Newly Developed Topical Cefotaxime Sodium Hydrogels: Antibacterial Activity and In Vivo ...

16 Pages·2016·2.96 MB·English

Hindawi Publishing Corporation
BioMed Research International
Volume 2016, Article ID 6525163, 15 pages
http://dx.doi.org/10.1155/2016/6525163
Research Article
Newly Developed Topical Cefotaxime Sodium Hydrogels:
In Vivo
Antibacterial Activity and Evaluation
AzzaS.Zakaria,1,2SamarA.Afifi,1,3andKadriaA.Elkhodairy4
1DepartmentofPharmaceutics,CollegeofPharmacy,KingSaudUniversity,P.O.Box22452,Riyadh11495,SaudiArabia
2DepartmentofMicrobiology,FacultyofPharmacy,AlexandriaUniversity,P.O.Box21500,Alexandria21527,Egypt
3DepartmentofPharmaceutics,NationalOrganizationforDrugControlandResearch,P.O.Box35521,Giza12561,Egypt
4DepartmentofIndustrialPharmacy,FacultyofPharmacy,AlexandriaUniversity,P.O.Box21500,Alexandria21527,Egypt
CorrespondenceshouldbeaddressedtoKadriaA.Elkhodairy;[email protected]
Received18January2016;Revised6April2016;Accepted13April2016
AcademicEditor:KamlaPathak
Copyright©2016AzzaS.Zakariaetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Inanattempttoreachbettertreatmentofskininfections,gelformulationscontainingCefotaxime(CTX)wereprepared.Thegel
wasformulatedusingCarbopol934(C934),HydroxypropylMethylcellulose4000(HPMC4000),CarboxymethylcelluloseSodium
(NaCMC),Pectin(PEC),XanthanGum(XG),orGuarGum(GG).Thirteendifferentformulaswerepreparedandcharacterized
physicallyintermsofcolor,syneresis,spreadability,pH,drugcontent,andrheologicalproperties.Drug-excipientscompatibility
studieswereconfirmedbyFTIRandtheninvitrodrugreleasestudywasconducted.Invitroandinvivoantibacterialactivitiesof
CTXwerestudiedagainstwoundpathogenssuchas,Staphylococcusaureus(S.aureus),Escherichiacoli(E.coli),andPseudomonas
aeruginosa(P.aeruginosa),usingeitherpuredrugorFucidin(cid:2)creamascontrol.F13providesbetterspreadabilitycomparedto
F1(XG)orF11(HPMC).Moreover,thereleaseofthedrugfromhydrogelF13containingC934wasslowerandsustainedfor8h.
Stabilitystudyrevealedthat,uponstorage,therewerenosignificantchangesinpH,drugcontent,andviscosityofthegels.Also,F13
showedthelargerinhibitionzoneandhighestantibacterialactivityamongotherformulations.Histologicalanalysisdemonstrated
thataftersingletreatmentwithF13gelformulation,anoticeablereductioninmicrobialbioburdenoccurredincaseofbothGram
positiveandGramnegativebacterialisolates.
1.Introduction Controlled release of antibiotics at site of infection is a
newstrategybeingemployedtotreatchronicinfections[4,5].
Several antimicrobial agents are available in the market in Localized delivery systems, based on biodegradable poly-
different pharmaceutical dosage forms for the treatment of mers,arecapableofslowingandcontrollingdrugreleasefor
skin and soft tissue infections (SSTIs). These preparations acertainperiodoftime,withinitialbursteffecttocircumvent
aredesignedtobeadministeredeitherorallyorparenterally theinfection.
ortopically.Topicalpreparationshaveseveralbenefitscom- Hydrogels, swollen three-dimensional networks of
paredwithsystemictherapy[1,2].Initially,therequiredcon- hydrophilicpolymersheldtogetherbyassociationbondsor
centrationforantibioticactivityattheskintargetsitemaybe cohesive forces, are of special interest in controlled release
moreeasily achieved after topicaldosing. Moreover,topical applications, because of their soft tissue biocompatibility,
administrationresultsinmuchlowerundetectablesystemic the ease with which drugs are dispersed in matrix, and the
levels[3].Additionally,itcanavoidanunnecessaryexposure high degree of control achieved by selecting the physical
ofgutflorathatmayexertselectionforresistance.Therefore, and chemical properties of polymer network. Hydrogels
topical application of antimicrobial agents is considered an havebeeninvestigatedextensivelyforapplicationascarriers
importantalternativeusingthestratumcorneaasthetarget in diffusion-controlled release devices [6]. Hydrogels are a
organofvariousantibioticdrugtreatments[1]. commonformoftopicalapplication.Theyachievesustained
2 BioMedResearchInternational
release by diffusion from a reservoir through microporous clinicalisolateandonestandardPseudomonasaeruginosa(P.
membraneintotheskin[5]. aeruginosa) (ATCC 27853) and one clinical isolate. Finally,
Cefotaxime (CTX) sodium as an antimicrobial agent onestandardStaphylococcusaureus(S.aureus)(ATCC29213)
is a semisynthetic broad spectrum third-generation cepha- and one methicillin resistant S. aureus clinical isolate were
losporin against Gram positive and Gram negative bacteria studied in this research. The strains were collected from
and some pseudomonal species as well as some anaero- wound specimens from outpatient departments of King
bic bacteria [7]. It has activity in presence of some beta- KhalidHospital,Riyadh,SaudiArabia.
lactamases, both penicillinases and cephalosporinases of
Gram negative and Gram positive bacteria. Its spectrum of (2)MediaandCultureConditions.Allclinicalsampleswere
activityincludesmoststrainsofbacterialpathogensresponsi- first inoculated onto Sheep Blood Agar (SPML Co. Ltd,,
∘
bleforsepticaemia,respiratorytractinfections,urinarytract Riyadh, Saudi Arabia), The plates were incubated at 37 C
infections, soft tissue infections, bone and joint infections, for24–48h.Identificationofisolateswasdoneaccordingto
obstetric and gynaecological infections, and other various standard methods described elsewhere [12, 13] and Clinical
typesofinfections[8].Cephalosporinsasanantibioticgroup Laboratory Standards Institute [14]. Isolates were stored in
iswellknowntohaveminimalsideeffectscomparedtoother brain heart infusion broth containing 16% (w/v) glycerol at
antibioticgroups. −80∘Cuntilfurtheruse.
Toourknowledge,thereisneithermarketedtopicalfor-
mulationofthisessentialgroupnorresearchstudyperformed (3) Growth on Mannitol Salt Agar. Staphylococcal isolates
to investigate topical cephalosporin preparations. Based on were reinoculated onto Mannitol Salt Agar (Oxoid, Hamp-
∘
thisknowledge,itwasofinteresttoformulateCTXastopical shire,UK)andthentheplateswereincubatedat37 Cfor24–
preparation to treat bacterial skin and soft tissue infections 48h.Mannitolfermentationwasobservedandrecorded.
moreefficiently.
(4)GrowthonMacConkeyandCetrimideAgar.Gramnegative
The present study was conducted to formulate topical
isolates were reinoculated onto MacConkey Agar (Oxoid,
hydrogel formulations of CTX. Natural polymers such as
Hampshire, UK) and then the plates were incubated at
Pectin (PEC), Xanthan Gum (XG), Guar Gum (GG), syn-
∘
37 C for 24–48h. Lactose fermentation was observed and
theticpolymer,namely,Carbopol934(C934),andsemisyn-
recorded.P.aeruginosastrainswerefurtherreinoculatedon
thetic polymers such as Hydroxypropyl Methylcellulose
∘
cetrimideagar(Oxoid,Hampshire,UK)at37 Cfor24h.
(HPMC) and Carboxymethylcellulose Sodium (Na CMC)
were used as gelling agents. Effect of propylene glycol (PG)
used as a penetration enhancer on the release had been 2.2.Methods
studied.Hydrogelswereevaluatedfortheirphysicalappear-
ance, rheological behavior, spreadability, drug release, and 2.2.1.FourierTransmittedInfraredSpectroscopy(FTIR). FTIR
antimicrobial activity. Since different microorganisms can spectra were performed to find out any possible drug-
exist on the margins of chronic wounds [9], in vitro and polymerinteractions.SamplesofCTXandofeachpolymer
in vivo antibacterial activity of CTX was studied against used (XG, GG, PEC, HPMC, Na CMC, and C934) as
commonlyisolatedwoundpathogenssuchasStaphylococcus well as samples of their physical mixtures in the ratio of
aureus(S.aureus),Escherichiacoli(E.coli),andPseudomonas 1:1 were grounded separately and mixed thoroughly with
aeruginosa(P.aeruginosa)[10,11]. potassiumbromide.Atotalof1mgofeachsampleand100mg
of potassium bromide (KBr) were ground uniformly and
compressedtoformaKBrfilmdisc.Potassiumbromidediscs
2.Experimental
werepreparedbycompressingthepowdersatapressureof5
tonesfor5mininahydraulicpress.Datawerescannedover
2.1.Materials −1
a spectral region from 400 to 4000cm and the obtained
2.1.1.MaterialsUsedforGelPreparation. Cefotaxime(CTX) spectrawereanalyzed[15].
sodium and Hydroxypropyl Methylcellulose 4000 (HPMC
4000)werepurchasedfromSigma,USA.XanthanGumwas 2.2.2. Preparation of Cefotaxime (CTX) Sodium Gel Formu-
purchasedfromUltrafine(India).GuarGumwaspurchased lations. Drug-loaded hydrogels were prepared according to
from Premcem Gums Ltd. (India). Sodium carboxymethyl previouslydemonstratedprocedure[6,16].Ingeneral,there
cellulose (Na CMC) and Pectin (Pec) were obtained from arefourdifferentclassesofexcipientsusuallyincorporatedin
Shinestu Chemical Co., France. Carbopol 934 (C934) and ordertopreparetopicalgelswhicharegellingagents,humec-
Triethanolamine(TEA)werepurchasedfromLobaChemie, tants, preservatives, and vehicles [17]. Different concentra-
Mumbai,India.Propyleneglycol(PG)andsodiumbenzoate tions of various polymers (gelling agents) were postulated.
areobtainedfromMerck,Germany.Fucidinisaproductof NaturalpolymerssuchasGG(inrangeof2–5%w/w),XG(in
LEOPharmaceuticalproducts,Denmark. rangeof2-3%w/w),PEC(inrangeof6-7%w/w),semisyn-
theticpolymersHPMC4000(inrangeof3-4%w/w),andNa
2.1.2.MaterialsUsedforMicrobiologicalStudies CMC (in 4 and 7% w/w) as well as the synthetic polymers
C934 in concentration of 2% w/w were used. Drug-
(1)BacterialIsolates.Atotalofsixbacterialisolateswereused, loaded hydrogels were formulated by dispersing the gelling
one standard E. coli (E. coli) (ATCC 25922) and one E. coli agentsslowlyinanaqueous-basedsolutioncontainingCTX
BioMedResearchInternational 3
Table1:CompositionofCTXtopicalhydrogels(%w/w).
Ingredients F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13
CTX 1 1 1 1 1 1 1 1 1 1 1 — —
XG 2 3 — — — — — — — — — — —
GG — — 2 3 4 5 — — — — — — —
PEC — — — — — — 6 7 — — — — —
NaCMC — — — — — — — — 4 7 — — —
HPMC — — — — — — — — — — 3 4 —
C934 — — — — — — — — — — — — 2
PG 5 5 5 5 5 5 5 5 5 5 5 5 5
Sodiumbenzoate 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Purifiedwaterto 100 100 100 100 100 100 100 100 100 100 100 100 100
(1% w/w), PG (5% w/w, as a humectant), and sodium (4)DrugContentDetermination.Aspecificquantity(100mg)
benzoate(0.25%w/w,asapreservative),withthehelpofan ofeachpreparedhydrogelwastakenanddissolvedin100mL
overheadmechanicalstirreratamoderatespeed.pHofC934 ofphosphatebuffer(pH6.8).Thevolumetricflaskcontaining
hydrogelwasadjustedusingTEAwithstirringuntildesired the gel solution was shaken for 2h on mechanical shaking
pH value was approximately reached (6.8–7). The prepared waterbathinordertocompletedrugsolubility.Thissolution
hydrogelswerepackedinwidemouthjarcoveredwithscrew wasfilteredandestimatedspectrophotometricallyat254nm
cappedplasticlidandwerekeptindarkandcoolplace[18]. [7] using phosphate buffer (pH 6.8) as blank [23]. This test
Sampleswereallowedtoequilibrateforatleast24hatroom wasperformed3timesandmeanvalue±SDwascalculated.
temperature prior to performing rheological measurement
(5) Determination of Viscosity. Viscosity of CTX topical
[3,19].ThecompositionofdifferentpreparedCTXhydrogel
hydrogelswasdeterminedusingRheometer(BrookfieldR/S,
formulationsisgiveninTable1.
USA) with spindle # C 50-1 having a speed of 50rpm. All
measurements were done in triplicate at room temperature
2.2.3.PhysicochemicalEvaluationofthePreparedHydrogels [21].
(1)VisualExamination.Cefotaximehydrogelswereinspected
(6) In Vitro Drug Release Study. In vitro drug release study
visually for their color, homogeneity (appearance and pres- was done as mentioned before with some modifications
ence of any aggregates), grittiness (presence of particles or [16]. Drug release from the prepared topical hydrogels was
grits),andsyneresis(phaseseparation). determined using dialysis tubing (MWCO of 12400 D;
99.99%retention,Sigma-Aldrich,USA)placedintherelease
(2) pH Determination. The pH of various CTX gel formu-
mediumunderconstantstirringusingdissolutionapparatus
lations was determined using pH meter (Mettler Toledo,
USPII(ErwekaDT600,Germany).Aquantityof5gofeach
Switzerland), which was calibrated before each use with
hydrogelformulationswereindividuallypackedintodialysis
standard buffer solutions. A quantity of 1g of CTX gel was tube with the ends being tightly fastened. Release medium
dissolved in 100mL freshly prepared distilled water and was 500mL phosphate buffer (pH 6.8). The medium was
stored for two hours. The electrode was inserted into the maintainedat37∘C±0.5andstirredcontinuouslyat50rpm.
samplesolution10minpriortorecordingthereadingatroom Aliquots of 5mL of the release medium were withdrawn at
temperature.Eachmeasurementwascarriedoutintriplicate predeterminedtimeintervals(15,30,45,60,90,120,180,240,
andaveragepHwascalculated[3,20].Resultsareshownin 300, 360, and 480min) over a period of 8h and replaced
Table2. by fresh phosphate buffer to provide sink condition. Each
withdrawnsamplewasfurtherdilutedwithphosphatebuffer
(3) Spreadability Test. Spreadability (g⋅cm/sec) is expressed
(pH6.8)anditsabsorbancewasmeasuredusingUV-visible
in terms of time taken in seconds by two slides to slip off spectrophotometer (Biochrom Libra S22, UK) at a 𝜆max of
from the gel placed between them, under certain load [21].
254nm. Absorbance was converted to drug concentration
Thestandardizedweighttiedontheupperplatewas20gand
using a linear calibration curve and then the cumulative
length of the glass slide was 7.5cm [22]. Spreadability was
percentage of CTX released was calculated with the help
calculatedbyusingthefollowingformula:
of dilution factor. All measurements were performed in
(Weight×Length) triplicate(𝑛=3).
Spreadability= . (1)
Time
(7) Drug Release Kinetic Study. The data obtained from the
According to the above equation, it is clear that the lesser in vitro release study were analyzed using various kinetic
thetimetakenforseparationofthetwoslides,thebetterits modelstodescribethemechanismofdrugreleasefromthe
spreadability. hydrogels. Three kinetic models including zero-order (2),
4 BioMedResearchInternational
first-order (3), and Higuchi square root models (4) were 2.2.5. Determination of the Isolates Sensitivity. Sensitivity of
appliedonthereleasedata[6]: the standard strains and the clinical isolates under test to
𝑄=𝐾0𝑡, (2) various CTX formulations was determined by Kirby-Bauer
welldiffusionsusceptibilitytestdescribedinCLSI[28].The
where𝑄istheamountofdrugreleaseattime𝑡,𝐾0 iszero- exponential phase cultures of the bacterial isolates under
orderrateconstantexpressedinunitsofconcentration/time, test were made in sterile normal saline and adjusted to 0.5
and𝑡isthetime.Consider McFarland’sstandardapproximatelycorrespondingto1-2×
𝐾 𝑡 8
log𝑄=log𝑄0− 2.3103, (3) H10inCtoFnUA/mgaLr.(MThHeAc)upltluatreess(Owexroeidt,hUeKn)suwnaibfobremdlyobnyMmueallners
where𝐶0 istheinitialconcentrationofdrugand𝐾1 isfirst- ofsterileswab.Equidistancewellswerecutintheplateswith
help of 8mm borer. In each of these wells the gel solutions
orderconstant.Consider
andpuredrugwereplacedandtheplateswereleftatambient
𝑄=𝐾 𝑡1/2, (4)
𝐻 temperaturefor30mintoallowprediffusionpriortoincuba-
∘
where𝐾𝐻istheconstantreflectingthedesignvariablesofthe tionat37 Cfor24h.Antibacterialactivitywasestimatedby
system. measuring the diameter of inhibition zones. Well diffusion
To find out the mechanism of drug release, the release testswereperformedintriplicatesandantibacterialactivity
datawasfittedinKorsmeyer-Peppasmodelasfollows: was expressed as the mean diameter of inhibition zone ±
𝑀 standarddeviation.
𝑡 =Ktn, (5)
𝑀
∞ 2.2.6. In Vitro Wound Model for Assessing Effectiveness
where𝑀𝑡/𝑀∞isfractionofdrugreleasedattime𝑡,𝐾isthe of Antibiotic Gel. Susceptibility of the bacterial strains in
releaserateconstantincorporatingstructuralandgeometric planktonicformwastestedagainstdifferentgelformulations
characteristicsofthetablet,and𝑛isthereleaseexponent.The usingthemodifiedquantitativemethodoriginallyintroduced
𝑛value(diffusionexponent)isusedtocharacterizedifferent by Hammond et al. [29, 30]. Each hydrogel was challenged
releasemechanisms[24].TheresultsareshowninTable3. against one of the strains tested. The concentration of CTX
was 1% in each formulation tested. Three sterile 6mm
(8)StabilityStudy.HydrogelsF1,F11,andF13whichshowed
cellulosediskswereplacedonapetridishwithLuriaBertani
apromisingsustaineddrugreleasewerepackagedinairtight agar(LBagar)(Oxoid,Hampshire,UK)and10𝜇Lbacterial
plasticcontainerandsubjectedtostabilitystudy.Theywere 2 3
∘ suspension containing 10 –10 CFU was dropped on each
stored at room temperature (25 C) and in the refrigerator
∘ disk.Thesurfaceoftheplatewascoveredwithasterilegauze
(4 C) over a period of three months [23, 24]. Physical
square(5×5cm)saturatedwithgelweightequivalentto1%
evaluationofthesampleswascarriedoutbyvisualinspection
CTX.Asterilegauzesquarewithouttopicalantimicrobialgel
forphaseseparationandchangeincolorandodor.Rheolog-
was used as a control. A sterile small glass petri dish was
ical properties were also examined. Furthermore, chemical
placedonthegauzetomaintaindirectcontactbetweenthe
stability was evaluated by spectrophotometric analysis of
agentandtheinoculateddisks.Theplateswereincubatedat
drugcontentandpHmeasurement[25].
∘
37 C for 24h, after which the gauze squares were removed
andeachdisktransferredintoasteriletubecontaining1mL
2.2.4. Determination of Minimum Inhibitory Concentration
phosphate buffered saline (PBS). The tubes were vigorously
(MIC). MICsofCTXandthedifferentgelformulationswere
performed in cation-adjusted Mu¨ller Hinton broth (Oxoid, vortexed three times for 2min to detach bacteria from the
Hampshire, UK) (MHB) by means of microdilution broth disks.Suspendedcellswereseriallydilutedfourtimes(a10-
method in accordance to National Committee for Clinical fold dilution was used) in PBS and 10-𝜇L aliquots of each
laboratory Standards [26] and CLSI documents [27]. Stock dilutionwereinoculatedonLBagarplates.Theseplateswere
∘
solutionsofCTXpuredrugoreachoftheformulationsunder incubated at 37 C for 16h and the numbers of CFU were
testwaspreparedinsteriledistilledwatertoreachaninitial counted.
concentrationof2mg/mL.Preparationofinoculaforbroth Thefinalresultrecordedrepresented3×108CFU/diskif
microdilution testing was performed in accordance with thebacterialgrowthwassomassivethatthenumberofCFU
CLSIstandardprocedures.Briefly,0.5McFarlandequivalent obtainedwasuncountableeveninthehighestdilution.
inoculumofeachstrainwaspreparedinnormalsalinefrom
18–24hagarplateculture.Thesuspensionwasfurtherdiluted 2.2.7. Topical Antimicrobial Efficacy of C934-CTX Hydrogel
5
toachievedesiredinoculumsconcentrationof10 CFU/mL. UsingaMouseModel. Toevaluatethepharmaceuticalpoten-
100𝜇Lofaliquotofeachstrainwasthenaddedtoa96-well tialofC934-CTXhydrogel(F13)asanovellocaltreatmentfor
microtiterplatecontaininggradientconcentrationsofeither SSTIs,antimicrobialeffectofthishydrogelwasinvestigatedin
CTXpuredrugorthegelformulationsundertestdilutedin amurinesurgicalsiteinfectionmodel.
∘
doublestrengthMHB.Theplateswerethenincubatedat37 C
for24h.Theturbidityofeachwellwasmeasuredat490nm (1)ExperimentalAnimals.Theanimalsusedforinvivoexper-
withamicroplateELISAreader.TheMICwasdefinedasthe imentswere200gspecific-pathogen-freemaleWistaralbino
lowest concentration of the antibiotic or the hydrogel that rats. All animals were obtained from Experimental Animal
preventedbacterialgrowth. Care Center, College of Pharmacy, King Saud University
BioMedResearchInternational 5
(KSU), Riyadh, Saudi Arabia. Rats were housed in stainless 3.ResultsandDiscussion
steel cages (5 animals/cage). Animals were acclimated with
free access to tap water and standard pellet diet (Purina 3.1. Fourier Transmitted Infrared Spectroscopy (FTIR). FTIR
Chow) in a facility with controlled temperature (22–24∘C) spectrumoffreeCTX(Figure1(a))showedthatthecharac-
−1
and humidity (50–60%), on a 12h light/12h dark cycle, teristicspeaksareat3430and3347cm duetoNHstretching
−1
for at least 1week before the experiments. The protocol of (NH2) and the peaks at 2938, 1760, 1729, and 1647cm
this study was carried out according to the regulations and are attributed to aliphatic C–H stretching, C=O (𝛽-lactam)
recommendationoftheAnimalResearchEthicsCommittee stretching,C=O(carboxylicester),andC=O(amide)stretch-
ofCollegeofPharmacy,KSU,SaudiArabia. ing,respectively[35].Inaddition,thespectrumshowedother
−1
characteristic peaks at 1610 and 1536cm which are cor-
(2)InVivoWoundModel.Inthismodel,twobacterialclinical responded to C=C and C=N stretching, respectively. Other
isolates were chosen based on being the most common peaks appear at 1386 and 1355cm−1 due to aliphatic C–H
pathogen in SSTIs [31, 32]. These strains were P. aeruginosa bond, whereas the peak at 1062cm−1 is attributed to C–O
isolateandMRSAisolate.TheBacterialisolateswereinitially stretching[15,36].
incubatedintrypticasesoyagarwith5%glucosefor24hat Figures1(b)and1(c)showedcharacteristicpeaksofC934
∘
37 C and re-suspended in saline adjusted to 0.5 McFarland
and the physical mixture of CTX and C934 in 1:1 ratio.
turbidity.
FTIRspectrumofthemixturerevealedthatthereisneither
Rats of each group were intraperitoneally anesthetized
appearanceofnewpeaksnordisappearanceofexistingpeaks,
withxylazine(8mg/kgbodyweight)andketamine(30mg/kg
whichindicatedthatthereisnointeractionbetweenthedrug
bodyweight),thehaironthelowerbackwasshavedandthe
andthegellingagentused.Characteristicpeaksofthedrug
skin was cleansed with antiseptic solution (10% povidone-
groupswereidentifiedinallthespectraofpreparedphysical
iodinesolution).A1-cm-long,fullthicknessincisionwound
mixturesofthedruganddifferentpolymersusedasgelling
was created on the dorsal side of the rat. Approximately
agents (not included). This study revealed that the drug
1cm of silk suture infected with either P. aeruginosa isolate
or MRSA isolate (approximately 5 × 103 cells/cm of suture) wascompatiblewitheachoftheselectedgellingagentsand
indicatedthesuitabilityoftheselectedpolymersforprepara-
was placed into the wound and secured in the skin by
tionoftopicalCTXgelformulation.
knotting.Onesinglesuturewasattachedoverthemiddleof
the incision. The animals were returned to individual cages
3.2.CharacterizationofthePreparedCTXHydrogels. Results
[33].
of the visual inspection of the prepared CTX hydrogels are
(3) Experimental Protocol. Rats were randomly allocated presented in Table 2. Most of the prepared hydrogels were
into six groups. For each bacterial strain, three challenged yellowishincolor,eitheropaqueortranslucentortranspar-
groupsreceivedthefollowing;GroupI(GI);Negativecontrol ent. All the prepared hydrogels showed good homogeneity
group(challengedwiththebacteriaanddidnotreceiveany withabsenceoflumpsandsyneresis.Nosignsofgrittiness.
antibiotic).GroupII(GII):Positivecontrolgroup(challenged The hydrogels had smooth homogenous texture and glossy
with the bacteria and treated with a Fucidin cream). This appearanceexceptF10whichshowedclumps.
cream was used as an available marketed topical antibiotic pHvaluesofthepreparedformulaswereintherangeof5-
formulation containing 2% fusidic acid for the sake of 6whichisconsideredacceptabletoavoidskinirritationupon
comparison with the formulated hydrogel and group III application[37].PEC-basedgelwasanexceptionaspHwas
(GIII):Challengedgroupwiththebacteriaandtreatedwith about4.Additionally,therewasnosignificantchangeinpH
C934-CTXhydrogel1%(F13). valuesasafunctionoftimeforallformulations.Resultsare
Antibiotictreatmentswerestarted24hafterincisionand illustratedinTable2.
2
suturing. After 24h, the animals were sacrificed and 1cm ThedatapresentedinTable2showedthatthepercentage
tissuewascutfromthewoundareainordertoexaminethe drugcontentofpreparedhydrogelformulationsrangedfrom
effectivenessoftheformulatedgelF13forinfectiontreatment. 95 to 98.9% which are within the official limits (100 ± 5%)
Equal sections of the isolated skin tissues were used for [38].Thisindicatedthatthedrugwasuniformlydistributed
reading bacterial bioburden. Viable counts of bacteria per throughoutthegel.Therefore,themethodusedinthepresent
sectionwereanalyzedintissuehomogenate. study seems to be reproducible for the preparation of CTX
Quantificationofviablebacteriainthehomogenatewas hydrogels.
done by culturing serial 10-fold dilutions of the bacterial Good spreadability is one of the criteria for gel to meet
suspension onto nutrient agar plates. The plates were incu- idealqualities.Itisthetermexpressedtodenotetheextentof
∘
batedat37 Cfor24handtheorganismswerequantifiedby areatowhichgelreadilyspreadsonapplication.Therapeutic
countingthenumberofCFU/section. efficacy of a gel formulation also depends on its spreading
Other tissue sections were further used for histologi- value[22].Additionally,spreadabilityisveryimportantasit
cal study. They were fixed with formalin and embedded showsthebehaviorofthegelwhenitcomesoutfromthetube
in paraffin. Consequently, the sections were stained using [39]. Spreading coefficient of different hydrogels is shown
Hematoxylin and Eosin (H&E), and then they were dried in Figure 2. The determined spreadability values indicated
overnight.Thestainedtissuesweremicroscopicallyexamined thatthepolymersusedprovidedhydrogelswhichcanspread
andthemostdetailedandcleartissueslideswerechosen[34]. by shearing force of low magnitude. It was observed that
6 BioMedResearchInternational
e
% transmittanc −2110000 CTX 3346.64 3100.40 2936.72 2821.48 1759.61 1729.74 1648.261535.251458.72 1354.711281.091242.36 1046.73
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers(cm−1)
(a)
e 20
c
n
% transmitta −11000 C934 3118.02 2361.37 1708.701648.59 1458.04 1170.44
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers(cm−1)
(b)
20
e
nc 6
% transmitta −200 CTX and C934 3100.62 2364.3 1727.32 1655.071560.321535.181451.44 1242.471182.55
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers(cm−1)
(c)
Figure1:FTIRspectraofCTX(a),C934(b),andphysicalmixtureofCTXandC934(c).
Table2:PhysicalpropertiesofCTXtopicalgelformulations(F1–F13).
Formulations Color Homogeneity Grittiness pH %drugcontent
F1 Opaque,yellowish +++ — 6.6 96±0.3
F2 Opaque,yellowish +++ — 6.5 98.8±0.1
F3 Translucentyellowish +++ — 5.9 95.4±0.2
F4 Translucentyellowish +++ — 5.6 98.9±0.3
F5 Translucentyellowish +++ — 5.7 97.5±0.4
F6 Translucentyellowish +++ — 5.6 97.8±0.1
F7 Opaque,buff +++ — 4.0 98±0.2
F8 Opaque,buff +++ — 4.0 98.3±0.1
F9 Shiny,transparent,yellowish +++ — 6.2 97.5±0.2
F10 Shiny,transparent,yellowish Clumpy — 6.3 95±0.1
F11 Transparent +++ — 5.7 98±0.3
F12 Transparent +++ — 5.9 97.8±0.4
F13 Transparent +++ — 6.3 96±0.2
++:good;+++:verygood;—:nogrittiness.
BioMedResearchInternational 7
30 120
100
25 d
e
m/sec) 20 % releas 80
·bility (gc 15 mulative 4600
ada 10 Cu 20
e
pr
S 0
5
0 100 200 300 400 500
Time (min)
0
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F1
Formulations F2
Figure2:Spreadingcoefficientofdifferenthydrogelformulations Figure4:DrugreleaseprofilesofhydrogelformulationsF1andF2.
F1–F13.
120
60000
100
d
e
50000 eas 80
el
% r
cosity (cps) 3400000000 Cumulative 246000
Vis 20000
0
0 100 200 300 400 500
10000
Time (min)
0 F3 F5
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F4 F6
Formulations
Figure5:DrugreleaseprofilesofhydrogelformulationsF3–F6.
Figure3:Viscosityincentipoisesofdifferenthydrogelformulations
F1–F13.
all hydrogels that when the rate of shear is increased the
viscosity decreases which indicated that the formulation is
spreadability of CTX hydrogels decreased by increasingthe shearthinningpseudoplasticinnature.
polymer concentration and the values were in the range of
10.9to27.4gm⋅cm/sec.Maximumspreadabilitywasobserved 3.3. InVitroReleaseStudy. ThereleaseprofilesofCTXfrom
for hydrogels containing C934 (2% w/w), followed by that differenthydrogelformulationsareshowninFigures4–7.It
containingHPMC4000(3%w/w)andthatformulatedwith isobviousthatthereleaseofCTXfromhydrogelswasvaried
2%w/wXG.ThelowestspreadabilitywasforNaCMC(4and according to concentration of the polymer and viscosity of
7%). thehydrogels.Generally,anincreaseinthevehicleviscosity
Itcanbeconcludedthatthepreparedhydrogelsfulfilled would cause a more rigid structure with a consequent
therequirementofgel-basedformulationsfordermatological decrease in the rate of drug release [16]. Hydrogels F7 that
use which should have several favorable properties such as exhibited the lowest viscosity (1522 centipoises) released its
greaselessandeaseofspreadability[3,17]. drugcontentwithin30min,whilehydrogelsF10whichhad
It was reported that viscosity is an important physical thehighestviscosity(59042centipoises)showed100%drug
property of topical formulations, which affects rate of drug releasewithin6h.
release [20]. It was found that viscosity of the prepared Thereleaseofthedrugfromthepreparedhydrogelswas
hydrogels ranged from 1522 to 59042 centipoises. The vis- ranked in case of hydrogels containing XG in the order F1
cosityincreaseduponincreasingthepolymerconcentration > F2 and the release was more than 95% after 6h. In case
(Figure3).Thelowestviscositywasobservedwithhydrogels of GG-based hydrogels, the order of release was F3 > F4 >
containingPECwhilehydrogelscontainingNaCMCshowed F5 > F6, depending on the amount of CTX released after
the highest viscosity. Viscosity of hydrogels prepared with 5h.Thisresultcanbeexplainedbytheincreaseinpolymer
GG was changed with time. Generally, it was observed in concentrationfrom2to5%w/wwhichresultedinanincrease
8 BioMedResearchInternational
120 example, the viscosities of F2 (XG-based hydrogel) and F11
(HPMC-basedhydrogel)arenearlyinthesamerange(9537
ed 100 and9817centipoises)butF2releasedabout93%ofitsdrug
as
ele 80 content within 6h, whereas F11 released its drug content
% r completelyafter4h.Ontheotherhand,hydrogelsF5(GG-
ve 60 based hydrogel) and F12 (HPMC-based hydrogel) with a
ulati 40 viscosity of 10788 and 10981 centipoises, respectively, both
m
u releasedabout95%oftheirdrugcontentafter4h.Increasing
C 20
viscosityto59042centipoisesasincaseofhydrogelF10(Na
0 CMC-based formulation) released the same amount of the
0 100 200 300 400 500 drugatthesametime(4h).
Time (min)
Among all hydrogel formulations, C934 (F13) showed
F7 F9 superior sustained drug release followed by HPMC 4000
F8 F10 (F11) and XG (F1). C934 is a hydrophilic polyacrylic acid
polymeranditscarboxylgroupsbecomehighlyionizedafter
Figure6:DrugreleaseprofilesofhydrogelformulationsF7–F10.
neutralization with TEA, forming a gel due to electrostatic
repulsionamongchargedpolymerchains.IncreasingpHof
the prepared hydrogel to be suitable for skin application
resultedinuncoilingofthepolymerchainsduetoionization
120
of its carboxyl groups and subsequently forming a rigid gel
d 100 [40]. The colligative results revealed that Carbopol was a
e
eleas 80 goodgellingagentascomparedtootheragentsforhydrogels’
% r preparation. These results were consistent with previously
e 60 reportedwork[41–43].
v
ulati 40 Results of in vitro release study verified that the most
m importantfactorsinfluencingCTXreleasefromtheprepared
Cu 20 hydrogelsarepolymertypeandconcentration.Polymertype
0 provedtohavearemarkableandpredominantinfluenceon
0 100 200 300 400 500 600 the drug release from the prepared hydrogels followed by
Time (min) the polymer concentration.Many studies showed that drug
releasewasdecreasedwithanincreaseingellingagentcon-
F11
centration.Asthepolymerconcentrationincreases,viscosity
F12
F13 increases[3,20,39].
Theprogressivedecreaseintheamountofdrugreleased
Figure7:DrugreleaseprofilesofhydrogelformulationsF11–F13.
fromhydrogels,asthereleasetestproceeded,wasattributed
to gradual increasein concentrationof the eroded polymer
which increased the viscosity of the system. Consequently,
thediffusionofthedrugthroughthemembranedecreased.
in the hydrogel viscosity from 5766 to 19582 centipoises.
Hydrogel F3 released about 80% of its drug content within
3.3.1. Kinetic Modeling of Drug Release. Different mathe-
one hour but hydrogel F6 released about 56% of its drug
matical models were used to describe the kinetics of CTX
content after the same time. It is clear that as the viscosity
increased by approximately 4-fold, a decrease in the drug hydrogels. The values of the release exponent (𝑛) and the
releasebyapproximately1.5-foldwasobserved. regression values of zero-order, first-order, and Higuchi
Sameresultswereobtainedfortheotherpreparedhydro- releasemodelsfordifferent formulationsare representedin
gels. Drug release from PEC-based hydrogels, namely, F7 Table3.
andF8,wasveryfastasmorethan95%ofthedrugcontent TheHiguchikineticplotswerefoundtobefairlylinearas
wasreleasedwithin45min.HydrogelF7showedfasterand indicatedbytheirhighestregressionvalues.Thecorrelation
higherdrugreleasethanformulationF8.Incaseofhydrogels coefficientsvalues(𝑅2)wereintherangeof0.843–0.995for
containingNaCMC,thereleasewasrankedintheorderF9> all hydrogel formulations. In general, it was observed that
F10andcompletedrugreleasewasattainedwithin4and5h, themechanismofdrugreleaseofallformulationswaseither
respectively. Drug release behavior of hydrogels containing anomalousdiffusionornon-FickiansupercaseII.
HPMC4000wasintheorderofF11>F12andcompletedrug The release exponents (𝑛) for optimized formulations
releasewasachievedafter4and6h,respectively,whereasthe F1, F11, and F13 were found to be 1.330, 1.069, and 0.860,
drugreleasefromhydrogelF13containingC934wasslowand respectively. The diffusion exponents 𝑛 for F1 and F11 were
sustainedfor8h. morethan1,whichindicatesanon-FickiansupercaseII.On
Itwasalsoobservedthatthedrugreleasedidnotdepend theotherhand,formulationF13showedadiffusionexponent
only on hydrogels’ viscosity but it was also influenced by of 0.860 (0.5 < 𝑛 < 1), indicating anomalous diffusion
the type and network structure of each polymer used. For coupledwitherosion.
BioMedResearchInternational 9
Table3:KineticstudyoftheinvitroreleasedataofpreparedCTXhydrogels.
Zero-order First-order Higuchimodel Korsmeyer-Peppasmodel
Formulationcode
Correlationcoefficient(𝑅2) (𝑛)
F1 0.848 0.652 0.976 1.330
F2 0.913 0.663 0.990 0.834
F3 0.689 0.644 0.843 2.510
F4 0.785 0.603 0.922 1.597
F5 0.875 0.647 0.975 1.256
F6 0.881 0.648 0.978 1.330
F7 0.805 0.241 0.936 8.730
F8 0.812 0.179 0.940 11.15
F9 0.896 0.589 0.980 0.629
F10 0.927 0.661 0.993 0.844
F11 0.820 0.579 0.943 1.069
F12 0.857 0.655 0.967 1.421
F13 0.969 0.693 0.995 0.860
3.4.StabilityStudyfortheFormulatedCTXHydrogels. CTX Table4:MIC(𝜇g/mL)ofdifferentCTXgelformulationsagainst
hydrogels(F1,F11,andF13)whichshowedpromisingresults GrampositiveandGramnegativestandardandclinicalisolates.
assustainedreleaseformulationsweresubjectedtostability
BacterialStrains CTX F13 F11 F1
studies at refrigerator and ambient room conditions for 3
E.coli(ATCC25922) 0.06 0.06 1 2
months.Afterstoragefor3months,hydrogelsdidnotshow
E.coli 1 1 >2 >2
anychangeincolor,odor,pH,drugcontent,andrheological
P.aeruginosa(ATCC27853) 31.25 31.25 62.5 31.25
properties.Additionally,nophaseseparationoccurred.This
P.aeruginosa 62.5 125 250 62.5
indicatedthatthedrugwasstableingelsevenafter3months
ofshorttermstorageandthegelformulationswerephysically S.aureus(ATCC29213) 0.25 0.25 0.5 1
andchemicallystable. MRSA 7.8 15.6 31.25 31.25
ItisclearfromabovediscussionthatF1,F11,andF13are
the best formulations among all the prepared formulations;
therefore, they were subjected for further investigation for
microbiologicalactivities.
their MIC against some different bacteria isolated from
wound and SSTIs. A standard strain of each of the tested
3.5. Microbiological Studies. SSTIs are considered as one of
Gram negative bacteria was studied as well for the sake of
the serious problems worldwide, especially due to bacterial
fair judgment to the selected formulas’ effect. Furthermore,
resistance. Shortage in marketed topical dosage forms con-
standard S. aureus was tested with one MRSA strain for
taining different groups of antibiotics aroused the need of
a better indication of the antimicrobial coverage of CTX
oralandparenteralapplicationswiththeiraccompaniedside
gel formulas. CTX pure drug was tested along with the gel
effects.
formulas as a control (Table 4). Results revealed that the
Cefotaxime(CTX)sodiumasoneofcephalosporinbroad
MICvaluesofthestandardstrainsobtainedwereconsistent
spectrum antibiotics has a high safety margin and was withtheinterpretedCLSI2011values.F13showedcomparable
reportedtobeeffectiveagainstdifferentmultidrugresistant MICs to the control drug and showed better efficacy than
pathogenasbytheFDA. F1 and F11. The MICs against standard strains of E. coli
In the present study, effect of the prepared CTX topical and P. aeruginosa were from 0.06–2 to 31.25–62.5𝜇g/mL,
gelformulationswasinvestigatedthroughdifferentmicrobi- respectively,whileitwasfrom1–>2andfrom62.5–250𝜇g/mL
ologicaltechniques. against clinical isolates of the same strains, respectively. S.
aureus showed much lower MICs of all formulations tested
3.5.1.EvaluationofBacterialSusceptibilityto whencomparedtotheisolatedMRSAwheretherangewas
CTXGelFormulations from0.25–1to7.8–31.25,respectively.Analysisoftheresults
indicated that isolated P. aeruginosa gave the highest MIC
(1)DeterminationofMinimalInhibitoryConcentration(MIC). values but it is still considered as having an intermediate
Results of broth microdilution method with different CTX sensitivity to CTX as previously reported [44, 45]. These
formulations were represented in Table 4. The formulas F1, obtained minimum inhibitory values against the bacterial
F11, and F13 which showed considerable good spreadability isolateswereconsistentwithpreviouslyreportedresults[46,
andreleasecharacteristicswereselectedfurthertodetermine 47].Thegelbasedidnotshowanyantibacterialactivity.
10 BioMedResearchInternational
50 45
45 42.6 43 43 40 37.25 38
m) 40 m) 35.25 36.25
40
m m 35
n zone ( 3305 32.25 32 32.25 33.14 n zone ( 30 26.5 26 25.75 28.4
o o 25
biti 25 biti
hi hi 20
er of in 1250 er of in 15
et et
m m 10
a 10 a
Di Di
5 5
0 0
F13 F11 F1 CTX F13 F11 F1 CTX
E. coli (ATCC 25922) P. aeruginosa (ATCC 27853)
E. coli P. aeruginosa
(a) (b)
40
35.3 35.8
34.6
35 33
m)
m 30
e ( 25 25
n
n zo 25 21.75 23
o
biti 20
hi
n
of i 15
er
et
m 10
a
Di
5
0
F13 F11 F1 CTX
S. aureus (ATCC 29213)
MRSA
(c)
Figure8:AgarwelldiffusionassayforantibacterialactivitiesofdifferentCTXgelformulationsagainstselectedpathogens.
(2) Agar Well Diffusion Assay. Agar well diffusion method be attributed to the great spreadability characteristic of F13
wasusedforscreeningtheantimicrobialpotentialofCTXgel gelwhichaffectstherapeuticefficiencyofthedrug.
formulationsandtheresultswereshowninFigure8.Effect Tested formulas showed significant results (𝑃 < 0.05)
ofthethreechosenformulasontheselectedbacterialstrains against Gram negative standard strains tested (Figures 8(a)
was represented as the mean of three readings (mm ± SD). and8(b))comparedtoisolatedone(Ca.10mmdifference).
Along with the formulas, control pure drug was tested as Although the tested formulations demonstrated better
well for better comparison. Figure 8 clearly demonstrated effect against E. coli isolate as compared with P. aeruginosa
thattherewasnosignificantdifferenceinzoneofinhibition isolate,theobtainedP.aeruginosaresultswereintheaccept-
betweenF1,F11,andF13.Diameterofinhibitionzonesshown ablesusceptibilityvaluestoCTXasrevisedinCLSI[49].
byformulationF13wassimilartothatofcrudedrugagainst Figure 8(c) showed the effect of three formulations
any of the strains tested which is in agreement with the against Gram positive S. aureus. Two strains were tested, S.
fact that incorporation of the drug into gel base does not aureusandMRSA.AgainstS.aureus,thezonesofinhibition
decreaseitsantibacterialactivity[48].Additionally,thiscan weresignificantlybetterthanMRSA,givingmorethan10mm