propranolol-loaded vesicle formula-tions were examined over a period of 14 days

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using phase contrast microscope (Leica, DMLB,Heebrugg, Switzerland).

Characterization of Elastic Liposomal Formulations

Vesicles Shape and Type

Elastic liposomal formulations were visualizedusing a Philips transmission electron microscope,with an accelerating voltage of 100 kV. A drop ofthe sample was placed on to a carbon-coatedcopper grid to leave a thin film on the grid. Beforethe film dried on the grid, it was negativelystained with 1% phosphotungastic acid (PTA). Adrop of the staining solution was added on to thefilm and the excess of the solution was drained offwith a filter paper. The grid was allowed tothoroughly dry in air and samples were viewed ona transmission electron microscope.

Vesicles Size and Size Distribution

The vesicles size and size distribution weredetermined by Dynamic Light Scattering method(DLS), in a multimodal mode using a computer-ized inspection system (Malvern Zetamaster,ZEM 5002, Malvern, UK). For vesicles sizemeasurement, vesicular suspension was mixedwith the appropriate medium (PBS pH 6.5) andthe measurements were conducted in triplicate.

Entrapment Efficiency

For determination of entrapment efficiency theunentrapped drug was separated by the use ofthe minicolumn centrifugation method.13,14 Theamount of drug entrapped in the vesicles was thendetermined by disrupting the vesicles using 0.1%Triton X-100 followed by filtration and amountof drug was quantified spectrophotometrically at289.5 nm.

Elasticity of Vesicle Membrane

Elasticity of vesicle membrane is an importantand unique parameter of elastic liposomal for-mulations because it differentiates elastic lipo-somes from other vesicular carriers like liposomesthat are unable to cross the stratum corneum,intact. The deformability study was done for theelastic liposomal formulation against the stan-dard liposome preparations using a home-builtdevice as described.9,11 In this study the flux ofvesicle suspensions through a large number ofpores of known size (a sandwich of polycarbonatefilters with pore diameter between 50 and 200 nm,

depending on the starting vesicle suspension),was driven by an external pressure of 2.5 bar. Theamount of vesicle suspension, which was extrudedduring 5 min, was measured and vesicle size andsize distribution were monitored by DLS mea-surement before and after filtration. The experi-ment was performed in triplicate and each samplewas analyzed twice. The elasticity of vesiclemembrane was calculated by using the followingformula as reported by Vanden Bergh et al.:15

D ¼ J � rvrp

� �2

where D is the elasticity of vesicle membrane; J,amount of suspension, which was extruded dur-ing 5 min; rv, size of vesicles (after passes); and rp,pore size of the barrier.

Turbidity Measurement

The turbidity of different elastic liposomal for-mulations was determined using PBS (pH 6.5) asblank (Nephelometer, Superfit, Mumbai, India).

Animals

All investigations were performed after approvalfrom the Institutional Animals Ethics Committeeof Dr. H. S. Gour University (Sagar, India). Albinorats (Sprague Dawley strain), 5–6 weeks old,weighing 80–100 g were kept under laboratoryconditions. The animals were prepared for theplasma concentration and confocal microscopicstudy, by manually trimming the hair of rats to amaximum length of 2 mm. The formulation wasapplied topically with a precision, positive dis-placement glass pipette in the case of elasticliposomes, conventional liposome and plain drugsolution. The designated application site on theanimal back or hind extremity was also marked.The area of treated animal was protected by usingnylon mesh, which was supported by the plasticsquares having small pores kept above the treatedarea. Treated animals were kept in a separatecage and maintained under laboratory conditions.Food and water were allowed ad libitum.

Confocal Laser Scanning Microscopy (CLSM)

Depth of skin penetration of Rhodamine Red (RR)loaded elastic liposomes and rigid liposomes wereassessed using CLSM. The probe-loaded vesicleswere first passed through the Sephadex G-50mini

TRANSDERMAL DELIVERY OF AN ANTI-HYPERTENSIVE AGENT 147

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007

column to separate the unentrapped probe andthen about 200 mL of the formulations was appliednonocclusively for 8 h to the dorsal skin of 5- to6-week-old nude albino rats (Sprague Dawleystrain). The rat was sacrificed by heart puncture;dorsal skin was excised and washed with distilledwater. Adhering fat and/or subcutaneous tissuewas gently removed from the excised skin. It wasthen cut out in the pieces of 1 mm2 and tested forprobe penetration. The full skin thickness wasoptically scanned at different increments throughthe z-axis of a CLS microscope (LSM 510 with anattached universal Zeiss epifluoroscence micro-scope). Optical excitations were carried out with a488-nm argon laser beam and fluorescence emis-sion was detected above 560 nm for RR. Theparameters those were set up before the experi-ment and were not changed throughout themeasurements were: pin hole size, electron gain,neutral density filters, and background levels.The quantitative analysis was performed by Zeissprogram.

Skin Permeation Study

The in vitro skin permeation of propranololhydrochloride from different formulations wasstudied using locally fabricated Franz diffusioncell with an effective permeation area andreceptor cell volume of 1.0 cm2 and 10 mL,respectively. The temperature was maintainedat 32� 18C. The receptor compartment contained10 mL PBS (pH 6.5) and was constantly stirred bymagnetic stirrer (Expo India Ltd., Mumbai, India)at 100 rpm. Dermatomed (500 mm thickness)human cadaver skin from abdominal area wasobtained from District Hospital, Sagar, India, andstored at �208C. The skin was carefully checkedthrough a magnifying glass to ensure thatsamples were free from any surface irregularitysuch as tiny holes or crevices in the portion thatwas used for transdermal permeation studies.After assurance, the skin was mounted on areceptor compartment with the stratum corneumside facing upward into the donor compartment.Two hundred microliters of different composi-tions, (a) elastic liposomal formulation, (b) rigidliposomal formulation, and (c) plain drug solutionwas applied to the epidermal surface of the ratskin. Samples were withdrawn through thesampling port of the diffusion cell at predeter-mined time intervals over 24 h and analyzed. Thereceptor phase was immediately replenished withequal volume of fresh diffusion buffer. Triplicate

experiments were conducted for each study.In vitro drug release study from all formulationswas repeated with dialysis membrane using thesame method as described above.

The amount of propranolol hydrochlorideretained in the skin was determined at the end ofin vitro permeation experiment (24 h). The skinwaswashed 10 times with a cotton cloth immersedin methanol. A sample of skin was weighed, cutwith scissors, positioned in a glass homogenizercontaining 1 mL of methanol, and homogenizedfor 5 min with an electric stirrer. The resultingsolution was centrifuged for 10 min at 7000 rpm.The supernatant was analyzed for drug by HPLC.

Calculation of Permeation Parameters

The cumulative amount of drug permeated perunit area was plotted as a function of time, thesteady-state permeation rate (Jss) and lag time(LT, h) were calculated from the slope andX-intercept of the linear portion, respectively.

The enhancement ratio (ER) was calculatedfrom following equation

ER ¼ Transdermal flux fromvesicular formulation

Transdermal flux fromplain drug

Plasma Concentration Studies

Albino rats (Sprague Dawley strain), 5–6 weeksold, weighing 80–100 g were divided into threegroups. First group served as control, received thetopical application of plain drug solution; thesecond and third groups received optimizedelastic liposomal formulation (EL-Sp803) andrigid liposomal formulation, respectively. Twohundred microliters of all formulations wasapplied at the dorsal side of rats to the area of1.0 cm2. At specific time intervals rats weresacrificed and blood was collected from cardiacpuncture in heparinized centrifuge tubes andcentrifuged at 3000 rpm for 10 min (RemiCentrifuge, Mumbai, India) and drug concentra-tion was determined by HPLC assay.

Estimation of Drug Plasma Concentration

The quantitative determination of drug in plasmaand in skin permeation study was performed byHPLC using methanol, aqueous 60% acetonitrile(50:50) as mobile phase delivered at 1.8 mL/minby LC 10-AT vp pump (Shimadzu, Tokyo, Japan).

148 MISHRA ET AL.


Twenty microliters of injection volume waseluted in LUNA 54, C18, 4.6� 150 mm column(Phonomex, Torrence, CA) at room temperature.The column eluant was monitored at 320 nmusing SPD-M10A vp diode array UV detector(Shimadzu).15 A calibration curve with a concen-tration range from 10.0 to 0.05 mg/mL was used tomeasure the propranolol hydrochloride concen-tration of the samples and to validate theanalytical technique.

Statistical Analysis

Data are expressed as the mean� standarddeviation (SD) of themean and statistical analysiswas carried out employing the Student’s t-testusing the software PRISM (Graph Pad). A value ofp< 0.005 was considered statistically significant.

RESULTS AND DISCUSSION

Propranolol hydrochloride-loaded elastic lipo-somes were prepared using rotary evaporationsonication method as reported by Cevc et al.12

Formulations prepared using different types andconcentrations of surfactant are listed in Table 1.The basic composition of the elastic liposomes

comprises phospholipid (stabilizing agent) andsurfactant (destabilizing agent) in optimum ratioto impart elasticity to vesicle membrane of elasticliposomes. In the present study we used threesurfactants: Span 80, Span 60, and Span 40.

The elastic liposomes were initially character-ized by electron microscopy where samples werenegatively stained using PTA and the elasticliposomes appeared as spherical lamellar vesicles(figure not shown).

For optimization of drug amount the elasticliposomes were prepared using varying concen-trations of drug, followed by morphological char-acterization and determination of entrapmentefficiency. The maximum concentration of propra-nolol hydrochloride that could be incorporatedinto the vesicle formulations was found to be6.0 mg/mL with percentage entrapment efficiencyof 51.2� 4.8; on further increasing the amount ofdrug, drug crystalswere precipitated probably dueto the saturation of the vesicular formulation. Itshould be stressed that these saturation concen-trations are the total concentrations of propranololhydrochloride that could be incorporated in thevesicular system and not the entrapment value16

(Tab. 2).The mean diameter of all elastic liposomal

formulations ranged between 70 and 136 nm.

Table 1. Composition and Characterization of Elastic Liposomal Formulation

FormulationCode PC:Sa (%w/w)

Particle Size(nm)

EntrapmentEfficiency (%)

Turbidity(N.T.U.)

PolydispersityIndex Elasticity

El-Sp401 95:5 110� 10 47.6� 4.2 16.2� 1.2 0.042 21.6� 2.0El-Sp402 90:10 124� 11 49.2� 4.5 18.4� 1.5 0.054 29.6� 3.0El-Sp403 85:15 136� 14 52.8� 5.0 22.6� 2.0 0.059 40.1� 3.8El-Sp404 80:20 88� 10 40.1� 4.1 15.0� 1.8 0.082 13.5� 1.2El-Sp405 75:25 75� 8 35.3� 3.8 13.8� 1.4 0.097 9.9� 1.0El-Sp601 95:5 106� 10 51.6� 4.9 19.6� 2.1 0.037 24.5� 2.5El-Sp602 90:10 117� 11 53.7� 5.1 20.0� 2.1 0.045 30.7� 3.2El-Sp603 85:15 132� 13 56.4� 5.5 23.8� 2.4 0.057 39.1� 4.0El-Sp604 80:20 86� 9 42.4� 4.4 16.2� 1.8 0.072 14.1� 1.5El-Sp605 75:25 73� 8 38.2� 4.0 14.4� 1.5 0.084 10.1� 1.2El-Sp801 95:5 102� 10 54.1� 5.2 22.4� 2.0 0.036 25.0� 2.7El-Sp802 90:10 114� 11 58.6� 6.0 23.2� 2.2 0.041 31.1� 3.2El-Sp803 85:15 128� 13 60.2� 6.0b 25.0� 2.6 0.047 51.2� 4.8El-Sp804 80:20 82� 8 46.2� 4.5 18.6� 2.0 0.073 15.5� 1.7El-Sp805 75:25 70� 8 42.3� 4.4 16.7� 1.7 0.081 10.3� 1.2Liposome 104� 9 50.2� 5.0 24.5� 2.5 0.038 6.4� 0.5

Values represent Mean�SD (n¼3).El-Sp40, El-Sp60 and El-Sp80: elastic liposomal formulations containing Span 40, Span 60, and Span 80, respectively as

formulation variable.aPhosphatidyl choline: Surfactant.bDenotes maximum entrapment efficiency.



The particle size distribution with a very lowpolydispersity index of less than 0.1 indicates anarrow size distribution of the elastic liposomalsuspension and consequently a homogeneous dis-tribution (Tab. 1). Though with increasing surfac-tant concentration polydispersity index increasedsuggesting formation of other structures.

Turbidity of different formulations was deter-mined employing nephalometer. Formulationscontaining Span-80 exhibited higher values ofturbidity, indicating good population of vesicles,in general. Lower values of the turbidity areindicative of the fact that the vesicles might haveruptured or were converted into other structures(Tab. 1).

Entrapment efficiency is the percentage frac-tion of the total drug incorporated into the elasticliposomes. The maximum entrapment efficiencyobtained was 60.2� 6.0% for elastic liposomalformulation EL-Sp803. It was observed that withincreased surfactant concentration in the lipidcomponents of the vesicles, the entrapment effi-ciency of the propranolol hydrochloride decreased(Tab. 1). This is due to the possible coexistence ofmixed micelles and vesicles at higher concentra-tion of surfactant, with the consequence of lowerdrug entrapment in mixed micelles.

The formation of micellar structure at higherconcentration of surfactant is an established fact.The studies by Lasch et al.17 and Lopez et al.18

prove this fact. They studied the direct formationof mixed micelles in the solubilization of phospho-lipid liposomes by nonionic surfactants like TritonX-100, octyl glycoside by a variety of techniqueslike turbidity measurement, DLS, freeze fractureelectron microscopy, carboxyfluorescein fluores-cence dequenching techniques, and reported thattransformation of liposome to mixed micelles wasconcentration-dependent process. Vesicle solubili-zation was produced by the direct formation ofmixed micelles without the formation of complexaggregates. Thus, vesicle to micelles transforma-

tion was mainly governed by the progressiveformation of mixed micelles within the bilayer.A subsequent separation of mixed micelles fromliposome surface (using vesicle perforation by theformation of surfactant-stabilized holes on thevesicle surface) led to complete solubilization ofliposome. To support the above fact we performedturbidity measurement studies. Elastic liposomalformulations were prepared at five different con-centration levels (5, 10, 15, 20, and 25%w/w of PC)and turbidity was measured in a nephalometer(Superfit, India) taking PBS pH 6.5 as blank. Theresults of the turbidity measurement studiessupport the fact of micelles formation at higherconcentration of surfactant as turbidity increaseswith increasing the surfactant concentrationwhereas at low (15% w/w) concentration of surfac-tant partition coefficient favors the lipid phase andcauses expansion of lipid bilayers resulting inincreased turbidity of vesicle dispersion (Tab. 1).At the same time the surfactant also causesfluidization of the bilayer that is responsible forincrease in elasticity of vesicle membrane. Athigher surfactant concentration the turbiditycurve shows peak and then rapidly decreasesbecause after sublytic concentration of detergent(optimum concentration), conversion of lipid vesi-cles into mixed micelles begins. Mixed micelleshave a diameter below 10 nm and show negligibleturbidity. These mixed micelles are reported to beless deformable in nature and also have less skinpermeationability across the skin in comparison toelastic liposomes.12,17–19

The crucial feature of elastic liposomal formula-tions in comparison with the standard liposomesandother types of thedrug-laden lipid suspensionsis the flexibility of the vesicle membrane andstress-dependent adaptability. Table 1 shows theresults of elasticity measurement studies. Theresults indicate that elasticity of vesicles dependson both surfactant concentration and type. Withincrease in the surfactant concentration from 5%to 15% w/w, the elasticity value increases from25.0� 2.7 to 51.2� 4.8 for formulation EL-SP801and EL-SP803, respectively, but with furtherincrease in surfactant concentration elasticityvalue of vesiclemembranesdecreases significantly(from 51.2� 4.8 to 10.3� 1.2 for formulation EL-SP803 and EL-SP805, respectively) (p< 0.005).This type of behavior may be attributed to theformation of mixed micelles at higher concentra-tion of surfactant.

CLSM studies were performed to assess theintensity and extent of penetration of RR-loaded

Table 2. Optimization of Propranolol HydrochlorideConcentration in Elastic Liposomal Formulation

FormulationCode

Drug(% w/v)

MicroscopicObservation

(Drug Crystal)EntrapmentEfficiency (%)

El-Sp803 0.2 Not appeared 44.8� 3.5El-Sp803 0.4 Not appeared 60.2� 6.0El-Sp803 0.6 Not appeared 51.2� 4.8El-Sp803 0.8 Appeared 42.9� 3.8El-Sp803 1.0 Appeared 41.1� 2.8

150 MISHRA ET AL.


elastic liposomes and conventional liposomes. Theuse of RR-loaded elastic liposome resulted in anincrease in both, the depth of penetration (170 mm)and fluorescence intensity (Max FI¼ 160 AU), ascompared to rigid liposomes that remained con-fined only to few micrometers (50 mm) with a lowmaximumfluorescence intensity (MaxFI) of 40AU(Fig. 1). The fluorescence intensity versus skinpenetration graph clearly delineates the transder-mal potential of elastic liposomes. It was wellobserved that classic liposomes did not facilitate

probe penetration into the skin rather remainedconfined to upper layers of the skin.

In vitro permeation studies give us valuableinformation about the product behavior in vivo.The drug permeated dictates the amount of drugavailable for absorption. PBS pH 6.5 (10 mL) wasused as a receptor fluid for the in vitro drugpermeation studies based on solubility considera-tion of propranolol hydrochloride. In vitro Franzdiffusion cell measurements were used to investi-gate the transport enhancement potential ofelastic and rigid vesicles. Thismethod is commonlyused in skin research and normally gives a goodcorrelation with the in vivo data. For the differentelastic liposomal formulations drug release pro-files were studied in triplicate and standarddeviation, % cumulative drug release, transder-mal flux, enhancement ratio and lag time werecalculated from the data (Tab. 3, Fig. 2).

The diffusion of entrapped molecule fromvesicular system is governed by the transfer ofthe molecules from the disperse system to theexternal aqueous phase and diffusion of themolecule through the dialysis membrane fromthe external phase to receptor fluid. The diffusionof thedrug from thebilayers of the vesicles appearsto be the main rate-limiting step for the releaseof propranolol hydrochloride and the differentrelease rates from solution and elastic liposomescan thus be attributed to the entrapment of

Figure 1. Fluorescent intensity (AU) versus skindepth profile (mm) study revealing comparative skinpermeation profile of elastic liposomes (El-Sp 803), rigidliposomes, and blank. AU, arbitrary unit.

Table 3. Skin Permeation Parameters of Propranolol Hydrochloride FormulationsAcross Human Cadaver Skin (After 24 h)

FormulationCode

TransdermalFlux (mg/cm2/h)

Lag Time(h)

% SkinDeposition

EnhancementRatio (ER)

El-Sp401 5.89� 0.6 2.1 4.91� 2.1 3.2El-Sp402 9.33� 1.1 1.7 6.44� 0.9 5.1El-Sp403 11.12� 1.2 1.3 8.42� 1.4 6.1El-Sp404 5.19� 0.6 2.0 4.91� 1.7 2.8El-Sp405 4.60� 0.5 2.3 3.98� 0.6 2.5El-Sp601 6.56� 0.7 1.8 5.47� 1.3 3.6El-Sp602 11.31� 1.2 1.6 7.72� 0.8 6.2El-Sp603 12.34� 1.5 1.1 9.11� 2.1 6.7El-Sp604 5.97� 0.7 1.9 5.21� 1.4 3.2El-Sp605 4.96� 0.6 2.1 4.12� 2.4 2.7El-Sp801 8.24� 0.9 1.5 7.12� 2.1 4.5El-Sp802 11.71� 1.2 1.1 8.32� 1.4 6.4El-Sp803 16.19� 1.5 0.7 11.41� 5.2 8.8El-Sp804 6.65� 0.7 1.4 6.21� 1.6 3.6El-Sp805 5.24� 0.7 1.9 5.13� 2.8 2.8Liposome 3.24� 0.6 2.4 6.56� 1.4 1.4Plain drug 1.82� 0.2 2.6 3.84� 1.9 —

Values represent mean�SD (n¼ 3).



propranolol hydrochloride in elastic liposome.Significant prolongation of propranolol hydro-chloride release across the dialysis membranewas achieved with the elastic liposomal formula-tion in comparison with the plain drug solution.%Cumulative amount of propranolol hydrochlor-ide released from the elastic liposomal formulationwas higher than conventional liposomal formula-tion but significantly lower than the plain drugsolution (Fig. 2). A higher release in case of elasticliposomes in comparison to conventional liposomalsystem could be attributed to the high fluidityprovided by the edge activator incorporated in theelastic liposomes.

The results of skin permeation study throughhuman cadaver skin from elastic liposomal for-mulations, liposomal formulation, and plain drugat the same drug concentration has been depictedin Table 3 and Figure 3. The value of steady-statetransdermal flux for different elastic liposomalformulations were observed between 4.60�0.5 and 16.19� 1.5 mg/cm2/h. The maximumflux obtained from El-Sp803 was substantially8.8 times higher than that of plain drug (1.82�0.2 mg/cm2/h) and nearly five times higher thanrigid liposomal formulation (3.24� 0.6 mg/cm2/h).Also, aminimumlag time (0.7h)was obtainedwithEl-Sp803 formulation. The enhancement in trans-dermal flux of propranolol hydrochloride by elasticliposomal formulation is comparable to thosereported in previous finding.3,4 Further, formula-tion El-Sp803 showed the highest percent skindeposition of 11.41� 5.2 as compared to otherformulations. Better transdermal flux and lower

lag phase with our formulation was perhaps dueto combination of one or more of followingmechanisms: (1) increase in thermodynamic activ-ity, (2) increased skin vehicle partitioning of drug,(3) altering the barrier properties by interactingwith skin component, and (4) elasticity of vesiclemembrane.

With respect to drug delivery from the vesicles(Tab. 3), transdermal flux first increased withincreasing surfactant concentration and thendecreased, a common phenomenon seen with allthree surfactants. As concentration of surfactantincreased, transdermal flux of propranolol hydro-chloride increased up to 15% w/w of phospholipids(from 8.24� 0.9 to 16.19� 1.5 mg/cm2/h) and withfurther increase in the surfactant concentration,transdermal flux decreased (from 16.19� 1.5 to5.24� 0.4 mg/cm2/h). These results suggested thattoo low or too high concentration of edge activators(surfactants) is not beneficial in vesicular deliverythrough skin and also indicated that the possiblepenetration-enhancing effect of surfactants is notmainly responsible for improved propranololhydrochloride skin delivery from deformable vesi-cles. These findings are in agreement with thepublished data.7,9–11 A possible explanation forlesser drug delivery at high surfactant concentra-tion may be that surfactant at high concentrationdecreased theentrapment efficiencyanddisruptedthe lipid membrane so that it becomes more leakyto the entrapped drug. This will, in turn, reducethe delivery especially if we consider the possiblecarrier function of these elastic liposomes.Anotherpossible explanation for the obtained lower trans-

Figure 2. % Cumulative propranolol hydrochloriderelease after 24 h via dialysis membrane from elasticliposomal formulation, liposomal formulation and plaindrug solution. Values represent mean�SD (n¼ 3).

Figure 3. Cumulative propranolol hydrochloride per-meated after 24 h via human cadaver skin from elasticliposomal formulation, liposomal formulation, and plaindrug solution. Values represent mean�SD (n¼ 3).

152 MISHRA ET AL.


dermal flux at higher surfactant concentration intransfersomal formulation is that at higher con-centration of surfactant, vesicles coexisted withmixedmicelles (surfactant concentration 20%w/wof PC) whereas only mixed micelles existed atconcentration greater than 20% w/w of PC. Thesemixed micelles are reported to be less effectivein transdermal drug delivery as compared withelastic liposomal system because micelles aremuch less sensitive to water activity gradientthan elastic liposomes.Discussing surfactants, thepossible reason for better performance of Span-80in the case of entrapment efficiency, vesicularelasticity, transdermal flux and formation of fairernumber of vesicles, is its lower HLB value (4.3),which provides better partitioning with the lipidlayers.

The use of elastic liposomes for noninvasivedrug delivery is widely reported. Better skinpenetration ability of these elastic liposomes iswell supported by many authors, for example,recently Honeywell et al.16 reported the enhanceddelivery of pergolide using elastic liposomes. ElMaghraby et al.20,21 reported better skin permea-tion ability of elastic liposomes using estradiol and5-Fluorouracil as a model drug; Trotta et al.22

reported the elastic liposomes for skin delivery ofdipotassium glycyrrhizinate; Guo et al.23 reportedthe elastic liposomes for systemic delivery ofcyclosporin A; Hofer et al.24 reported the elasticliposomes for systemic transdermal delivery ofimmunomodulatory proteins, Interleukin-2 andInterferon-a; VandenBerghet al.15 in their studiesalso proved the better skin permeation ability ofthese elastic liposomes. We also have evaluatedthe better skin permeation ability of elasticliposomes using dexamethasone, diclofenac, andnorgestrel as model drugs.8–10 All the above-mentioned studies have established the betterskin permeation ability of elastic liposomes incomparison to plain liposomes.

The in vivo study of different formulations wascarried out bymeans of plasma drug concentrationmeasurement. Three formulations: control, lipo-somal, and elastic liposomal were applied on backof the rat skin. The blood concentration wasdetermined for 24 h. The elastic liposomal for-mulation, the rigid liposomal formulation, andplain drug solution showed the blood level con-centration 92� 8.9 ng/mL, 35� 7.1 ng/mL, and25� 5.0 ng/mL, respectively (Fig. 4). Elasticliposomal formulation showed nearly three timesbetter blood level concentration representinggreater permeation (Tab. 4).

Plasma levels of propranolol hydrochlorideafter application of control were low (Cmax¼25� 5.0 ng/mL) while those after applicationof the elastic liposomal formulation graduallyincreased and reached a peak level of 92� 8.9 ng/mL at 8 h post administration and maintained forlonger times. Theareaunder the curve (AUC0–24 h)was found to be 1659� 95 ng �h/mL and wasmuchlarger than 315� 28 ng �h/mL being AUC0–24h ofcontrol. The liposomal formulation also showedhigher drug level (579� 59 ng �h/mL) in compar-ison to control, yet lower as compared to elasticliposomal formulation.

The results of the present investigation showedthat the problems associatedwith the transdermaldelivery of propranolol hydrochloride could beovercome by incorporating it into the new ultra-flexible drug carrier, elastic liposomes. Elasticliposomes are specially optimized vesicles, whichcan respond to an external stress by rapid andenergetically inexpensive shape transformations.Elastic liposomes differ from conventional gelstate niosomes and liposomes by their character-istic fluid membrane with high elasticity. Theelasticity of these vesicles is attributed to thesimultaneous presence of different stabilizing

Table 4. Pharmacokinetic Parameters of PropanololHydrochloride

Formulations Cmax (ng/mL) AUC (ng �h/mL)

Plain drugsolution

25� 5.0 315� 28

Liposomes 35� 7.1 579� 59Elastic

liposomes92� 8.9 1659� 95

Values represent mean�SD (n¼6).

Figure 4. Plasma concentration of propranololhydrochloride after administration in optimized elasticliposomal formulation (EL-Sp803), rigid liposomes andplaindrug solution.Values representmean�SD (n¼ 6).



(phospholipids) and destabilizing (surfactant)molecules and their tendency to redistribute inbilayers. Such highly deformable vesicles can thusbe used to bring drugs across the biologicalpermeability barriers such as skin. The lowerpenetration ability that is associated with the useof vesicular carriers such as liposomes andniosomes can be overcome by entrapment of thedrugs in the elastic liposomes.

ACKNOWLEDGMENTS

One of the authors (Dinesh Mishra) is grateful tothe Electron microscopy section, All India Insti-tute of Medical Sciences, New Delhi for Transmis-sion Electron Microscopy, Institute Of NuclearMedicine And Allied Sciences (INMAS) NewDelhifor Confocal microscopy, the University GrantsCommission New Delhi India for awarding thefellowship to carry out this work and M/s SunPharma, Vadodara, India for providing the giftsample of propranolol hydrochloride.

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