Jafar M osafer,Am ir-Hossein Sabbaghi,A li Badiee,Solm az Dehghan ,M ohsen Tafaghodi,?

a Research Cen ter ofAdvanced Technologies in M edicine,Torbat Heydariyeh University ofM edica l Sciences,Torbat Heydariyeh,Iran

b SchoolofPharm acy,Mashhad University ofM edical Sciences,Mashhad,Iran

c Student Research Comm ittee,Mashhad University ofMedical Sciences,Mashhad,Iran

d Nanotechnology Research Center,Pharm aceutical Technology Institute,Mashhad University ofM edical Sciences,Mashhad,Iran

Keywords:Ch itosan Trim ethy l ch itosan Alginate PR8 in f luen za virus Nasa l im m un ization

A B S T R A C TFo r eff icien tm ucosa l vaccine delivery,nanoparticu late an tigens are better taken by m ic rofo ld cells in the nasal associated lym phoid tissue and also dend ritic cells.Nanoparticles based on po lym ers such as ch itosan(CHT)and its w ater so lub le derivative,trim ethy lch itosan(TMC),cou ld be successfu lly used as carrier/ad juvan t for th is pu rpose.Sod ium a lginate,a negative ly charged biopo lym er,cou ld m od ify the im m unostim u latory p roperties of CHT and TMCNPs and in crease their stability.Sod ium alginate(ALG)-coated ch itosan(CHT)and trim ethy lch itosan(TMC)nanoparticles(NPs)loaded w ith inactivated PR8 in f luen za virusw ere successfu lly p repared by d irect coating of the virusw ith CHT or TMC po lym ers to evaluate their im m unoad juvan t poten tia la fter nasa l im m un ization.A fter nasa l im m un ization s in BALB/c m ice,PR8-CHT form u lation elicited h igher IgG2a and IgG1 an tibody titers com pared w ith PR8-TMC.ALG coating of th is fo rm u lation(PR8-CHT-ALG)sign if ican tly decreased the an tibody titers and a less im m une respon se w as in duced than PR8-TMC-ALG fo rm u lation.PR8-TMC-ALG fo rm u lation show ed sign if ican tly h igher IgG2a/IgG1 ratio,as criteria for Th1-type im m une response,com pared w ith PR8-CHT-ALG and PR8 virus a lone.A ltogether,the PR8-TMC-ALG fo rm u lation cou ld be con sidered as an eff icien t in tranasa lan tigen delivery system fo r nasa l vaccines.

1. Introduction

A lthough alum inum in virtually all cu rren tly ad juvan thum an vaccines is used to help in crease the hum ora l o r ce llu lar imm une responses to an an tigen,it usua lly induces a Th2 an tibody dom inated response.Accord ing ly,a lum inum-based vaccines are not su itab le fo r in tracellu lar pathogens,ch ron ic infection s o r can cers.There fo re,eno rm ous effo rts have been done to develop new ad juvan ts that a re ab le to induce bo th hum ora land cellu lar imm une responses.

In tram uscu lar(Im)o r subcu taneous(SC)are them ost comm on ly adm in istration rou tes of cu rren tly availab le vaccines that e licit robust and strong system ic im m une responses.Sin ce,these vaccines create pain upon in jection and are not ab le to e licit m ucosa l im m une responses,a desirab le noninvasive im m un ization is necessary[1-7].Am ong the noninvasive rou tes,nasa l adm in istration is of especia l in terest due to induction of m u cosa l imm un ity[8,9].Un fortunately,there are som e d raw backs associated w ith nasa l adm in istration that shou ld be considered in order to elicit an accep tab le m u cosa land system ic response.Fo r exam p le,them ucociliary c learan ce,the to lerogen ic natu re ofm ucosalep ithelium s and the large size of an tigen are som e vita l facto rs that lead to the decrease of the residen ce tim e of an tigen and its up take th rough nasa l ep ithe lium and even tua lly com p licate a robust im m une response.A com m on w ay to tack le these p rob lem s is to in corpo rate an tigens in to them ucoadhesive po lym eric NPs[10].Th is stra tegy p ro longs the residen ce tim e of an tigens and also p rotects against enzym es[11].Add itionally,nanoparticu lated an tigens are m o re taken by dend ritic ce lls(DCs)and cou ld cross the ep ithe lia l barrier m ore easily th rough a fam ous k ind of ce llsw h ich are ca lled m icrofo ld cells in the nasa l associated lym phoid tissue(NALT)[12,13].

Ch itosan(CHT)is a biodegradab le,biocom patib le and sa fe cation ic m ucoadhesive po lym er that is m ade by treating the ch itin shells of sh rim p w h ich num erous stud ies have show n its poten tialas an enhan cer po lym er for nasal vaccine de livery acrossm ucosa lsu rfaces[14-17].CHT can in crease the perm eability of the m ucosa l barrier possib ly th rough d isruption of in trace llu lar tigh t jun ctions.Sin ce,CHT has a lim ited aqueous solubility at alkaline or neu tral pH,a derivative of CHT,N,N,N-trim ethy l ch itosan(TMC)has been deve loped as a m u coadhesive po lym er for nasa l vaccination to overcom e the p rob lem,w h ich has an im p roved so lubility over a w ide range of pH[18,19].Sod ium a lginate(ALG)is a lso a negatively charged po lym er that cou ld be sim p ly coated around the positive ly charged CHT or TMC[20].ALG cou ld m od ify the imm unostim u lato ry p roperties of NPs,in crease their stability[21,22]as w ell as p reven t a bu rst release of loaded an tigens[23].

Hum an in f luenza A/Puerto Rico/8//1934(H1N 1)virus(PR8)w as them ost comm on cause ofhum an in f luenza at2009.That is the sub type of in f luenza A virus w ith a negative charge and cou ld be easily associa ted w ith positive ly charged po lym ers such as CHT o r TMC[24,25].

In th is study,w e investigate w hether PR8-loaded TMC or CHTNPs,w ith orw ithou t ALG coat,are su itab le an tigen carrier system s to enhan ce in vivo transm u cosa l an tigen de livery.

2. M ateria ls and methods

2.1. M aterials

IgG2a and IgG1 secondary an tibod ies w ere ob tained from Zym ed In c.(USA).Coating and detection m Ab an tibod ies fo r IFN-γand IL-4 asw e llas strep tavid in-HRPw ere obtained from M abtech(Sw eden).Con canava lin A and ALG(low m o lecu lar w eigh t)w ere pu rchased from Sigm a A lderich(USA).RPM I1640 cu ltu re m ed ium,pen icillin-strep tom ycin so lu tion and fetal ca lf serum(FCS)w ere pu rchased from Sigm a A ld rich(USA).CHT w as pu rchased from Fluka(USA).TMC w as syn thesized and characterized from abovem en tioned ch itosan as repo rted in ou r p revious stud ies[1].

BALB/c m ice and PR8 an tigen w ere ob tained from the Pasteu r Institu te of Iran.Experim en ts w ere perform ed in accordan ce w ith the gu idelines and regu lation s fo r the care and use of an im als im p lem en ted by the eth ics comm ittee of M ashhad Un iversity of M ed ica l Scien ces(App rova l number:IR.MUMS.REC.1392.23).A lso the an im a l stud ies w ere perform ed based on the Eu ropean Com m un ity gu ide lines as accep ted p rincip les for the use of experim en tal an im als.

2.2. Preparation ofPR8-CHT-ALG and PR8-TMC-ALG NPs

NPsw ere p repared w ith d ifferen tw eigh t ratio of various componen ts includ ing PR8 an tigen,CHT or TMC po lym ers and ALG coating for f ind ing the best resu lts in clud ing the sm a llest particle size and po lyd ispersity index(PDI)as w ell as the h ighest su rface charge(zeta po ten tia l).The 1:4:6(w/w/w)ratios of the PR8:CHT/TMC:ALG show ed the bestm en tioned physicochemica lp roperties and used fo r fu rther stud ies.First,the PR8-CHT and PR8-TMC NPs w ere p repa red by add ing PR8 so lu tion to CHT or TMC po lym ers(d ispersed in phosphate bu ffer(PB),pH 6)at the ratio of 1:4(w/w)and gen tly vortex-m ixed for abou t 5 s[1,9,26].Nex t,the fo rm u lations con tain ing(1:4)/6(w/w)ratios of PR8-CHT(o r TMC)/ALG w ere a lso p repared.A ll of the ob tained fo rm u lations w ere p repa red in PB and stored at 2-8°C.The f ina l PR8 con cen tration in the form u lations w as set as 15μg/dose/m ouse for in vivo adm in istration.

2.3. Characterization ofNPs

Dynam ic ligh t scattering analysis(NANO-Zetasizer,Malvern,UK)w as used to determ ine the zeta poten tia l,m ean partic le size and PDIof NPs.A lso,to eva luate the stability of NPs,each 5 d,the zeta po ten tia l,m ean particle size and PDIof d ifferen t form u lations(in PBbu ffer,pH 6)w ere evaluated at4°C for 30 d

2.4. In vivo vaccination protocol

The in vivo eva luation of im m unoad juvan t poten tia l of p repared NPs w as investigated in fem ale BALB/c m ice.Seven groups w ere im m un ized in tranasa lly(In)w ith:1.PBS so lution as a negative con tro l,2 and 3.PR8 an tigen(15μg/m ouse,given in tram uscu larly(Im)or in tranasa lly,4 and 5.PR8-CHT and PR8-TMC NPs(15μg PR8 an tigen+64μg CHT or TMC/m ouse)and 6 and 7.PR8-CHT-ALG and PR8-TMCALG NPs(15μg PR8 an tigen+64μg CHT or TMC+96μg ALG/m ouse).Six m ice in each group w ere in jected th ree tim es in 2-w eek in terva ls w ith these NPs.For nasa l im m un ization,an in traperitoneal in jection of xy lazine and ketam in(10 and 100μg/g body w eigh t,respective ly)w ere used to anesthetize the m ice.Fina lly,a to ta l vo lum e of 5μl of each fo rm u lation w as adm in istered in to the tw o separated nostrils[1].Fo r Im imm un ization,a total vo lum e of 100μl of each form u lation w as adm in istered.

Tab le 1-Ze ta po ten tia l,particle size,PDIof NPs.

2.5. An tibody isotype assay

Ten days a fter the last booster in jection,the m ice b lood samp les w ere obtained by heart pun ctu re and retro-orbitalb leeding.The b lood w as a llow ed to coagu late at 4°C and then the serum w as co llected by cen trifugation fo r 10 m in at 14 000 rpm.The serum sam p les w ere kep t at-20°C[27].The sera of vaccinated BALB/c m ice w ere used to titrate bo th of IgG2a and IgG1 an tibod ies using ELISA techn ique[28].Brie f ly,96-w ell p lates w ere coa ted w ith 0.5μg/50μl PR8 an tigen in bicarbonate bu ffer(pH 9.6)and in cubated overn igh tat4°C.A fter w ash ing the p lates,they w ere b locked w ith 300μl of 2.5%BSA in PBS-tw een per w ell fo r 1 h at 37°C.Differen t d ilu tion s of serum w ere added to the p lates fo r 75m in at 37°C.A fterw ashing w ith PBS-tw een so lu tion,p lates w ere treated w ith IgG1 and IgG2a secondary an tibod ies based on the m anu factu rer's instru ctions(Zym ed In c.,USA).Op tica ldensity w asm easu red using a m icrop late reader(StatFax?4200 m icrop late reader,NEOGEN?Corporation,USA)at 450 nm and a referencew avelength of 630 nm.

2.6. Statisticalanalysis

Graph Pad Prism version 6 w as used to perform the statistical analysis.A lso,tw o-w ay analysis of varian ce(ANOVA)and Tukey'sm u ltip le com parison testw ere used to ana lyze the obtained data.Data w ere show ed asm ean±standard deviation(SD).

3. Resu lts and d iscussion

3.1. Characterization ofNPs

In the p resen t study,NPsw ere p repared by a sim p le in cubation m ethod in w h ich the d ifferen t com ponen tsw ere gen tlym ixed to each other[1].As a resu lt,the coating of NPs w ith ALG sign if ican tly in creased the particle size to m o re than 100 nm that suggested the p resen ce of an ALG coating layer.A lso,ALG has a negative charge,thus resu lting in sign if ican t decrease in zeta poten tial for the ALG-coated NPs as com pared w ith noncoated NPs.The characteristic featu res of ob tained NPs w ere sum m arized in Tab le 1.In ou r p revious study,The TMC and CHT NPs loaded w ith hepatitis B su rface an tigen(HB)show ed m ore positively charged of 14.6 and 13.9m V,respectively[1].Add itiona lly,Fig.1 show s that these NPs have great stability and no sign if ican t d ifferen ces w ere found in particle size,PDI as w e ll as zeta poten tia l du ring the period of 30 d[20].However,the negative resu lts of PR8-CHT-ALG form u lation cou ld be attribu ted to the agg lom eration of these NPs du ring the p reparation p rocess.To ta lly,the p repared NPsw ith th is sim p le and sca lab le methodshow ed su itab le physicochem ica l p roperties,stability and cou ld be used in vivofo r their im m unoadjuvan t po ten tia l.

3.2. An tibody response

The ad juvan t poten tial of p repared NPs w as investigated by the im m un ization of BALB/c m ice[1].An im a ls w ere im m un ized w ith d ifferen t fo rm u lations,th ree tim esw ith tw o w eeks in terva ls.10 d a fter the last im m un iza tions,the sera from the fresh ly killed m ice w ere co llected.The sera of vaccinated BALB/c m ice w ere used to titrate IgG1 and IgG2a an tibod ies using ELISA techn ique.In th is study,Fig.2A and B show s that CHT or TMC-adm in istrated PR8 virus,as e ff icien t im m unoadjuvan ts,cou ld sign if ican tly increase imm uno logica l p rotection against PR8 w ho le virus a fter In adm in istration.In ou r p revious study,TMC and CHT NPs loaded w ith hepatitis B su rface an tigen(HB)show ed sim ilar po ten tia l as m ucosa l imm unoad juvan ts for HB[1].Xie et al.show ed that H py lori-CHT particlesm anaged to create an sign if ican tly h igher im m unologica lp ro tection in 60%m ice com pared w ith H py lo rian tigen a lone(P＜0.05).

Add itiona lly,the am oun ts of IgG2a and IgG1 secretions are usua lly considered in favo r of Th1 ce llu lar im m une response and Th2 hum ora l im m une response,respective ly[29].Ou r resu lts show ed that the p repared NPs cou ld create a m ixed Th1/Th2 response[30].Fo r exam p le,Fig.1A show s that sign if ican t h igher IgG1(P＜0.001)and IgG2a(P＜0.01)an tibody titers in sera of m ice im m un ized w ith PR8-CHT NPs m igh t be considered as an ind icator of a m ixed Th1/Th2 p rof ile.In con trast,the ALG-coated PR8-TMCNPs(PR8-TMC-ALG)generated a sign if ican tly h igher IgG2a an tibody titer than the PR8-CHT-ALG ones(P＜0.001)that ind icate a Th2 p rof ile fo r these NPs.Accord ing to ou r resu lts,the ALG coating cou ld increase the IgG2a an tibody titer,how ever,the negative resu lts on PR8-CHT-ALG NPs(P＜0.001)cou ld be attribu ted to the agg lomeration of th is NP du ring the p reparation p rocess and subsequen tly low er up take of th is form u lation by M cells and APCs.A jdary etal.show ed that the bacille calm ette-guerin(BCG)encapsu lated in ALGm icrospheres indu ced equa l or better Th1 im m une responses than standard BCG vaccination by o ra ladm in istration[31].

Fig.1-Stability of p repa red NPs in PB bu ffer(p H 6).The particle size(A),PDI(B)as w e ll as zeta po ten tia l(C)of NPs w ere m easu red,each 5 d,du ring the period of 30 d in 4°C.Data rep resen ted asm ean±SD(n=3).

Fig.2-The leve l of an ti-PR8 specif ic IgG1 and IgG2a(A);and the ratio of IgG2a/IgG1 an tibody titers(B)of BALB/c m ice im m un ized by In and Im rou tes,10d a fter the last booster in jection w ith d ifferen t fo rm u lation s.The assays w ere perfo rm ed using an ELISA m ethod in trip licate at d ifferen t d ilu tion s of serum sam p les.Sign if ican t d ifferen ces betw een the IgG1 titers of ALG-coated NPs(In)and none-coated ones w as m a rked as?(P＜0.05)and???(P＜0.001).Sign if ican t d ifferen ce of IgG2a titers betw een ALG-coated NPs(In)and none-coated ones w as labe led w ith###(P＜0.001).Data rep resen ted as m ean±SD(n=6).

It also has been show n that in f luenza an tigens are poten t inducers of IgG2a an tibody responses in m ice and resu lts in a Th1-type im m une respon se tha t is genera lly considered as a critica l facto r fo r p rodu cing a variety of p rophy lactic and therapeu tic vaccines,especia lly those are to p reven t o r treat vira l in fections and in tracellu lar pathogens[32,33].Therefo re,it is bene f icia l to e licit a h igher ratio of IgG2a/IgG1 an tibody titer for imm un ization against in f luenza virus.The ratio of IgG2a/IgG1 an tibody titer is usua lly considered as an ind icato r of Th p rof ile[34].W hen the IgG2a/IgG1 ratio is h igher,it is in favor of Th1 im m une response[35].Fig.2B show s that the IgG2a/IgG1 ratio w as sign if ican tly h igher in in tranasally adm in istered PR8-TMC-ALG NPs than those indu ced w ith o ther form u lation s(P＜0.001).Th is no tab le ach ievem en t show ed that TMC-ALG ad juvan ted PR8 vaccines are po ten t inducer of Th1 imm une responses after In delivery.

4. Con clu sion

It is con cluded that the CHT and TMC NPs are eff icien t imm unoad juvan ts for imm un ization against PR8 w ho le inf luenza virus.Th is e ffect w as p roved a fter im m un ization by the In rou te.Genera lly,the PR8-TMCNPs in du ced less im m une responses com pared w ith PR8-CHT NPs after In adm in istration.How ever,the ALG-coated NPs cou ld generate superior im m une respon se to the non-coated ones,especia lly in the PR8-TMC-ALG NPs.

Con f licts of in terest

The au thors declare that there is no con f licts of in terest.

Acknow ledgm en ts

The data p resen ted a re part of Am ir-Hossein Sabbagh i Pharm.D.thesis(Gran t num ber:911042)suppo rted by Vice Chan ce llor fo r Research,M ashhad Un iversity of Med ica l Scien ces.