Antivenom Production against Bothrops jararaca and Bothrops erythromelas Snake Venoms Using ...

15 Pages·2017·15.17 MB·English

toxins
Article
Antivenom Production against Bothrops jararaca and
Bothrops erythromelas Snake Venoms Using
Cross-Linked Chitosan Nanoparticles as
an Immunoadjuvant
KarlaSamaraRochaSoares1,FiammaGláucia-Silva1,AlessandraDaniele-Silva1,
ManoelaTorres-Rêgo1 ID,NatháliaKellydeAraújo1,YamaraArrudaSilvadeMenezes1 ID,
IgorZumbaDamasceno2,DeniseVilarinhoTambourgi3 ID,ArnóbioAntôniodaSilva-Júnior1,*
andMatheusdeFreitasFernandes-Pedrosa1,* ID
1 DepartmentofPharmaceuticalSciences,FacultyofPharmacy,FederalUniversityofRioGrandedoNorte,
Natal59012-570,Brazil;[email protected](K.S.R.S.);fi[email protected](F.G.-S.);
[email protected](A.D.-S.);[email protected](M.T.-R.);
[email protected](N.K.d.A.);[email protected](Y.A.S.d.M.)
2 DepartmentofMaterialsEngineering,TechnologyCenter,UniversityCampus,FederalUniversityofRio
GrandedoNorte,Natal59078-970,Brazil;[email protected]
3 LaboratoryofImmunochemistry,InstitutoButantan,SãoPaulo05503-900,Brazil;
[email protected]
* Correspondence:[email protected](A.A.d.S.-J.);[email protected](M.d.F.F.-P.);
Tel.:[phone](A.A.d.S.-J.);[phone](M.d.F.F.-P.)
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:12March2018;Accepted:5April2018;Published:16April2018
Abstract:InBrazil,envenomationbysnakesofthegenusBothropsisclinicallyrelevant,particularlyfor
thespeciesBothropsjararacaandB.erythromelas. Themosteffectivetreatmentforenvenomationby
snakesistheadministrationofantivenomsassociatedwithadjuvants. Noveladjuvantsarerequired
toreducesideeffectsandmaximizetheefficiencyofconventionalserumandvaccineformulations.
Thepolymerchitosanhasbeenshowntohaveimmunoadjuvantproperties,andithasbeenused
as a platform for delivery systems. In this context, we evaluated the potential immunoadjuvant
propertiesofchitosannanoparticles(CNPs)loadedwithB.jararacaandB.erythromelasvenomsinthe
productionofseraagainstthesevenoms. StableCNPswereobtainedbyionicgelation,andmicewere
immunizedsubcutaneouslyfor6weekswith100µLofeachsnakevenomatconcentrationsof5.0or
10.0%(w/w),encapsulatedinCNPsorassociatedwithaluminiumhydroxide(AH).Theevaluationof
proteininteractionswiththeCNPsrevealedtheirabilitytoinduceantibodylevelsequivalenttothose
ofAH,evenwithsmallerdosesofantigen. Inaddition,theCNPswerelessinflammatoryduetotheir
modifiedreleaseofproteins. CNPsprovideapromisingapproachforpeptide/proteindeliveryfrom
snakevenomandwillbeusefulfornewvaccines.
Keywords: Bothropsvenoms;antivenom;adjuvants;nanoparticles;chitosan;nanovaccines
KeyContribution:WepresentabiotechnologicalapproachtoproduceantivenomagainstBothropsjararaca
and Bothrops erythromelas snake venoms. The potential immunoadjuvant properties of chitosan
nanoparticlesloadedwiththesevenomswereevaluated;theresultsshowthatthisapproachcanbe
appliedtoobtainpolyclonalserum.
Toxins2018,10,158;doi:10.3390/toxins10040158 www.mdpi.com/journal/toxins
Toxins2018,10,158 2of15
1. Introduction
Snakebiteenvenomingisahighlyrelevantglobalpublichealthissuethatisconsideredamajor
occupationalhealthproblemandhasbeenaperennialcauseofdeathorchronicdisabilityformany
active young people [1,2]. The global incidence of snakebites is approximately 5,400,000 per year,
andapproximately125,000ofthesecasesarefatal[1,3]. Snakebiteenvenomingisestimatedtooccur
mostlyinAsia,Africa,andLatinAmerica,wherebetween20,000and94,000fatalitiesarereported
eachyear[2–4]. Generally,snakebitesaremostcommonintropicalandsubtropicalareas,buttheyalso
occurinregionswithtemperateclimes[3]. InBrazil,accidentalsnakebiteenvenomingrepresentsan
importantpublichealthhazard. Ninetypercentoftheyearlyestimated20–30,000snakebiteaccidents
arecausedbyspeciesoftheBothropsgenus. B.jararaca(SouthandSoutheast),B.moojeni(Centre-West),
B.atrox(North),andB.erythromelas(Northeast)areresponsibleformostoftheseaccidents[5–10].
Systemic signs of envenomation are observed in patients bitten by Bothrops snakes, such as
haemostatic disturbances, signs at the site of bite, haemorrhage, myonecrosis, dermonecrosis,
andinflammatoryreactions(oedema,leukocyteinfiltration,andpain)[9,11–13]. Thepathogenesisof
Bothropsenvenomationiscomplex,butlocalmanifestationshavebeendirectlyassociatedwiththe
activityofmetalloproteases,phospholipaseA ,orserineproteasespresentinthevenom[11,12,14–16].
2
Parenteral administration of animal-derived antivenoms is the mainstay treatment for snakebite
envenoming[17–20]. InBrazil,thetreatmentforenvenomationbybothropicspeciesinvolvestheuse
ofanequinepolyspecificantibothropic(orantibothropic-lachetic)F(ab’)2antivenom, preparedby
conventionalimmunizationschedulesusingapoolthatincludesthevenomsoffiveBothropsspecies:
B.jararaca,B.jararacussu,B.moojeni,B.alternatus,andB.neuwiedi[10,17].
Theserumsroutinelyproducedaresafeandeffectivefortreatment. However,someproblems
havearisenrelatedtotheapplicationofimmunoadjuvantsforvaccinesandsera,suchasthetoxic
potentialandcollateraleffectsoftheseformulations[21–23]. Aluminium-containingadjuvantsare
approvedbytheUnitedStatesFoodandDrugAdministrationforhumanuseandarewidelyappliedin
immunobiologicalproduction[23]. Therearetwomainaluminiumcontainingadjuvants: aluminium
hydroxide(AH)andaluminiumphosphate[23].
Unfortunately,someevidenceaboutthetoxicityofaluminiumsaltshasbeenreported[24–27]. The
effectsofthesesaltsincludelatehypersensitivity[28,29],severegranulomatousinflammation[22,27,30]and
pruriticsubcutaneousnodules[28].Othersideeffectsofaluminiumincludeincreasedimmunoglobulin
Etitres,allergenicity,andpotentialneurotoxicity[22,30,31]. Normally,aluminiumisexcretedbythe
kidneys;however,undercertainconditions,suchasreducedrenalfunction,aluminiumaccumulates
inthebodyandcanbecometoxic[30].
Consequently,severalstudieshavefocusedonthedevelopmentofnewadjuvantsforhuman
vaccines to generate stronger vaccines capable of inducing protective and long-lasting immunity
in humans with reduced side effects and toxicity compared with conventional formulations [30].
Forexample, onestudyevaluatedthedevelopmentofanewcarrierforvaccinedeliverythatwas
aimedattreatingscorpionenvenoming[32]. Sustaineddrugreleasethroughnanocarriershasalready
beenintroducedinpreviousstudies[33]. Hydrophilicnanoparticleshavereceivedmuchattention
regardingtheirabilitytodelivertherapeuticpeptides,proteins,antigens,oligonucleotides,andgenes
byintravenous,oral,andmucosaladministrationroutes[34,35]. Forproteinincorporation,theideal
diameterofananoparticleshouldbebetween100and200nm[21,36,37]. Nanoparticlescanpotentially
circulateforalongtimeinthebody,releasingtheirloadsovermultiplehoursordays,dependingon
theirproperties[33,38,39]. Hence,proteindeliverythroughnanoparticlesisaneffectivewaytocontrol
drug release as well as to design an efficient protein delivery system [33]. In this context, several
polymersthathaveshowninterestingproperties,suchaschitosan,havebeeninvestigatedasboth
deliverysystemsandadjuvantsforvaccinepreparations[34,40–42].
Chitosan(CN)isanaturallyoccurringpolysaccharideobtainedbypartialN-deacetylationof
chitin.Itisusedasananocarrierbecauseofitsuniquebiodegradability,biocompatibility,hydrophilicity,
non-toxic,andimmunomodulatorypropertiesaswellasitslowcost[34,35,41,43–45]. CNhasbeen
Toxins2018,10,158 3of15
extensivelyinvestigatedforcarrieranddeliverysystemformulationsfortherapeuticmacrosolutes.
Particularly,geneandproteinmoleculeformulationshavebeenexploredduetothepositivecharge
ofchitosan,whichcanbeeasilycomplexedwithnegativelychargedDNAsandproteins[21,46,47].
ChitosancaneffectivelybindDNAandproteins,protectingthesemoleculesfromnucleaseandprotease
degradation,respectively[47].
Effectiveimmuneresponseshavebeenobservedwhenchitosanwasusedasanimmunoadjuvant
invaccinesforimmunizationagainstHelicobacterpylori[48],diphtheria[49],hepatitisB[50],T.serrulatus
venom[21],andNajanajaoxianasnakevenom[36];moreover,CNhasrecentlybeenappliedingene
therapystrategiesforthecontrolofAedesaegyptimosquitoproliferation[43].
Amoredetailedapproachtotheuseofthisnon-toxicandnon-inflammatoryimmunoadjuvant
asadeliverysystemwouldprovideapowerfulpublichealthtoolaswellasaninnovativeapproach
for the development of a new serum against toxins from the B. jararaca and B. erythromelas snake
venoms. This system could generate an effective immune response with low loads of antigens,
thusminimizingtheadverseeffectscausedbytraditionalimmunoadjuvants. Thisstudyevaluated
thepotentialimmunoadjuvanteffectsofchitosannanoparticles(CNPs)loadedwithB.jararacaand
B.erythromelasvenomsintheproductionofserumagainstthesevenoms,andcomparativelyassessed
theinflammatoryprocessgeneratedbyCNPsversusaconventionaladjuvant.
2. Results
2.1. PreparationofChitosanNanoparticles
Titrations of a tripolyphosphate (TPP) solution into a chitosan solution formed cross-linked
chitosannanoparticlesspontaneouslythroughintra-andintermolecularbondsbetweenthephosphate
groupsofthepolyanionTPPandtheaminegroupsofchitosan[35,51,52]. Thechitosannanoparticles
weregeneratedviaanionicgelationtechniqueandhadanaveragesizeof167.5nm,azetapotential
(ZP)of+24.5mV,andapolydispersityindex(PdI)of≤0.3(Table1). TheZPandPdIvaluesofdifferent
sampleswerenotsignificantlydifferent.
Table1.Physicochemicalpropertiesofdifferentcross-linkednanoparticlescontainingB.jararaca(BJ)
andB.erythromelas(BE)venomsatconcentrationsof5,10,and15%.
Sample Size(nm) ZetaPotential(mV) PdI EncapsulationEfficiency(%)
CNPs 159.6±2.2 24.50±3.64 0.272±0.01 -
CNPs/BJ5 179.3±9.4** 23.03±3.09 0.188±0.08 76.7
CNPs/BJ10 174.7±5.0** 24.91±2.91 0.203±0.07 67.7
CNPs/BJ15 187.3±7.5*** 31.40±3.86 0.185±0.07 74.0
CNPs/BE5 189.4±1.0*** 20.71±2.93 0.305±0.05 97.2
CNPs/BE10 160.0±2.3 19.00±2.76 0.302±0.01 87.6
CNPs/BE15 200.3±5.6*** 27.21±1.50 0.328±0.05 88.1
Valuesarethemean±standarddeviation(S.D.),n=3;***p<0.001and**p<0.01forthevenom-groupcompared
totheCNPgroup.
2.2. ProteinLoadingEfficiencyoftheBothropsjararacaandBothropserythromelasVenoms
Thesnakevenomproteinswereloadedintothenanoparticlesusinganincorporationmethodin
whichtheproteinsweredissolvedintheTPPsolution. Theproteinsdidnotimpairtheformationof
thechitosannanoparticles,whichoccurredspontaneouslyduringthedropwiseadditionofTPPinto
thechitosansolution.
Thesuccessfulproductionofcross-linkednanoparticleswasconfirmedbytheiraverageparticle
size. Aslightincreaseinparticlesizeoccurredwiththeadditionofvenomsintothesystem,butasmall
sizeof200nmwasmaintained. TheCNPsloadedwithbothB.jararacaandB.erythromelasvenom
exhibitedsimilarbehaviour. Allformulationshadapolydispersityindexof≤0.3(withnosignificant
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differencesamongthesamples),andtheproteinloadingdidnotchangetheircationiccharacteristics
(Table1).
The nanoparticles demonstrated a great protein loading capacity with the ability to maintain
a particle size of approximately 200 nm for all of the different venom and chitosan ratios used.
Thedatashowedanencapsulationefficiencygreaterthan87%foralltestedformulationscontaining
B.erythromelasvenom. ForB.jararaca,theproteinencapsulationefficiencyreachedlevelsgreaterthan
67%(Table1). Electrophoresisassaysofprotein-freenanoparticlesandprotein-loadednanoparticles
wereusedtovisualizetheseproteinloadingevents.
The protein profiles of the B. jararaca and B. erythromelas venoms as well as the distinct
protein-loadedcross-linkedchitosannanoparticleswereanalysedbySDS-PAGEandareshownin
Figure1. TheB.jararaca(BJ)andB.erythromelas(BE)venomproteinfractionsweredistributedwithin
amolecularmassrangeofapproximately14to66.4kDa. Apoolofproteinswascharacterizedfor
mediumtolowmolecularweightproteins. Highlightingthebandsbetween55.5and42.7kDa,aswell
asthoseat14.3and6.5kDa,waspossibleinbothvenomsanalysed. Comparingtheelectrophoretic
profilesoftheprotein-freenanoparticles,thefreevenomproteins(BJandBE),andtheprotein-loaded
nanoparticles with the B. jararaca protein-loaded nanoparticles (CNPs/BJ 5 and CNPs/BJ 10) and
the B. erythromelas protein-loaded nanoparticles (CNPs/BE 5 and CNPs/BE 10) demonstrated the
abilityoftheCNPstobeananocarrierfortheexaminednegativelychargedproteins. Thebandsof
venomproteinswerenotdetectedinthenanoparticlesamples,thusconfirmingformationofvenom
protein–nanoparticlecomplexes(Figure1,lanesCNPs/BJ5,CNPs/BJ10,CNPs/BE5,CNPs/BE10
andCNPs). Theseresultsreinforcethehighprotein–nanoparticleloadingcapacityoftheCNPs.
Figure1. ElectrophoreticprofileofBothropsjararacavenomat10%(BJ),chitosannanoparticleswith
Bothropsjararacaat5%(CNPs/BJ5)and10%(CNPs/BJ10),Bothropserythromelasvenomat10%(BE),
chitosannanoparticleswithBothropserythromelasvenomat5%(CNPs/BE5)and10%(CNPs/BE10),
andCNPs.
Toxins2018,10,158 5of15
The morphology of the nanoparticles and protein-loaded nanoparticles was examined using
atomicforcemicroscopy(AFM)andscanningelectronmicroscopy(SEM).Theirsurfacesandaspectsare
showninFigure2;theypresentedasizeofapproximately200nmwithasphericalshape,homogeneous
aspect,roughsurface,andgreatencapsulationefficiency.
Figure2. 2Dand3DatomicforcemicroscopyandscanningelectronmicroscopyimagesofBothrops
protein-loadedchitosannanoparticleformulations.(A)Protein-freechitosannanoparticles,(B)Bothrops
jararacaprotein-loadedchitosannanoparticlesataconcentrationof10%and(C)Bothropserythromelas
protein-loadedchitosannanoparticlesataconcentrationof10%.
Fouriertransforminfrared(FT-IR)spectroscopyanalyseswereusedtoevaluatetheinteractions
betweenchitosanandthevenomproteins. Thisevaluationwasbasedonthefactthatthisinteraction
leadstofrequencyshiftsorabsorptionbandsplitting[21]. Figure3showsthespectrarecordedfor
theCNPsandprotein–proteinnanoparticlescontainingdistinctsnakevenomsloadedata10%w/w
concentration. IntheanalysisoftheCNPs,N-HbendingandC=Ostretchingofamidegroupswere
observedat1561and1649cm−1, respectively, indicatingpurechitosan[53]. Moreover, thehigher
intensityofthesebandsintheCNPs/BJandCNPs/BEisduetotheformationofnewamideinteractions
byionicgelation. ThisfactisfurthersupportedbytheremarkableP=Obandstretchingat1214cm−1
and the P-O-P stretching at 892 cm−1, which is characteristic of TPP [53,54]. These characteristic
intermolecularinteractionshavebeenestablishedinpreviousstudiesofionicgelationandaredue
tochitosanchaincross-linking[55,56]. Theprotein-loadednanoparticlesamplehadC=Obandsof
increasedintensityforthecarbonylgroupsofthesecondaryamides(1651cm−1)andprimaryamides
(1540cm−1)ofproteins.
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Figure3.InfraredspectroscopyofCNPs,chitosannanoparticlesloadedwithBothropsjararacavenom
protein (CNPs/BJ) and chitosan nanoparticles loaded with Bothrops erythromelas venom protein
(CNPs/BE).
2.3. StabilityAssay
The physical stability of the protein-loaded nanoparticles and protein-free nanoparticles was
assessed using samples stored at 4 ± 2 ◦C for 40 days (Figure 4). Particle size measurements,
which were taken every 5 days, revealed the incredible performance of the protein-loaded CNPs.
VenomloadingimprovedthestabilityoftheCNPs, asnanoparticleswithoutaddedvenombegan
to increase in size after the fifth day (*** p < 0.001). No differences were observed among the
protein-loadednanoparticleformulations.
Figure 4. Evaluation of the stability of cross-linked chitosan nanoparticle samples loaded with
BothropsjararacaorBothropserythromelasvenom(5%and10%w/w).***p<0.001comparedtothesize
oftheCNPsonday1.CNPs:protein-freechitosannanoparticles;CNPs/BE5:chitosannanoparticles
loadedwithB.erythromelasproteinataconcentrationof5%; CNPs/BE10: chitosannanoparticles
loadedwithB.erythromelasproteinataconcentrationof10%;CNPs/BJ5:chitosannanoparticlesloaded
withB.jararacaproteinataconcentrationof5%; CNPs/BJ10: chitosannanoparticlesloadedwith
B.jararacaproteinataconcentrationof10%.
Toxins2018,10,158 7of15
2.4. AntibodyTitres
Mice were immunized subcutaneously for 6 weeks with 100 µL of each snake venom at
concentrationsof5.0or10.0%(w/w),encapsulatedinCNPsorassociatedwithAH[21]. Oneweek
afterboostervaccination,serumwasobtainedviaocularplexusbloodcollection. Theserumsamples
weresubjectedtoserialdilutionswithastandarddiluent(PBS/0.1%w/wBSA),startingatadilution
of 1:6400 and continuing to 1:204,800, and the antibody titres were evaluated by enzyme-linked
immunosorbentassay.
Theantibodytitresweredetectedintheserumofmiceimmunizedwithvenomfrombothsnakes,
B.jararaca(Figure5A)andB.erythromelas(Figure5B),uptothe1:102,400dilution. Theseresultswere
statisticallyequaltothoseoftheAHimmunizedgroupsanddemonstratedthatthenanoparticles
canstimulaterefinedandhightitresofproductionevenatlowconcentrations. Similarresultswere
foundinexperimentsperformedwithcross-linkedchitosannanoparticlesloadedwithproteinfrom
thescorpionTityusserrulatus[21].
Figure 5. Evaluation of antibody titres from mice immunized subcutaneously for 6 weeks with
Bothropsjararaca(A)orBothropserythromelas(B)venomatconcentrationsof5.0or10.0%encapsulated
inCNPsorassociatedwithAH,asdeterminedbyenzyme-linkedimmunosorbentassays.
3. Discussion
Inthepresentstudy,cross-linkedchitosannanoparticlesweresuccessfullyproducedusingthe
establishedparametersoftheionicgelationtechnique. ChitosannanoparticlescontainingB.jararaca
orB.erythromelasvenomformedspontaneouslywhenasolutionofproteinandTPPwasaddedto
achitosansolution. Intheincorporationprocess,theproteinsareentrappedandembeddedintothe
chitosan–proteinmatrix,withsomeproteinmoleculesalsoadsorbingtotheparticlesurface[51,57].
Thisphenomenonexplainsthehighencapsulationefficiencyofnanoparticlesof<200nm,evenafter
theadditionofsnakevenom. Thisfactisfurthersupportedbytheintermolecularinteractionsofthe
carboxylgroupsfromproteinswiththefreeaminegroupsofchitosanobservedintheFT-IRspectra.
Thesespecificinteractionscanbemonitoredbytheenhancementoftheintensitiesofthesebandsin
thespectraofprotein-loadednanoparticles. Theionicgelationtechniquehasbeensuccessfullyapplied
toobtainedchitosannanoparticlesformedicalproposes,suchasanti-staphylococciagents[58]and
letrozoledelivery(anticancerdrug)[59].
Loadingproteinintonanoparticlespreservestheirphysicalstabilitybykeepingtheiraverage
diameter in the nanometre range and their particle size distribution uniform for parenteral
administration [60]. Their characteristics, including an average size of 167.5 nm, a zeta potential
of+24.5mV,andapolydispersityindexof≤0.3,areinaccordancewithotherchitosannanoparticles
obtained in other studies. Costa et al. (2017) reported an average size of 244 (±11.64) nm with
a polydispersity index of 0.358 and a zeta potential of +17.3 9 (±1.41) mV [58]. The FT-IR spectra
recordedforthechitosannanoparticlesareconsistentwithpreviouslypublisheddatabyourgroup[54].
Theseresultsconfirmthepresenceofpurechitosanaswellasnewamideinteractionsfromtheionic
gelation process. Costa et al. (2017) related that chitosan–TPP crosslinking is realized through
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interactionsbetweenTPPandtheaminogroupspresentinchitosan,whichcanbeconfirmedbythe
peakat1586cm−1[58]. AlterationsintheNH bendingvibrationbandofchitosanwerealsodescribed
2
byGomathiandcolleagues[59]. Inaddition,theexperimentaldatademonstratedthattheelectrostatic
interactionsofthevenomproteinswithchitosanalsoinducecrosslinking,whichisabarrierforrapid
proteinreleaseinthemedium.
The high encapsulation efficiency (greater than 87% for all tested B. erythromelas protein
concentrations) observed for the cross-linked chitosan nanoparticles can be attributed to venom
dissolution in the TTP solution. Thus, these venom proteins are fully trapped inside the polymer
nanomatrix at the instant of nanoparticle cross-linking. Furthermore, that Bothrops venom has
negatively charged peptides [9,61] can also explain the high loading efficiency. These results
demonstrate the substantial ability of the optimized nanoparticle formulation to carry negatively
charged proteins, maintaining colloidal stability, which is not common for particle-encapsulated
proteins. The 3D conformational structure of nanoparticles is complex and dependent on
environmental conditions, such as pH, ionic strength, electrostatic interactions and composition.
Thegreatabilityofthispolymericnanocarriertointeractwithnegativelychargedbiomoleculesisdue
tothecationiccharacterofchitosan[54,60,62].
AneffectivepolymericnanocarriershouldbeabletocondenseDNAorproteintoprotectagainst
denaturation [63]. This ability of cross-linked chitosan nanoparticles was confirmed for distinct
proteinsviathegelretardationassay. Thedatarevealedthatwhennanoparticle–proteincomplexes
formed,theproteinbandswerenolongervisible,indicatingentrapmentofthevenomproteininthe
polymericmatrix.
The increased physical stability of the CNPs induced by protein loading suggests a possible
perturbationoftheelectrostaticinteractionsamongthepositivelychargedgroupsofchitosanduring
theformationofnanoparticlesthatresultedinfurtherstericstabilization[47,51].
Asuitableantigen-presentingsystemisrequiredforvaccinesuccessandisdirectlyrelatedto
the adjuvant or vehicle used [48]. The treatment for envenomation by bothropic species includes
antiophidic serum administration. Although this approach has been efficient, studies must be
performedtoidentifyalesstoxicadjuvantthatisabletoinduceantibodytitresgreaterthanthose
producedbythealreadyestablishedapproach.
MicewereimmunizedwiththechitosannanoparticleorAHadjuvantaloneorinconjunction
withB.jararacaorB.erythromelasvenom. Intermsofimmuneprotection,thedatarevealedthatanimals
immunizedwithCNPscross-linkedwithB.erythromelasvenomatconcentrationsof5.0or10.0%were
statisticallyequalwhencomparedwiththeantibodytitresofgroupsvaccinatedwiththeadjuvant
AH and the venom at the same concentrations. For animals immunized with CNPs loaded with
B.erythromelasvenom,theantibodytitreswerenotsignificantlydifferentwhencomparedwiththose
oftheAHgroupsimmunizedwiththesamevenom. TheapplicationofCNPsasanimmunoadjuvant
invaccinescansupportanefficientimmuneresponseandmayleadtoantibodyproductionsimilarto
thatinducedbyaluminiumhydroxide. However,thispolymerdisplaystheadvantageofbeingless
inflammatoryornon-inflammatoryandprovidesmodifiedantigenrelease,whichpromotesgreater
antibodytitresinserumwiththeadministrationofasmalleramountofantigen.
Theexperimentaldatademonstratedthatchitosannanoparticlescaninduceantibodyproduction
equivalent to that of AH, reinforcing data previously published by our group [21]. Other studies
haveshownefficientimmunizationusingCNPs; forexample,thestrongadjuvanteffectofγ-PGA
and chitosan nanoparticles for toxin B from Clostridium difficile was reported [64]. Thus, chitosan
nanoparticlesprovideanefficientandsecureapproachforthedeliveryofpeptides/proteinsfrom
snakevenom.
Toxins2018,10,158 9of15
4. MaterialsandMethods
4.1. Materials
Chitosan(85%deacetylated;molecularweight,90–190kDa),AH,andTTPwerepurchasedfrom
Sigma-Aldrich® (Saint Louis, MO, USA). The B. jararaca and B. erythromelas venoms were kindly
suppliedbytheButantanInstituteinSãoPaulo. Thebicinchoninicacid(BCA)ProteinAssaykitswere
purchasedfromPierce(Woburn,MA,USA),andtheMouseIgGtotalELISAkitswerepurchasedfrom
eBioscience(SanDiego,CA,USA).Allotherreagentsandsolventsusedwereofanalyticalgrade.
4.2. Cross-LinkedChitosanNanoparticles
The cross-linked chitosan nanoparticles used for the incorporation of the B. jararaca and
B. erythromelas venoms were acquired using the ionic gelation technique. A solution of 0.1% w/v
TPPinwaterobtainedbyreverseosmosis(<1.3µScm−1)wasaddeddropwisetochitosan(0.1%w/v
in0.175%w/vaceticacid)undermagneticstirring. Afteranopalescentsuspensionspontaneously
formed,themixwasmaintainedunderagitationatroomtemperaturefor30min.
4.3. LoadingoftheB.jararacaandB.erythromelasVenomsintoCross-LinkedChitosanNanoparticles
To load protein into the chitosan nanoparticles, different amounts of proteins (B. jararaca or
B. erythromelas venoms) in different proportions (5, 10 and 15% w/w) were solubilized in the TPP
solution in a pre-mixing procedure under magnetic stirring (700 rpm) in a thermostatized bath at
20±2◦C[51]. ThesesolutionscontainingTPPandeachspecificproteinsampleweretitratedinto
thecopolymersolutionusingthesameoptimizedproceduredescribedabovetoobtaincross-linked
chitosannanoparticles. Protein-freenanoparticleswerealsoproducedtoevaluatetheeffectofprotein
loadingonthephysio-chemicalpropertiesoftheparticles. Allexperimentalprocedureswerecarried
outintriplicate,andthedataisexpressedasthemean±standarddeviation(SD).
4.4. Protein-LoadingEfficiencyAssay
Distinctsamplesofprotein-loaded(withtheB.jararacaandB.erythromelasvenoms)cross-linked
chitosan nanoparticles were carefully transferred to 1.5-mL centrifuge tubes and then centrifuged
at20,000×gfor30minat4◦C.Theproteinconcentrationofeachsupernatantwasanalysedusing
aBCAProteinAssayKitaccordingtothemanufacturer’sinstructions. Theencapsulationefficiency
(EE)wascalculatedusingEquation(1)[47,60]. Allanalyseswerecarriedoutintriplicate,andthedata
isexpressedasthemean±standarddeviation(SD).
(Total protein−supernatantprotein)
EE%= ×100 (1)
Total protein
4.5. Electrophoresis
Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)wasusedtodetermine
theproteinprofilesoftheB.jararacaandB.erythromelasvenomsandtheirresultantprotein-loaded
cross-linked chitosan nanoparticles. For this, a mini-gel electrophoresis system (Mini-Protean® II,
BIO-RAD,Hercules,CA,USA)wasused[65]. Themigrationofastandardproteinmixture(Gibco-BRL
LifeTechnologies,Gaithersburg,MD,USA)wasusedtodeterminetherelativemolecularmassesof
thetestproteins. TheobtainedgelswerestainedwithasolutionofCoomassieBrilliantBlueR-250[66].
4.6. PhysicochemicalPropertiesoftheNanoparticles
Scanning electronic microscopy images (SSX550, Shimadzu, Tokyo, Japan) and atomic force
microscopy(AFM,SPM-9700,Shimadzu,Tokyo,Japan)wereusedtoassesstheshapeandsurface
aspectsofthenanoparticles. FortheAFManalysis, onedropofeachnanoparticledispersionwas
Toxins2018,10,158 10of15
placed on a washed microscope slide and dried under a desiccator for 24 h prior to observation.
Themeasurementswereperformedatroomtemperatureincantilevernon-contactmode.
ThephysicochemicalpropertiesoftheCNPssuchastheirzetapotential(ZP)andpolydispersity
index(PdI)weredetermined. Thepotentialoftheslipping/shearplaneofacolloidparticlemoving
underanelectricfieldcorrespondstotheZP.Theelectricdoublelayer(EDL)ofelectrophoretically
mobileparticlesandthelayerofdispersantaroundthemshowdifferentpotential,whichistheZP[67].
ThePdIprovidesinformationaboutthehomogeneityoftheparticlesizedistributioninasample[68].
Particlesizemeasurementsofdistinctnanoparticleswereassessedat25◦Cusingacumulative
method of analysis in which the intensity of the light scattered (DLS) was measured in a particle
sizeanalyser(ZetaPlus-BrookhavenInstruments,NewYork,NY,USA)atawavelengthof659nm
witha90◦ detectionangle. Thecorrelationwasruninparallelmode,anddatawasanalysedusing
ZetaPlus®ParticleSizingversion3.95software. Zetapotential(ZP)measurementswereperformed
usingthesameequipmentbyapplyingafieldstrengthapproximately5.9Vcm−1. Fiverunswere
performedforeachsampletodeterminetheZPvaluewithPALSZetaPotentialAnalysersoftware
usingelectrophoreticmobilityaccordingtotheHelmholtz-Smoluchowskiequation. Thesampleswere
diluted1:100(v/v)withpurifiedwater. Allmeasurementswerecarriedoutintriplicate,andthedata
isexpressedasthemean±standarddeviation(SD)[21,54].
Fourier transformed infrared absorption spectra (FT-IR) were recorded using a Prestige 21
FT-IRspectrophotometer(Shimadzu,Tokyo,Japan). Spectraofcross-linkedchitosannanoparticles
were compared with protein-loaded cross-linked chitosan nanoparticles containing the B. jararaca
and B. erythromelas venoms. The samples were dried using a Centrivap Labconco speed vacuum
concentrator(KansasCity,MO,USA),mixedwithpotassiumbromide(KBr)inameltingpotandthen
compressedinhydraulicpress. TheweightratiobetweenthepowderandKBrwasapproximately
1:200w/w[21,54].
4.7. StabilityAssay
Samplesofprotein-loadednanoparticlescontainingtheB.jararacaandB.erythromelasvenoms
(5 and 10% w/w) were stored at 20 ± 2 ◦C for 40 days; every 5 days, the particle diameters were
assessedat25◦Cusingacumulativemethodofanalysisinwhichtheintensityofthelightscattered
(DLS)wasmeasuredinaparticlesizeanalyser(ZetaPlus-BrookhavenInstruments,NewYork,NY,
USA)atawavelengthof659nmwitha90◦ detectionangle. Thecorrelationwasruninparallelmode,
andthedatawasanalysedusingZetaPlus®ParticleSizingversion3.95software.
4.8. Animals
Male and female BALB/c mice (25–35 g, 6–8 weeks of age) were received from the “Animal
FacilityattheCenterforHealthSciences”attheFederalUniversityofRioGrandedoNorte(UFRN).
The animals were kept under controlled temperature (22 ± 2 ◦C) conditions with free access
to commercial feed and water ad libitum, and they spent at least one week in the experimental
room prior to the adaptation test. The animal care and experimental assays were performed in
accordancewiththeguidelinesestablishedforthecareoflaboratoryanimals(CommitteeforEthicsin
AnimalExperimentationattheFederalUniversityofRioGrandedoNorte,ProtocolNo: 003/2012;
18April2012).
4.9. Immunization
OnehundredmicrolitersoftheB.jararacaandB.erythromelasvenomsatdifferentconcentrations
(5or10%w/w)cross-linkedwiththeCNPsorcombinedwithAHwereusedtoimmunizetheanimals.
Immunizationwasperformedsixtimes,onceperweek,viathesubcutaneousadministrationofthetest
compoundsinthelumbarregion(bilaterally)[21]. Asacontrol,animalswereimmunizedwiththe
adjuvantsintheabsenceofeithervenom.