Formulation and evaluation of topical niosomal gel of Erythromycin

Abstract

Erythromycin is macrolide antibiotic used commonly for the treatment of acne either single or in combination. But use of this drug some time shows unwanted side effects like skin redness, irritation, itching and edema. Niosomes, a vesicular formulation, has been explored extensively for topical application to enhance skin penetration as well as to improve skin retention of drugs. In the present investigation, Erythromycin was entrapped into niosomes by thin film hydration technique and various process parameters were optimized by partial factorial design. The optimized niosomal formulation was incorporated into carbopol gel and extensively characterized for Percentage Drug Entrapment (PDE) and in-vitro release performance. The stability of above formulation was studied at different temperatures. The present study demonstrates prolongation of drug release, an increase in amount of drug retention into skin and improved permeation across the skin after encapsulation of Erythromycin into niosomal topical gel.

Full text

ResearchArticle
FORMULATIONANDEVALUATIONOFTOPICALNIOSOMALGELOFERYTHROMYCIN

VYASJIGARa*,VYASPUJAb,SAWANTKRUTIKAa
PharmacyDepartment,TheM.S.UniversityofBaroda,Gujarat,India,SigmaInstituteofPharmacy,Baroda,Gujarat,India,Pharmaceutics
Department,SigmaInstituteofPharmacy,Baroda,Gujarat,IndiaEmail:jigs4u_80@yahoo.co.in
Received:03Oct2010,RevisedandAccepted:04Nov2010
ABSTRACT
Erythromycinismacrolideantibioticusedcommonlyforthetreatmentofacneeithersingleorincombination.Butuseofthisdrugsometimeshows
unwantedside effectslike skinredness,irritation,itchingandedema. Niosomes,a vesicularformulation,has beenexploredextensivelyfortopical
applicationtoenhanceskinpenetrationaswellastoimproveskinretentionofdrugs.Inthepresentinvestigation,Erythromycinwasentrappedinto
niosomes by thin film hydration technique and various process parameters were optimized by partial factorial design. The optimized niosomal
formulation was incorporated into carbopol gel and extensively characterized for Percentage Drug Entrapment (PDE) and in‐vitro release
performance.Thestabilityofaboveformulationwasstudiedat different temperatures. The present study demonstrates prolongati on ofdrug 
release,anincreaseinamountofdrugretentionintoskinandimproved permeation across the skin after encapsulation of Eryt hromycin int o
niosomaltopicalgel.
Keywords:Niosomes,Erythromycin,PercentageDrugEntrapment,NiosomalGelandSkinRetention.

INTRODUCTION
Drug delivery systems using vesicular carriers such as liposomes1
and niosomes2 have distinct advantages over conv entional dosage
forms because the vesicle can act as drug containing reservoirs.
Modificationofvesiclecompositionor surfacecan adjusttheaffinity
for the target site and / or the drug release rate, and the slowing
drug release rate may reduce the toxicity of the drug. Hence th ese
carriers play an increasingly important role in drug delivery.
Niosomes and liposome are unilamellar or multilamellar vesicles
whereinanaqueousphaseisencapsulatedinhighlyorderedbilayer
madeupofnonionicsurfactant(niosomes)orlipid(liposomes)with
or without other components like, cholesterol (chol) and Dicety l
phosphate3.
Bothniosomesand liposomesshow desiredinteractionwithhuman 
skinwhenappliedthroughtopicalpreparationbyimproving
especially the horny layer characteristics, which in turn due to
reductionintransdermalwaterlossandincreasein smoothnessvia
replenishingskin lipids4.Althoughniosomes andliposomespossess
moreor less same advantage, niosomes were preferred due tohigh
cost and lower stability of lipids which have been replaced by non
ionic surfactants. Niosomes loaded with drugs for dermal
application show interactions with epidermal tissue without
exerting immediate or strong systemic action4. Erythromycin is
macrolide antibiotic which may be either bacteriostatic or
bactericidal dependingon the sensitivity ofthe microorganism and
theconcentrationofthedrug.
Topicalapplicationof Erythromycinoften producesadverseeffects
likeskinredness,irritation,itching,etc.whichleadsto
inconvenienceand ignoranceof therapyandresults inno benefit or
emergenceofresistantstrainsofbacteria,sometimes.Presentstudy
isbasedonthehypothesis that incorporation of Erythromycin into
niosomeswillimprovetheamountandtimeofdrugretentionwithin
the skin;which in turn willincrease the therapeutic efficacy of the
drugandreducethetoxicity.
MATERIALSANDMETHODS
Materials
Erythromycin was obtained as gift sample from Recvina
PharmaceuticalsLtd.(Vadodara,India),Span20,Span60and Span80
were purchased from S.D. Fine Chemicals Ltd. (Mumbai, India),
Cholesterol, chloroform and methanol were purchased from Loba
Chem (Mumbai, India). All the reagents were used without furthe r
purifications. Phosphate Buffer Saline pH 7.4 (PBS pH 7.4) and
PhosphateBufferSalinepH6.8(PBSpH6.8)werepreparedas
described in the Indian Pharmacopoeia (1996) and necessary
chemicals were obtained from the Loba Chem (Mumbai, I ndia). All
the chemicals used were of Analytical Reagent (AR) grade unless 
otherwisespecified.Synigel® (5 % Erythromycin) was used as
marketedformulation.
All necessary permissions from ethical committee were procured
beforecommencementofthestudy.
Preparationandcharacterizationofniosomes
Niosomes were prepared by thin film hydration technique5.
Thorough review of the literature gives numerous data on variou s
parameters needed to be optimized, like type of non‐ionic
surfactant, drug:cholesterol:surfactant ratio, solvent system,
hydrationvolume,hydrationtemperature,hydrationtime,annealing
time,filmformationtime,etc.Themostcriticalparametersamong
these, type of surfactant was optimized separately using full 23
factorialdesignasshowninthetable1.
The type of surfactant was optimized, keeping
drug:cholesterol:surfactant molar ration at 1:1:1, and all other
parameters like, Solvent system (chloroform:methanol, 1:1),
temperatureof waterbath(60 °C),vacuumforsolventevaporation
(20mmHg),speedofrotation(100rpm),volumeofhydration(5ml),
time of hydration (1hr.) and annealing time (1hr.) constant. The
values of all these parameters were determined from thorough
review of literature. The prepared niosomes loaded with
Erythromycinwereanalyzedforpercentage drugentrapment(PDE)
by colorimetric method using UV‐Visible spectrometer after
separationoffreedrug;aswellastheparticlesizewasanalyzedby
Malvonparticlesizerandd
90wastakenasdataandtabulatedin
differentstudies.
Preparationofcarbopolgel
Sufficient quantity of Carbopol 934 (1% w/w) was weighed and
sprinkled onto warm distilled  water with continuous stirring. The
dispersion was allowed tohydrate for 1‐2 hours. Other ingredients
like Propylene Glycol (10 % w/w) and Glycerol (30 % w/w) were
added subsequently to the aqueous dispersion with continuous
stirring.Aplaindruggel(BatchC1)waspreparedbyadding
requiredquantityofdrug(2%w/w)anddispersedproperly.The
dispersionwasneutralizedtopH6using1%w/vofSodium
Hydroxidesolutionandthefinalweightwasadjustedwithdistilled
water.Thegelwassonicatedfor30minutesonbathsonicatorand
keptovernight to remove air bubbles. Niosomal gel (Batch C2) was
prepared by following the same procedure and adding niosomal
cakecontaininganequivalentamountofdruginsteadofplaindrug.

International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 3, Issue 1, 2011

Vyasetal.
IntJPharmPharmSci,Vol3,Issue1,123126
124
Table1:Tableshowsoptimizationexperimentsforselectionofsurfactant
Formulation

Code

Span20

Span60

Span80

Percentagedrug

entrapments

+

%

SD

A
verage


particlesize

+

%SD

F1 1:0
(
‐1
)
1:0
(
‐1
)
1:0
(
‐1
)
Noniosomesformedwithoutsurfactant
F2 1:0(‐1) 1:0(‐1) 1:1(+1) 82.26%(
±
1.89) 4.67(±0.088)
F3 1:0(‐1) 1:1(+1) 1:0(‐1) 52.55%
(
±
1.65
)
6.87
(
±0.317
F4 1:0(‐1) 1:1(+1) 1:1(+1) 70.62%
(
±
2.25
)
3.39
(
±0.078
)
F5 1:1(+1) 1:0(‐1) 1:0(‐1) 29.23%
(
±
0.96
)
6.87
(
±0.317
)
F6 1:1(+1) 1:0(‐1) 1:1(+1) 49.51%(+ 1.35) 6.13
(
±0.199
)
F7 1:1(+1) 1:1(+1) 1:0(‐1) 39.19 %
(
±
1.28
)
4.13
(
±0.170
)
F8 1:1(+1) 1:1(+1) 1:1(+1) 47.95%
(
±
1.96
)
6.87
(
±0.317
)
n=3
The two levels of study: ‐1 = 1:0, +1 = 1:1; are in the form of
drug:surfactant molar ratio. Hence, the final formulation would
contain drug:cholesterol:surfactant at 1:1:1 of either a surfac tant
alone or in combinations. In all further optimization study, all the
parameters other than considered for optimization were kept
constant as per the values taken from literature or as optimized
previously.
BatchF2issuccessful batchandhenceis carriedforwardforfurther
optimization of combination of drug:cholesterol:surfactant molar
ratio.Thedatawasrecordedintable2.
Batch F10 was found to be the best combination of
drug:cholesterol:surfactant (1:1:2) and was used for all further
study.Volumeofhydrationandtimeof hydrationwereoptimizedby
usinga32factorialdesignmodelastabulatedbelowintable3.
Other process parameters like, speed of rotation, intensity of
vacuum, temperature and annealing time were optimized by using
half24factorialdesignasshownintable4below.
Finallythesolventsystemwasalsooptimizedforproportionofboth
thesolventsaswellastotalvolumeofsolventsand recordedintable
5.
Final optimized batch was then preparedrepeatedly to check the
reproducibilityand to get final formulation insufficientamount for
furtherstudies.
Table2:Tablecontainsdataofoptimizationofsurfactant:ch olesterolratio
Formulation


Code

Drug

Cholesterol

Span80

Percentagedrugentrapments

+

%SD

A
verageparticlesize

+

%SD

F9 1 1 1 82.26%
(

±
1.89
)
4.67
(
±0.08
)

F10 1 1 2 86.35%(
±
2.77) 4.51(±0.31)
F11 1 2 1 56.55%
(
±
1.98
)
5.23
(
±0.22
)

F12 1 2 2 72.02%
(
±
3.25
)
6.68
(
±0.08
)

n=3;BatchF2wastakenandexperimentswereconductedbyvaryingtheproportionofcholesterolandsurfactant.
Table3:Tableexplainsoptimizationofvolumeofhydrationandtimeofhydration
Formulation

Code

Volumeo
f

h
y
dration

Timeof

h
y
dration

Percentagedrugentrapments

+

%SD

A
verageparticlesize

+

%

SD

F13 3(‐1) 0.5(‐1) 70.05%
(
±
0.80
)
7.77
(
±0.31
)
F14 3(‐1) 1.0(0) 75.26%
(

±
2.39
)
4.67
(
±0.09
)
F15 3(‐1) 2.0(+1) 79.55%(
±
2.10) 6.87(±0.32)
F16 5(0) 0.5(‐1) 70.62%
(
±
2.25
)
3.39
(
±0.08
)
F17 5(0) 1.0(0) 82.26%
(

±
1.89
)
4.67
(
±0.08
)
F18 5(0) 2.0(+1) 88.51%(+ 1.30) 4.11(±0.19)
F19 7(+1) 0.5(‐1) 69.19%
(
±
1.88
)
4.13
(
±0.17
)
F20 7(+1) 1.0(0) 77.95%
(
±
1.96
)
6.87
(
±0.30
)
F21 7(+1) 2.0(+1) 83.22%(+ 2.23) 5.78
(
±0.13
)
n=3;BatchF10wastakenwithallotherparameterconstantexceptparametersshownabove.
Table4:Tablereflectsdataofoptimizationofspeedofrotation,intensityofvacuum,temperatureandannealingtime
Formulation

code
Speedof

rotation
(
r
p
m
)

Intensityof

vacuum
(
mmH
g)

Temperature

(°C)
A
nnealingtime

(Hour)
Percentagedrug

entrapments+%SD
A
verageparticle

size+%SD
F22 100(‐1) 20(‐1) 60(‐1) 1(‐1) 82.26%
(

±
1.89
)
 4.67
(
±
0.08
)
F23 125(+1) 25(+1) 60(‐1) 1(‐1) 79.67%(+ 1.27) 6.67
(
±
0.22
)
F24 125(+1) 20(‐1) 70(+1) 1(‐1) 80.11%(+ 3.31) 7.81(
±
0.32)
F25 125(+1) 20(‐1) 60(‐1) 2(+1) 89.55%(+ 3.90) 2.43
(
±
0.03
)
F26 100(‐1) 25(+1) 70(+1) 1(‐1) 77.34%(+ 2.88) 5.66
(
±
0.11
)
F27 100(‐1) 25(+1) 60(‐1) 2(+1) 81.14%(+ 2.11) 5.22
(
±
0.14
)
F28 100(‐1) 20(‐1) 70(+1) 2(+1) 80.12%(+ 3.78) 4.06
(
±
0.16
)
F29 125(+1) 25(+1) 70(+1) 2(+1) 75.54%(+ 2.21) 4.43
(
±
0.13
)
n=3;BatchF18wastakenandoptimizedforabovementionedvari ables.
Table5:Tableshowsdataofoptimizationofsolventsystem
FormulationCode

Chloroform

Methanol

Volumeof

solvent


s
y
stem

Percentage

drug

entra
p
ments

+

SD

Av
erage

particlesize

+

SD

F30 1 1 10 82.26%
(

±
1.89
)
4.67
(
±0.09
)
F31 2 1 10 90.35%
(
±
2.77
)
4.51
(
±0.11
)
F32 1 2 10 56.55%
(
±
1.98
)
5.23
(
±0.21
)
F33 2 1 5 72.02%
(
±
3.25
)
6.68
(
±0.08
)
F34 2 1 15 80.35%
(
±
2.77
)
7.51
(
±0.33
)
n=3;BatchF25wastakenandstudiedforthebestsolventsystemtogetmaximumPDE.

Vyasetal.
IntJPharmPharmSci,Vol3,Issue1,123126
1
Drugleakagestudy
Sufficient quantity of niosomal suspension (after removal of free
drug) was sealed in 10 ml glass vial and the niosomal gel
formulation (Batch C2) was sealed in 10 gm collapsible aluminum
tube in triplicate, and stored at refrigerated temperature (2‐8˚ C)
and room temperature (25 +2˚C).Specimen(0.5gm)fromeach
samplewaswithdrawnatanintervalofoneweekandanalyzedfor
free drug content to determine the leakage rate. The results are
recorded in Table 6. The data were compared by applying ANOVA
(singlefactor)atp=0.05.

Table6:TablecontainsdataofdrugleakagestudyatRTandrefrigeratedtemperature
Timeinweeks
Percentagedrugretained(+S.D.)
NiosomalsuspensionNiosomalgelofCarbopol
4

˚C(NS)

RT(NS)

4

˚C(NG)

RT(NG)

1 98.90(+3.74) 89.11(+3.33) 99.89(+3.26) 94.20(+2.72)
2 97.30(+3.67) 78.83(+ 3.41) 99.22(+ 2.11) 87.09(+ 3.92)
3 95.89(+3.16) 68.22(+2.98) 98.73(+3.94) 81.82(+3.16)
4 93.55(+2.71) 61.19(+2.86) 98.32(+4.02) 76.90(+3.57)
5 89.48(+1.76) 54.40(+1.99) 98.02(+3.65) 72.10(+1.78)
6 86.88(+1.24) 46.21(+ 1.32) 97.77(+ 2.83) 68.89(+ 2.78)
7 82.77(+2.43) 39.11(+0.74) 97.56(+1.98) 65.11(+2.67)
8 78.92(+0.74) 33.38(+0.43) 97.38(+2.87) 61.08(+1.96)
9 75.45(+1.17) 28.39(+1.15) 97.22(+3.49) 58.12(+1.14)
10 73.29(+1.87) 23.45(+ 1.07) 97.07(+ 2.67) 56.23(+ 1.08)
11 70.67(+2.87) 20.04(+0.56) 96.97(+1.10) 53.55(+1.17)
12 65.89(+1.65) 17.12(+0.48) 96.85(+2.26) 51.07(+2.22)
n=3;RT=RoomTemperature(25+2˚C);NS=NiosomalSuspension;NG=NiosomalGel

Invitropermeationstudies
Preparationofmembraneforinvitrostudies
Human cadaver skin (HCS) was obta ined and stored at 0°C.Afull
thicknessHCSmembranewaspreparedbyshavingtheskin,
punchingoutatissueofapproximately2.5cm
2areawithsharp
blade, trimming away the excess fat and slicing to about 450μm
thickness. These slices were hydrated in pH 6.8 phosphate buffer
salineovernightpriortouse6.
TheverticaltypeofFranzdiffusioncellwasdesigned,fabrica tedand
validated7, 8 prior to diffusio n study. 50 mg of gel was applied on
2.00 cm2 area of epidermal surface of HCS tied tothe lower end of
donor compartment. The volume of the receptorcompartment was
kept20ml. The cell was assembled in such a way that,the dermal
surfacewas just flushedto the surfaceof permeationfluid (pH6.8
PBS)maintainedat 37+1˚Candstirred continuouslyona magnetic
stirrerat50rpm.Aliquotsof0.5mlwerewithdrawnandanalyzed
forthe drugcontent aftersuitabledilutions bycolorimetricm ethod.
The volume of fluid was replaced with the same volume of fresh
bufferaftereachsampling.Thecumulativepercentagedrugdiffused
acrossthe HCS was calculated ateachsamplingpointand recorded
inTable7.
Amountofdrugretainedintheskinwascalculatedbysubtracting
the amount of free drug content in the receptor compartment and
theamount ofdrug remainedonthe epidermalsurfaceof skinfr om
theinitial drugcontent oftheformulation applied,andresults were
recorded in Table 7. All the determinations were carried out in
triplicateandthedatawerecomparedbyANOVA(p=0.05).

Table7:Tableshowsdataofdiffusionstudyofdrugacrosshumancadaverskin(HCS)
Time

inhours
Percentagedrugrelease

(+

S.D.)

BatchC1

BatchC2

Market

preparation

0.5 ‐ ‐ ‐
1 07.97(+ 0.54) ‐ 9.98(+0.27)
2 17.34(+ 0.89) 09.24(+ 1.20) 18.84(+0.67)
3 24.45(+0.76) 15.11(+1.86) 26.33(+0.91)
5 36.63(+1.94) 21.76(+1.13) 39.08(+1.40)
8 48.43(+ 2.35) 28.83(+ 2.09) 51.23(+1.34)
12 59.42(+ 3.01) 32.31(+ 2.34) 63.67(+2.89)
 Percentagedrugretainedintohumancadaverskin(HCS)after12hours
12 21.45(+0.36) 41.53(+1.75) 24.88(+0.49)
n=3;Littleornoreleasewasobservedinfirsthourwhichlandeddifficultiesinthequantification

RESULTSANDDISCUSSION
Amongst many reported methods for the preparat ion of niosomes,
thin film hydration technique was selected as it is the most
documentedmethodwithgreaterentrapmentefficiencyandsmaller
particle size. An intense review of literature reveals that Tweens
showpoorentrapmentwithlipophilicoramphiphilicdrugswhereas
Spansgive higher entrapment withhighstability. This is dueto the
fact that hydrophilic surfactants (Tweens) owing to high aqueous
solubility do not form proper vesicular structure in aqueous
medium,whereasduetolipophilicinnature,Spansformvesicles
andentrapthelipophilicdrugoramphiphilicdrugs.
Table 1 reveals that Span 80 alone gave highest entrapment
(82.26%) which decreased when combined with either Span 20
(49.51%)orSpan60(70.62%).Dataoftable2suggeststhatthePDE
decreasedfrom 86.35%to56.55%as theproportionof Cholesterol
increased from 25% (1:1:2) to 50 %( 1:2:1). This indicates that the
characteristics of Cholesterol of decreasing leakage of bilayer 
structure and producing surface smoothness diminish at higher
proportions as it imparts crystalinity to the bilayer9, 10. Other
parameters were also optimized as recorded in table 3, table 4 &
table5togetthefinaloptimizedformulationwhichwasrecordedin
table8 below.Final optimizedbatch was thenprepared repeatedly
tocheckthereproducibilityandtogetfinalformulationinsufficient
amountforfurtherstudies.

Vyasetal.
IntJPharmPharmSci,Vol3,Issue1,123126
126

Table8:Tableshowsfinaloptimizedbatch
Sr.

No.
Parameters

Optimizedvalue

1 Nonionicsurfactant Span80
2 Drug:cholesterol:surfactant
molarratio
1:1:2
3 Solventsystem Chloroform:methanol,
2:1
4 Hydrationtemperature 60°C
5 Vacuum 20mmHg
6 Speedofrotation 125
7 Hydrationvolume 5
8 Hydrationtime 1hour
9 Annealingtime 2hour

AnalysisofdataofdrugleakagestudybyapplyingANOVAreveals
thatniosomaldruggelissignificantlymorestableascompared
niosomalsuspensionandalsoboththeformulationsaresignificantly
more stable at refrigerated temperature than room temperature.
Thereasonbehind higherleakageathighertemperaturemaybethe
higher fluidity of lipid bilayer at higher temperature11, 12. The
stabilityofniosomesimprovedafterincorporationintogelbasemay
beduetopreventionoffusionofniosomes.


Fig.1:FigureShowsdrugleakagestudyatroomtemperature
andrefrigeratedtemperature
NG at RfT=Niosomal Gel at Refrigerated Temperature; NS at
RfT=Niosomal Suspension at Refrigerated Temperature; NG at
RT=Niosomal Gel at Room Temperature; NS at RT=Niosomal
SuspensionatRoomTemperature.
The data of the in‐vitro drug release study suggests that all the
formulations followed Higuchi’s diffusion controlled model. When
thedatawascomparedbyANOVAtest(singlefactor,p=0.05), it
revealed a significant difference in drug release rate between
niosomalgelandplaindruggel.Thedataofdrugretentionintoskin
after24hourshaveshownmaximumdrugretention(41.53%)with
niosomalgel(BatchC2)ascomparedtoplaindruggel(21.45%)and
marketedgel(24.88%).
Prolongeddrugreleasewasobservedduringinvitrodiffusion study
across human cadaver skin from niosomal Erythromycin gel as
comparedtoplaindruggelandmarketpreparationwhichmaybe due
toslowerdiffusionofdrugintotheskinandcreationofreservoireffect
fordrugintheskin.Theothercomponentsofniosomesi.e.surfactant,
cholestreolalsodepositalongwithdrugintotheskinandthereby
increasingthedrugretentioncapacityintoskin.


Fig.2:Figureshowsdiffusionstudyofdrugacrosshuman
cadaverskin(HCS)

CONCLUSION
The finding of this investigation have conclusively demonstrated
thatencapsulationofErythromycinintoniosomalgelformulation
improves skin retention which may be reflected, based on prior
hypothesis,assignificantlyimprovedtherapeuticresponseand
considerably reduced adverse symptoms. However, the role of
niosomal Erythromycin gel of this study can only be settle da fter
clinicalevaluationoftheproductwithlargenumberofpatientwith
specialfocusontheadversesymptomsofthetherapy.
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