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Original Research Paper

Preparation, characterization and in vivo evaluation of alginate-coated chitosan and trimethylchitosan nanoparticles loaded with PR8 influenza virus for nasal immunization

Jafar Mosafer

^a^,^b

, Amir-Hossein Sabbaghi

^c

, Ali Badiee

^b^,^d

, Solmaz Dehghan

^c

, Mohsen Tafaghodi

^b^,^d^,^∗

aResearchCenterofAdvancedTechnologiesinMedicine,TorbatHeydariyehUniversityofMedicalSciences,Torbat Heydariyeh,Iran

bSchoolofPharmacy,MashhadUniversityofMedicalSciences,Mashhad,Iran

cStudentResearchCommittee,MashhadUniversityofMedicalSciences,Mashhad,Iran

dNanotechnologyResearchCenter,PharmaceuticalTechnologyInstitute,MashhadUniversityofMedicalSciences, P.O.Box9196773117,Mashhad,Iran

a r t i c l e i n f o

Articlehistory:

Received23November2017 Revised14March2018 Accepted6April2018 Availableonlinexxx

Keywords:

Chitosan

Trimethylchitosan Alginate

PR8influenzavirus Nasalimmunization

a bs t r a c t

Forefficientmucosalvaccinedelivery,nanoparticulateantigensarebettertakenbymicro- foldcellsinthenasalassociatedlymphoidtissueandalsodendriticcells.Nanoparticles basedonpolymerssuchaschitosan(CHT)anditswatersolublederivative,trimethylchitosan(TMC),couldbesuccessfullyusedascarrier/adjuvantforthispurpose.Sodiumalgi- nate,anegativelychargedbiopolymer, couldmodifytheimmunostimulatorypropertiesof CHTandTMCNPsandincreasetheirstability.Sodiumalginate(ALG)-coatedchitosan(CHT) and trimethylchitosan(TMC)nanoparticles (NPs)loadedwith inactivatedPR8 influenza virusweresuccessfullypreparedbydirectcoatingoftheviruswithCHTorTMCpolymersto evaluatetheirimmunoadjuvantpotentialafternasalimmunization.Afternasalimmuniza- tionsinBALB/cmice,PR8-CHTformulationelicitedhigherIgG2aandIgG1antibodytiters comparedwithPR8-TMC.ALGcoatingofthisformulation(PR8-CHT-ALG)significantlyde- creasedtheantibodytitersandalessimmuneresponsewasinducedthanPR8-TMC-ALG formulation.PR8-TMC-ALGformulationshowedsignificantlyhigherIgG2a/IgG1ratio,ascri- teriaforTh1-typeimmuneresponse,comparedwithPR8-CHT-ALGandPR8virusalone.Al-

∗Correspondingauthor.NanotechnologyResearchCenter,PharmaceuticalTechnologyInstitute,MashhadUniversityofMedicalSci- ences,P.O.Box9196773117,Mashhad,Iran.Tel.:+985131801337.

E-mailaddress:[email protected](M.Tafaghodi).

PeerreviewunderresponsibilityofShenyangPharmaceuticalUniversity.

https://doi.org/10.1016/j.ajps.2018.04.005

Pleasecitethisarticleas:JafarMosaferetal.,Preparation,characterizationandinvivoevaluationofalginate-coatedchitosanand

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together,thePR8-TMC-ALGformulationcouldbeconsideredasanefficientintranasalanti- gendeliverysystemfornasalvaccines.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense.

(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction

Althoughaluminuminvirtuallyallcurrentlyadjuvanthuman vaccinesisusedtohelpincreasethehumoralorcellularim- muneresponsestoanantigen,itusuallyinducesaTh2anti- bodydominatedresponse.Accordingly,aluminum-basedvac- cinesarenotsuitableforintracellularpathogens,chronicin- fections orcancers.Therefore, enormousefforts havebeen donetodevelopnewadjuvantsthatareabletoinduceboth humoralandcellularimmuneresponses.

Intramuscular(Im)orsubcutaneous(SC)arethemostcom- monlyadministrationroutesofcurrentlyavailablevaccines that elicit robust and strong systemic immune responses.

Since,thesevaccinescreatepainuponinjectionandarenot able to elicitmucosal immune responses,a desirablenon- invasive immunizationis necessary [1–7].Amongthe non- invasive routes,nasal administrationisofespecial interest due toinductionofmucosal immunity[8,9].Unfortunately, therearesomedrawbacksassociatedwithnasaladministra- tionthatshouldbeconsideredinordertoelicitanacceptable mucosalandsystemicresponse.Forexample,themucociliary clearance,thetolerogenicnatureofmucosalepitheliumsand the largesizeofantigenare somevitalfactors thatleadto thedecreaseoftheresidencetimeofantigenanditsuptake throughnasalepitheliumandeventuallycomplicatearobust immuneresponse.Acommonwaytotackletheseproblemsis toincorporateantigensintothemucoadhesivepolymericNPs [10].Thisstrategyprolongstheresidencetimeofantigensand alsoprotectsagainstenzymes[11].Additionally,nanopartic- ulatedantigensaremoretakenbydendriticcells(DCs)and could cross theepithelial barriermoreeasilythroughafa- mouskindofcellswhicharecalledmicrofoldcellsinthenasal associatedlymphoidtissue(NALT)[12,13].

Chitosan(CHT)isabiodegradable,biocompatibleandsafe cationic mucoadhesive polymer that is made by treating the chitin shells of shrimp which numerous studies have shownitspotentialasanenhancerpolymerfornasalvaccine deliveryacrossmucosalsurfaces[14–17].CHTcanincreasethe permeabilityofthemucosalbarrierpossiblythroughdisrup- tionofintracellulartightjunctions.Since,CHThasalimited aqueoussolubility atalkalineorneutral pH,aderivativeof CHT,N,N,N-trimethylchitosan(TMC)hasbeendevelopedas amucoadhesivepolymerfornasalvaccinationtoovercome the problem,which hasanimprovedsolubility overawide rangeofpH[18,19].Sodiumalginate(ALG)isalsoanegatively chargedpolymerthatcouldbesimplycoatedaroundthepos- itivelychargedCHTorTMC [20].ALG couldmodifytheim- munostimulatory properties ofNPs,increase their stability [21,22]as wellas preventaburstreleaseofloadedantigens [23].

HumaninfluenzaA/PuertoRico/8//1934(H1N1)virus(PR8) wasthemostcommoncauseofhumaninfluenzaat2009.That isthesubtypeofinfluenzaAviruswithanegativechargeand couldbeeasilyassociatedwithpositivelychargedpolymers suchasCHTorTMC[24,25].

Inthisstudy,weinvestigatewhetherPR8-loadedTMCor CHTNPs,withorwithoutALGcoat,aresuitableantigencarrier systemstoenhanceinvivotransmucosalantigendelivery.

2. Materials and methods

2.1. Materials

IgG2a and IgG1 secondary antibodies were obtained from ZymedInc.(USA).CoatinganddetectionmAbantibodiesfor IFN-γandIL-4aswellasstreptavidin-HRPwereobtainedfrom Mabtech(Sweden).ConcanavalinAandALG(lowmolecular weight)werepurchasedfromSigmaAlderich(USA).RPMI1640 culturemedium, penicillin–streptomycinsolution and fetal calfserum(FCS)were purchasedfrom SigmaAldrich(USA).

CHTwaspurchasedfromFluka(USA).TMCwassynthesized andcharacterizedfromabovementionedchitosanasreported inourpreviousstudies[1].

BALB/cmiceandPR8antigenwereobtainedfromthePas- teur Institute of Iran. Experiments were performed in ac- cordance with the guidelines and regulations for the care and use of animals implemented bythe ethics committee ofMashhadUniversityofMedicalSciences (Approvalnum- ber:IR.MUMS.REC.1392.23).Alsotheanimalstudieswereper- formedbasedontheEuropeanCommunityguidelinesasac- ceptedprinciplesfortheuseofexperimentalanimals.

2.2. PreparationofPR8-CHT-ALGandPR8-TMC-ALGNPs

NPswerepreparedwithdifferentweightratioofvariouscom- ponentsincludingPR8antigen,CHTorTMCpolymersandALG coatingforfindingthebestresultsincludingthesmallestpar- ticlesizeandpolydispersityindex(PDI)aswellasthehighest surfacecharge(zetapotential).The1:4:6(w/w/w)ratiosofthe PR8:CHT/TMC:ALGshowedthebestmentionedphysicochem- icalpropertiesandusedforfurtherstudies.First,thePR8-CHT andPR8-TMC NPswereprepared byaddingPR8solutionto CHTorTMCpolymers(dispersedinphosphatebuffer(PB),pH 6)attheratioof1:4(w/w)andgentlyvortex-mixedforabout 5s[1,9,26].Next,theformulationscontaining(1:4)/6(w/w)ra- tiosofPR8-CHT(orTMC)/ALGwerealsoprepared.Allofthe obtainedformulationswerepreparedinPBandstoredat2–

8°C.ThefinalPR8concentrationintheformulationswasset as15μg/dose/mouseforinvivoadministration.

Please citethisarticleas:JafarMosaferetal.,Preparation,characterizationandinvivoevaluationofalginate-coatedchitosanand

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2.3. CharacterizationofNPs

Dynamiclightscatteringanalysis(NANO-Zetasizer,Malvern, UK)wasusedtodeterminethezetapotential,meanparticle sizeandPDIofNPs.Also,toevaluatethestabilityofNPs,each 5d,thezetapotential,meanparticlesizeandPDIofdifferent formulations(inPBbuffer,pH6)wereevaluatedat4°Cfor30d

2.4. Invivovaccinationprotocol

Theinvivoevaluationofimmunoadjuvantpotentialofpre- pared NPs was investigated in female BALB/c mice. Seven groups wereimmunizedintranasally (In) with:1.PBS solu- tionasanegativecontrol,2and3.PR8antigen(15μg/mouse, given intramuscularly (Im) or intranasally, 4 and 5. PR8- CHT and PR8-TMC NPs (15 μg PR8 antigen + 64 μg CHT or TMC/mouse) and 6 and 7. PR8-CHT-ALG and PR8-TMC- ALG NPs (15μg PR8 antigen+ 64μg CHT or TMC +96 μg ALG/mouse).Sixmiceineachgroupwereinjectedthreetimes in2-weekintervalswiththeseNPs.Fornasalimmunization, anintraperitonealinjectionofxylazineandketamin(10and 100μg/gbodyweight,respectively)wereusedtoanesthetize themice.Finally,atotalvolumeof5μlofeachformulation wasadministeredintothetwoseparatednostrils[1].ForIm immunization,atotalvolumeof100μlofeachformulation wasadministered.

2.5. Antibodyisotypeassay

Tendaysafterthelastboosterinjection,themicebloodsam- pleswereobtainedbyheartpunctureandretro-orbitalbleed- ing.The blood was allowed to coagulate at4 °C and then the serumwascollected bycentrifugationfor10 minat14 000rpm.Theserum sampleswerekeptat−20°C[27].The seraofvaccinatedBALB/cmicewereusedtotitratebothof IgG2aandIgG1antibodiesusingELISAtechnique[28].Briefly, 96-wellplateswerecoatedwith0.5μg/50μlPR8antigeninbi- carbonatebuffer(pH9.6)andincubatedovernightat4°C.After washingtheplates,theywereblockedwith300μlof2.5%BSA inPBS-tweenperwellfor1hat37°C.Differentdilutionsof serumwereaddedtotheplatesfor75minat37°C.Afterwash- ing withPBS–tweensolution,plates weretreatedwithIgG1 andIgG2asecondaryantibodiesbasedonthemanufacturer’s instructions(ZymedInc.,USA).Opticaldensitywasmeasured usingamicroplatereader(StatFax® 4200microplatereader, NEOGEN® Corporation,USA)at450nmandareferencewave- lengthof630nm.

2.6. Statisticalanalysis

GraphPadPrismversion 6wasusedtoperformthestatisti- calanalysis.Also,two-wayanalysisofvariance(ANOVA)and Tukey’smultiplecomparisontestwereusedtoanalyzetheob- taineddata.Datawereshowedasmean±standarddeviation (SD).

3. Results and discussion

3.1. CharacterizationofNPs

Inthepresentstudy,NPswerepreparedbyasimpleincubation methodinwhichthedifferentcomponentsweregentlymixed toeach other [1]. Asaresult,the coating ofNPs withALG significantlyincreasedtheparticlesizetomorethan100nm thatsuggestedthepresenceofanALGcoatinglayer.Also,ALG hasanegativecharge,thusresultinginsignificantdecreasein zetapotentialfortheALG-coatedNPsascomparedwithnon- coatedNPs.ThecharacteristicfeaturesofobtainedNPswere summarizedinTable1.Inourpreviousstudy,TheTMCand CHTNPsloadedwithhepatitisBsurfaceantigen(HB)showed morepositivelychargedof14.6and13.9mV,respectively[1].

Additionally,Fig.1showsthattheseNPshavegreatstability andnosignificantdifferenceswerefoundinparticlesize,PDI aswellaszetapotentialduringtheperiodof30d[20].How- ever,thenegativeresultsofPR8-CHT-ALGformulationcould beattributedtotheagglomerationoftheseNPs duringthe preparationprocess.Totally,thepreparedNPswiththissimple andscalablemethodshowedsuitablephysicochemicalprop- erties,stabilityandcouldbeusedinvivofortheirimmunoad- juvantpotential.

3.2. Antibodyresponse

TheadjuvantpotentialofpreparedNPswasinvestigatedby theimmunizationofBALB/cmice[1].Animalswere immu- nizedwithdifferentformulations,threetimeswithtwoweeks intervals.10 dafterthe last immunizations,the sera from thefreshlykilledmicewerecollected.Theseraofvaccinated BALB/cmicewereusedtotitrateIgG1andIgG2aantibodies usingELISAtechnique.Inthisstudy,Fig.2AandBshowsthat CHTorTMC-administratedPR8virus,asefficientimmunoad- juvants,could significantly increaseimmunological protec- tionagainstPR8wholevirusafterInadministration.Inour previousstudy,TMCandCHTNPsloadedwithhepatitisBsur- faceantigen (HB)showed similar potentialasmucosal im- munoadjuvantsforHB[1].Xieetal.showedthatHpylori-CHT particlesmanagedtocreateansignificantlyhigherimmuno- logicalprotectionin60%micecomparedwithHpyloriantigen alone(P<0.05).

Additionally,theamountsofIgG2aandIgG1secretionsare usuallyconsideredinfavorofTh1cellularimmuneresponse and Th2 humoral immune response, respectively [29]. Our resultsshowedthatthe preparedNPscouldcreateamixed Th1/Th2response[30].Forexample,Fig.1Ashowsthatsig- nificanthigherIgG1(P<0.001)andIgG2a(P<0.01)antibody titersinsera ofmiceimmunizedwithPR8-CHTNPs might beconsideredasanindicatorofamixedTh1/Th2profile.In contrast,theALG-coatedPR8-TMCNPs(PR8-TMC-ALG)gener- atedasignificantlyhigherIgG2aantibodytiterthanthePR8- CHT-ALGones(P<0.001)thatindicateaTh2profileforthese NPs.Accordingtoourresults,theALGcoatingcouldincrease theIgG2aantibodytiter,however,thenegativeresultsonPR8- CHT-ALGNPs(P<0.001)couldbeattributedtotheagglom- erationofthisNPduringthepreparationprocessandsubse- quentlyloweruptakeofthisformulationbyMcellsandAPCs.

Pleasecitethisarticleas:JafarMosaferetal.,Preparation,characterizationandinvivoevaluationofalginate-coatedchitosanand

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Table1– Zetapotential,particlesize,PDIofNPs.

Formulations Ratio Z-averagemean Intensitymean Volumemean Numbermean PDI Zetapotentials

diameter(nm) diameter(nm) diameter(nm) diameter(nm) (mV)

PR8antigen – 284.3 284.3 284.3 284.3 0.41 −9.3

PR8-CHT 1:4 380.6 633.9 977.2 234.7 0.43 +5.9

PR8-TMC 1:4 318.2 607.9 1205.8 194.8 0.39 +7.0

PR8-CHT-ALG 1:4:6 471.1 439.8 371.9 239.5 0.66 −32.8

PR8-TMC-ALG 1:4:6 453.2 247.2 248.5 268.9 0.56 −29.6

Fig.1– StabilityofpreparedNPsinPBbuffer(pH6).Theparticlesize(A),PDI(B)aswellaszetapotential(C)ofNPswere measured,each5d,duringtheperiodof30din4°C.Datarepresentedasmean±SD(n=3).

Ajdaryetal.showedthatthebacillecalmette-guerin(BCG)en- capsulatedinALGmicrospheresinducedequalorbetterTh1 immuneresponsesthanstandardBCGvaccinationbyoralad- ministration[31].

Italsohasbeenshownthatinfluenzaantigensarepotent inducersofIgG2aantibodyresponsesinmiceandresultsina Th1-typeimmuneresponsethatisgenerallyconsideredasa criticalfactorforproducingavarietyofprophylacticandther- apeuticvaccines,especiallythosearetopreventortreatvi- Please citethisarticleas:JafarMosaferetal.,Preparation,characterizationandinvivoevaluationofalginate-coatedchitosanand

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Fig.2– Thelevelofanti-PR8specificIgG1andIgG2a(A);andtheratioofIgG2a/IgG1antibodytiters(B)ofBALB/cmice immunizedbyInandImroutes,10dafterthelastboosterinjectionwithdifferentformulations.Theassayswereperformed usinganELISAmethodintriplicateatdifferentdilutionsofserumsamples.SignificantdifferencesbetweentheIgG1titers ofALG-coatedNPs(In)andnone-coatedoneswasmarkedas^∗(P<0.05)and^∗∗∗(P<0.001).SignificantdifferenceofIgG2a titersbetweenALG-coatedNPs(In)andnone-coatedoneswaslabeledwith###(P<0.001).Datarepresentedasmean±SD (n=6).

ral infectionsandintracellularpathogens[32,33].Therefore, itisbeneficialtoelicitahigherratioofIgG2a/IgG1antibody titerforimmunizationagainstinfluenza virus.Theratio of IgG2a/IgG1antibodytiterisusuallyconsideredasanindica- torofThprofile[34].WhentheIgG2a/IgG1ratioishigher,itis infavorofTh1immuneresponse[35].Fig.2Bshowsthatthe IgG2a/IgG1ratiowas significantlyhigherinintranasallyad- ministeredPR8-TMC-ALGNPsthanthoseinducedwithother formulations (P< 0.001).Thisnotableachievement showed thatTMC-ALGadjuvantedPR8vaccinesarepotentinducerof Th1immuneresponsesafterIndelivery.

4. Conclusion

It is concluded that the CHT and TMC NPs are efficient immunoadjuvants for immunizationagainstPR8 whole influenzavirus.Thiseffectwasprovedafterimmunizationby theInroute.Generally,thePR8-TMCNPsinducedlessimmune responsescomparedwithPR8-CHTNPsafterInadministra- tion.However,theALG-coatedNPs could generatesuperior immuneresponsetothenon-coatedones,especiallyinthe PR8-TMC-ALGNPs.

Conflicts of interest

Theauthorsdeclarethatthereisnoconflictsofinterest.

Acknowledgments

The data presented are part of Amir-Hossein Sabbaghi Pharm.D.thesis (Grant number: 911042) supportedby Vice ChancellorforResearch,MashhadUniversityofMedicalSci- ences.

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Please citethisarticleas:JafarMosaferetal.,Preparation,characterizationandinvivoevaluationofalginate-coatedchitosanand