ContentslistsavailableatScienceDirect
Colloids
and
Surfaces
B:
Biointerfaces
j ou rn a l h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b
Protocols
Synergistically
enhanced
stability
of
laccase
immobilized
on
synthesized
silver
nanoparticles
with
water-soluble
polymers
M.N.M.
Cunha
a,
H.P.
Felgueiras
a,∗,
I.
Gouveia
b,
A.
Zille
aa2C2T,CentreforScienceandTextileTechnology,DepartmentofTextileEngineering,UniversityofMinho,CampusofAzurém,4800-058Guimarães,
Portugal
bUniversityofBeiraInterior,FibEnTech,FiberMaterialsandEnvironmentalTechnologies-R&DUnit,Covilhã,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received20October2016
Receivedinrevisedform6March2017 Accepted9March2017
Availableonline12March2017 Keywords: Silvernanoparticles Polymerstabilizer Laccase Synergisticeffect Enzymaticstability
a
b
s
t
r
a
c
t
Silvernanoparticles(AgNPs)weresynthesizedbycitratereductionmethodinthepresenceofpolymers, poly(ethyleneglycol)(PEG),poly(vinylalcohol)(PVA)andchitosan,usedasstabilizingagents,andan oxidoreductaseenzyme,laccase(Lac),withthegoalofexpandingtheNPsantimicrobialaction.
AgNPswerecharacterizedbyUV–visspectrometry,dynamiclightscatteringandtransmissionelectron microscopy.Asprotectingagents,PEGandPVApromotedtheformationofsphericaluniformly-shaped, small-sized,monodispersedAgNPs(≈20nm).HighMwpolymerswereestablishedasmosteffectivein producingsmall-sizedNPs.Chitosan’sviscosityledtotheformationofaggregates.Despitethedecreasein Lacactivityregisteredforthehybridformulation,AgNPs-polymer-Lac,asignificantaugmentinstability overtime(upto13days,at50◦C)wasobserved.Thisnovelformulationdisplaysimprovedsynergistic
performanceoverAgNPs-Lacorpolymer-Lacconjugates,sinceintheformertheLacactivitybecomes residualattheendof3days.Byenablingmanyionicinteractions,chitosanrestrictedthemasstransfer betweenLacandsubstrateand,thus,inhibitedtheenzymaticactivity.
ThesehybridnanocompositesmadeupofinorganicNPs,organicpolymersandimmobilized antimicro-bialoxidoreductiveenzymesrepresentanewclassofmaterialswithimprovedsynergisticperformance. Moreover,theLacandtheAgNPsdifferentantimicrobialaction,bothintimeandmechanism,mayalso constituteanewalternativetoreducetheprobabilityofdevelopingresistance-associatedmutations.
©2017ElsevierB.V.Allrightsreserved.
1. Introduction
Silvernanoparticles(AgNPs)displayuniqueoptical,electrical and thermal properties, makingthem exceptional agents tobe incorporatedintoproductsofelectrical,textile,cosmetic, biolog-icalorevenmedicaluses[1–3].Sincethe20thcentury,AgNPshave
beenusedinthebiomedicalfieldasantimicrobialagents[4].AgNPs acteffectivelyinthemicroorganismmetabolismbyattachingtothe cellmembraneanddrasticallydisruptingitsproperfunctions,such aspermeabilityandrespiration.Recentstudiessuggesttheprimary mechanismofantibacterialactionofAgNPstobetherelease of silverions(Ag+)[5].Ag+ ionscaninteractwiththethiolgroups
ofvitalenzymesinactivatingthemand,ultimately,affectingthe abilityofthecell’sDNAtoreplicate[6].AgNPspossess remark-ableantisepticproperties,broadrangeofactivityagainstbacteria (gram-negativeandgram-positive),fungiandvirus,are
bactero-∗ Correspondingauthor.
E-mailaddress:helena.felgueiras@2c2t.uminho.pt(H.P.Felgueiras).
static(growthinhibition)andbactericidal,arebiocompatibleand, atlowconcentrations,areharmlesstothehumancells[3,7].In com-parisontootherantibiotics,bacterialresistanttoAgNPshasbeen observedonlyrarelyanddoesnotconstituteacomplication[4].In addition,severalnewandcheapermethodstoproduceAgNPshave beendevelopedinthelastyears,whichisofgreatimportancefor largescaleproduction[3,7].
TheAgNPsantimicrobialnatureisdescribedassize-and shape-dependent.IthasbeenestablishedthatsmallerNPsdisplaybetter antimicrobialactivityastheycanpenetrateeasilythecell mem-brane and reach the nuclear content [6,8]. Concerning shape, althoughtruncated-triangular-shapedNPshavebeenreportedas themostbactericidal[9],sphericalNPsremainthebestsuitedfor practicalapplicationsineithercolloidalformorimmobilizedstate [10,11].Manymethods,chemical,physical,photochemicaland bio-logical,havebeenusedtosynthesizeAgNPswithcontrolledsize, shape,distributionandstability[12].Chemicalmethodsare per-hapsthemostcommonlyused,astheyprovideasimplewayto synthesizeAgNPsinsolution.Generally,thechemicalsynthesisof AgNPsrequiresthreemaincomponents:ametalprecursor,a reduc-http://dx.doi.org/10.1016/j.colsurfb.2017.03.023
ingagentandastabilizingagent[3].Thereducingagentreduces Ag+andpromotestheformationofmetallicsilver(Ag0).Thisis
fol-lowedbyagglomerationintooligomericclustersthateventually generatecolloidalAgNPs.TostabilizethedispersiveNPs,prevent aggregationorbinding,sedimentationand lossofsurface prop-erties,astabilizingagentisaddedduringAgNPsproduction[12]. Formanyyears,organicandinorganiccompounds,likecitratesor surfactants,wereusedtostabilizethedispersivenatureofAgNPs. However,problemswithcost,scalability,controlofparticlesize, sizedistributionand/orparticleaggregationwereverydifficultto solve[13].Recently,polymerssuchaspoly(ethyleneglycol)(PEG) orpoly(vinylalcohol)(PVA)havebeenrecognizedaseffective sta-bilizingagentsbyimplementingaprotectionmechanismbasedon stericrepulsion.Abalanceisestablishedbetweenattractiveand repulsiveforcesthatdependonthepolymer’schainlengthand adsorptionmode[4,14,15].ThesepolymersgovernnotonlytheNPs sizebutalsotheirsurfacechargeandbiologicalresponses, includ-ingadsorption,distribution,metabolism,andexcretionoftheNPs [12,16].Naturaloriginpolymerslikechitosan,whichexhibit bet-terbiocompatibilitythanthesyntheticreferred,havebeenusedas well[17,18].
Inthepresentstudy,thestabilityofAgNPsproducedby chem-icalreductionmethodusingPEG,PVAandchitosanasstabilizers wasfollowed.Thepolymerswereappliedatdifferentmolecular weights(Mw)andconcentrations,andtheirinfluenceontheAgNPs size,shapeanddistribution(formationofaggregates)was estab-lished.StabilizingAgNPswithwatersolublepolymersisveryuseful toimprovethedispersionstabilityandantimicrobialperformance oftheNPs[19,20].Also,sinceAgNPsiscytotoxicandgenotoxic inhighconcentrations, itis important tofabricateantibacterial surfaceswithcontrolledrelease of Ag+.Systems prepared with
polymericmatricescanpreventthedirectcontactofAgNPswith theskinandallowapreciseandlocalreleaseofAg+[21].
To enhance AgNPs antimicrobial action and safety, an oxi-doreductaseenzyme,laccase(Lac),wasalsocombinedwiththe polymer-stabilizedAgNPs.Lacisa multicopperoxidaseenzyme withgreat, but littleexplored, potentialas a therapeuticagent in thefightagainstmicrobial organisms and biofilmformation, mainlyduetoitsuniquespecificity,highactivityandselectivity [22].Lacisknowntocatalyzereactionsthatleadtothe genera-tionofantimicrobialspeciesinthepresenceofmediators,oxyacids, phenols,iodine,andbromine[23,24].Directantimicrobial activ-ity ofcrude Lac hasbeenobserved and primarilyattributedto theelectrochemicalmodeofaction,which consistsin penetrat-ingthecellwallofthemicroorganisms,therebycausingleakage ofessentialmetabolitesandphysicallydisruptingothermicrobial keycellfunctions[25–27].Themajorlimitationsofusingenzymes astherapeuticagentsaretheinabilityofkeepingthemactive,their rapidelimination,lowconcentrationatsiteandavailability[28]. ImmobilizingLaconacarrierislikelytosolvealltheseproblems [29].TherehavebeenmanyreportsontheimmobilizationofLac ona varietyofsupportmaterials,includingalginatebeads[30], magnetic-chitosan[31],Amberlite[32],SiO2nanocarriers[33]and
variousmetallicNPs[34–36],towhichLacrevealedgreat stabil-ity.However,noneofthoseresearchesinferredabouttheindirect antimicrobialeffectofLacinpresenceofAgNPsanditspotentialto thebiomedicalfield.Infact,verylittleresearchhasbeenconducted onthissubject.Lateefetal.haspublishedoneofthefewstudiesin whichtheantimicrobialcapacitiesofLacimmobilizedontoAgNPs wereassessed[37].Theyreportedeffectiveinhibitionagainst clini-calisolatesofEscherichiacoli,PseudomonasaeruginosaandKlebsiella pneumoniae.Inapreviouswork,wehavealsoshowntheindirect antimicrobialeffectofLacimmobilizedontobacterial nanocellu-loseagainstgram-positive(92%)andgram-negative(26%)bacteria and itsacceptable cytotoxicityfor wound dressing applications [38].TheeffectivenessofLacasantimicrobialreliesontheoxidation
ofasubstratethatisfurthertransformedinanantimicrobialagent. Thus,anenhancedactivityandstabilityinanimmobilizedenzyme isoneofthemostimportantfeaturestobeattained.Theinnovation behindthisresearchfocusonthecombinationoftheLacenzyme withpolymer-stabilizedAgNPs,whichtotheauthors’knowledge hasnotyetbeenresearched.TheactivityatdifferentpHs, temper-aturesand substrates,andthestabilitywithtime ofthecreated complexwerefollowedwiththefinalgoalofachievingimprove performances.Inthiscontext,hybridnanocompositesmadeupof inorganicNPs,organicpolymersand immobilized antimicrobial enzymesrepresentanewclassofmaterials[39,40].Thesenovel materialscombinethe benefitsof enhanced antibacterial prop-erties of polymer-stabilized NPs, that allow a better-controlled release ofionsratherthanthebulk material,withthesafeand unspecificactionofenzybiotics,andthusreducingthepotential hazardinfluenceofAgNPsinthehumanbody[41,42].
2. Materialsandmethods
2.1. Materials
Silver nitrate(AgNO3,Sigma), trisodium citrate(Na3C6H5O7,
Sigma)and sodiumhydroxide(NaOH, Merck)wereusedin the production of the AgNPs. The synthesis of polymer-stabilized AgNPs wasperformed using PEG (Sigma), PVA (Sigma) or chi-tosan(Chitoclear®,Primex,Iceland)atdifferentmolecularweights (Mw): PEGat Mw 1500 and 10,000,PVA at Mw 9000, 13,000, 31,000and85,000,andchitosanat800Cps(∼320Mw)and1600 Cps (∼350Mw). A commercial Lac (18g protein L−1) obtained fromMyceliophthorathermophila(NS51003,Novozymes,Denmark, kindlysuppliedbyProfessorMoldes’researchgroupfrom Univer-sityof Vigo,Spain)wasused asprovided bythemanufacturer. Thisenzymewasselectedbecauseofitswell-established antimi-crobial activity[43].The activityofthe enzymeLacwastested against the substrates 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)(ABTS),3,4-dimethoxyphenol(DMP)andcatechol usingthebufferscitrate-phosphate(citricacid,C6H8O7 and
dis-odium phosphate, Na2HPO4·7H2O), phosphate (Na2HPO4·7H2O
and sodium phosphate monobasic, NaH2PO4) and
carbonate-bicarbonate (sodium carbonate anydrous, Na2CO3, and sodium
bicarbonate,NaHCO3),allpreparedwithreagentsfromSigma(the
detailedbuffers’compositionisprovidedinTableS1fromthe Sup-portinginformation).Allwaterusedwasdistilled(dH2O).
2.2. SynthesisofAgNPs:bareandpolymer-stabilized
AgNPscolloidal dispersion,atan approximate concentration of0.02mgL−1,wassynthesizedinlaboratorybyamodified step-wisemethodoftheconventionalcitratereductiontechnique(also knownasTurkevichmethod[44]),aspreviouslydescribed[45].The aboveconcentrationwasestablishedforcomparisonpurposeswith thecommerciallyavailableAgNPs(Sigma),whichconcentrationis 0.02mgL−1.Fortheentireprocess,thesolutionswerestirredat constantspeedandthepHadjustedwith0.1MofNaOH.
2.2.1. BareAgNPs
A100mLof1mMAgNO3washeatedtoboilingpointina250mL
flask.Tothissolution,10mLof1%Na3C6H5O7wereaddeddropwise
(3.8mMfinalconcentration).ThepHofthesolutionwasadjusted to7.7 withnitricacid(HNO3)orNaOH.Becauseofevaporation,
dH2Owasaddedtorestoretheinitialvolume.Thesolutionwas
heatedagaintoboilingtemperatureuntilthecolourchanged(pale yellow).Then,itwasremovedfromtheheaterandstirreduntil reachingroomtemperature(RT).TheAgNPswerestoredat4◦C
for12hpriortousetoreducethethermo-instabilityofthecitrate emulsion.
2.2.2. Polymer-stabilized:PVA,PEGandchitosan
1%,3%and5%m/vsolutionsofPVAandPEG,atdifferentMw, werepreparedin dH2O andheated at 70–80◦C for a complete
homogenization.Eachsolutionwascombinedat 90/10v/vratio witha10mMAgNO3solutionindH2O(pH8),toavolumeof100mL.
Themixturewasheateduntiltheboilingpointwasreached.10mL of1%m/vNa3C6H5O7wereaddeddrop-by-drop.Thesolutionwas
lefttoboiluntilthecolorchanged.After,theheatingwasturned-off butthestirringwaskeptuntilthesolutionreachedRT.
ThefirststepsoftheAgNPssynthesisinchitosan(deacetylation degreeof84.8±1.2%)wereequaltothosedescribedinthe pre-vioussub-chapter(BareAgNPs):1mMAgNO3washeatedand1%
Na3C6H5O7 wasaddeddropwise.Then,1.14mLofacetonewere
addedtothemixturetogetherwith0.5,0.75or1gof chitosan, repeatedforeachMw,toproducea0.5%,0.75%and1%chitosan contentintheAgNPssolution.Theheatingwaskeptuntilthe chi-tosanwascompletelydissolved.ThemixturewasstirreduntilRT wasreached.
2.3. Spectrophotometricmeasurements
UV–visspectraofAgNPssynthesizedinthepresenceorabsence ofpolymerswereacquiredusingaUnicanUV–vis2equipment. Aliquotswerecollectedfromeachmixtureatdifferenttimepoints totesttheAgNPsstability.Theyweredilutedat1:2,1:4or1:8when necessarytopreventdeviationfromBeer’sLawlinearity.
2.4. Dynamiclightscattering(DLS)andzetapotential measurements
The AgNPs size distribution, polydispersity index and zeta potentialweremeasuredbydynamic light scattering(DLS)and electrophoretic light scattering (ELS) using a Zeta Sizer-Nano (MalvernInstruments).Datawascollectedafter30scansata con-stanttemperatureof25±1◦C.Zetapotentialsweremeasuredin solutionatamoderateelectrolyticconcentration.Eachvaluewas obtainedbyaveragingmeasurementsofthreesamples.
2.5. Transmissionelectronmicroscopy(TEM)
TheAgNPsmorphologywasobservedusingaJEOLJEM1400 transmissionelectronmicroscope(TEM,Japan)operatingat120kV. NPssamplesweresubmittedtoglowdischarged carbon-coated coppergridsfollowedbynegativestainingwith1%(w/v)uranyl acetate.Thissolutionwasused toimproveAgNPscontrast and toeasilyvisualizeweakerelectroncontrastcompounds,suchas theusedpolymersandthedegradedortransformedAgspecies. ImagesweredigitallyrecordedusingaGatanSC1000OriusCCD (USA)digitalcamera.AverageNPscorediameter,sizedistributions andstandarddeviations(SD)werecalculatedforeachNPssample byapplyingImageJsoftwareanalysis(developedattheNational InstitutesofHealth,USA)tothecollectedTEMimages.
2.6. Lacimmobilizationonbareandpolymer-StabilizedAgNPs Bareorpolymer-stabilizedAgNPs,at0.02mgL−1,were immo-bilizedwithLacdiluted1:1000intheappropriatebuffertoafinal protein concentration of 18mg protein L−1, by simple immer-sionfor15minandRT,understirringconditions.Priortoenzyme activitymeasures the non-immobilizedLac wasremoved from solution by centrifugation for 5min at 10,000rpm (Eppendorf, Centrifuge5415D)and re-suspended inthe appropriatebuffer
(citrate-phosphateatpH3forABTSandpH6forDMP).The the-oreticalratiobetweenLacandNPswasestimatedas≈0.9gofLac permgofAgNPs.
2.7. Enzymaticactivity
Lacactivity,relativeandspecific,wasdeterminedby measur-ing the slope of the initial linear portion of the kinetic curve usingABTS,DMPandcatecholassubstratesatthetemperatureof 50◦C. Thereactionwasstartedwiththeadditionof1mLofthe enzymediluted1:1000 intheappropriate 0.2Mbuffer (citrate-phosphatepH3–6;phosphatepH7–8;carbonate-bicarbonatepH 9–11) combinedwith1mLof 0.5mM substratesolution(ABTS, DMPor catechol) in a quartz cuvette(final enzyme concentra-tionof9mgmL−1ina2mLvolume).Thespectrophotometerwas zeroedwithbuffer, theappropriate amountofpolymer(0,1,3, 5%)andthesubstratewithoutenzyme[46,47].Substrateoxidation wasmonitoredbymeasuringtheabsorbanceat420nmforABTS (=36,000M−1cm−1),468nmforDMP(=49,600M−1cm−1),and 450nmforcatechol(=2211M−1cm−1),attheappropriate dilu-tionfactor(1:30000ABTS,1:60000DMPand1:1000catechol).The catalyticactivitywasdeterminedbymeasuringtheslopeofthe initiallinearportionofthekineticcurve.Oneunit(U)ofenzyme activitywasdefinedas theamountofenzyme requiredto oxi-dize1molof usedsubstrate. Lacactivitieswere expressedin molmin−1mL−1 of enzymesolution. RelativeLacactivity was expressedastheratiobetweentheactivityatagiventime and theinitialactivity.Allactivitieswerereportedasaverage±SDof threeindependentmeasurements.
2.8. Enzymaticstability
Free Lac stability at different pHs (2–9, using different buffers:citrate-phosphatepH3–6;phosphatepH7–8; carbonate-bicarbonatepH9–11)andtemperatures(20–70◦Cmeasuredevery 10◦C)wasdeterminedusingABTS,DMPandcatecholassubstrates. Thermalstabilityinfunctionoftimeforthefreeenzymewas inves-tigatedbyincubatingLacinABTS(pH3),DMP(pH6)andcatechol (pH7)for60minat50◦C(pHandtemperaturepre-establishedas optimal).Lacthermalstabilityinfunctionoftimeinthepresenceof bareAgNPs,polymerorpolymer-stabilizedAgNPswasmeasured inDMP(pH6)at50◦Cfor13days.Thesolutionswerekeptinan ovenat50◦Candtheactivitymeasuredeachdayatthesame tem-perature.Enzymaticactivitywasmeasuredfollowingthemethod alreadydescribedinthepreviouschapter.Datawerereportedas average±SDofthreeindependentmeasurements.
3. Resultsanddiscussion
3.1. BareAgNPscharacterization
ThesizedistributionandthecolloidaldispersionoftheAgNPsis determinedbytherateofNPsnucleation,subsequentgrowthand aggregation.Fig.1AshowstheUV–visspectraofthebareAgNPs. Theverydistinctandbroadpeakdetectedat412nmputstherange ofNPssizesbetween30and80nmandsuggestsahigh polydis-persityoftheAgNPs[10,48].DLSspectra(Fig.S1intheSupporting information)corroboratethesedata.ItestimatestheaverageAgNPs sizeat≈54nmandthepolydispersityindexat0.3,indicatinga mul-timodaldistribution[4].Thezetapotential()oftheAgNPs,which givesasuggestionofthepotentialstabilityofthecolloid,was deter-minedat−30mVand,thus,consideredrelativelystable.Indeed, iftheparticles’valuesaremorepositivethan+30mVormore negativethan−30mVthecolloidsareconsideredstable[49].Also, itindicatestheAgNPsrelativestabilityinthedispersiontoresult
Fig.1.(A)UV–visspectraofAgNPscolloidalsuspensionwithdilutionfactorof8(D8x).(B)TEMmicrographoftheAgNPs(scale200nm).
fromtheelectricrepulsionofthenegativechargeofthecitrateions adsorbedontheNPssurface[50].
TheTEMimagefromFig.1Bshowstwofamiliesofspherical AgNPs,oneformedofsmall-sizedNPs,≈30nm(≈85%),andanother formedoflarger-sizedNPs,≈80nm(≈12%).Athird,verylittle fam-ilyofrod-shapedAgNPsranging910nm(≈3%)wasalsodetected. DLSspectra(Fig.S1in theSupportinginformation)corroborate thesedata.Theaveragediametercalculatedby TEMwas deter-minedat34.5±30.1nm,demonstratingthehighpolydispersityof thecolloidalAgNPsdispersion.Thesizedistributionofcolloidal dispersionofsolidparticlesisdeterminedbytheratiobetweenthe ratesofnucleationofthesolidcores,theirsubsequentgrowth,and aggregation.Asthereagentsinitialconcentrationswereelevated,a considerablenumberofnucleiwerequicklygenerated,consuming themetallicspecimensinthesystemandresultingintheformation ofsmallNPs,withrelativeuniformsizedistribution[51].Yet,awide distributionofparticlesizeandshapes,fromspheresandcubesto rodsandpolygonsarealsoobserved.Thisfeatureistypicalforsilver colloidspreparedbycitratereductionusingweakreducingagents, liketrisodiumcitrate[10,52].
3.2. Polymer-stabilizedAgNPscharacterization
ToobtainhighlystableNPs,withsmalldiametersand unifor-mityofshape, theAgNPswere synthesizedwithPEG,PVA and chitosanatdifferentMwandconcentrations.Thesepolymerswere usedasstabilizingagentsbecauseoftheirpolymerizationdegree andchainlength,whichisknowntoprotecttheNPsfrom aggre-gating[53,54].Thelevelof interferenceofthepolymersonthe AgNPsadsorptionspectrawastakenintoconsiderationbyusing therespectivePEGandPVAMwandconcentrationasbackground duringUV–vismeasurements.ChitosanwasexcludedfromUV–vis testingsinceitshighviscositymadethereadingunfeasible.
Adsorptionpeaksweredetectedat≈420nm,indicatingthe for-mationofAgNPsbothinthepresenceofPEGandPVA(Fig.2).Data showsaclearinterferenceofthepolymersonthepositionofthe peaks,which havebeen shiftedfromtheoriginal412nm(bare AgNPs),but moreimportantlyin theirintensity.Insomecases, namelyPVA Mw85,000,nopeaksweredetected.Thecomplete disappearanceoftheAgNPscharacteristicpeaksisanindicatorof thefullyaggregatedstateinwhichthePVA-AgNPscanbefound. EqualobservationsweremadebyKvíteketal.[4].TEMcapturesin Fig.3confirmthisstatement.
The DLS and of the polymer-stabilized AgNPs were not obtainedduetothehighviscosityandscatteringeffectofthe poly-mersolution,whichinterferedwiththereading.Instead,TEMwas usedtoanalysetheNPssizeandshape.Formostcases,the associ-ationoftheAgNPswithPEGorPVAreducedtheNPsheterogeneity
Table1
Averagediameter(±SD,n=numberofNPsin3images/condition)oftheAgNPs synthesizedwithPEGandPVAatdifferentMwandconcentrationof5%m/v.
Polymer AverageDiameter±SD(nm)
AgNPs(Control) 34.5±30.1 PEGMw1500 25.9±6.2 PEGMw10,000 21.5±5.1 PVAMw9000 21.6±4.6 PVAMw13,000 22.3±4.6 PVAMw31,000 18.8±2.7
bothinsizeandshape,asexpected[16].Thepolymerssurroundthe
NPs(lightgreyareainturnoftheNPsdarkerspot,veryclearinthe firstimageofFig.3),protectingandconferringtheNPstheability toberecognizedindividually.Fig.3showsmanyofthePEG-and PVA-AgNPstobemonodispersedandtodisplayaspherical-like morphology;rod-shapedNPsarehardlyobserved.Withinthese groups,theaveragesizeandparticularlythepolydispersitywere reducedwhencomparedtothebareAgNPs(Table1).Generally, highMwledtotheformationofmoreuniformlyshapedNPswith smallersizes.Ithasbeenshownthatpolymerswithlargernumber offunctionalgroupshavetheabilitytoincreasetheinteractions withAg+ duringnucleation.Asmorebindingsitesareprovided
forAg(0)atomstoaggregateduringreduction,astrongerphysical barrierofpolymericchainssurroundingtheAgNPsisgenerated. Thispreventsuncontrolledaggregationandleadstothe forma-tionofmonodispersedAgNPs[55].Contradictingthisnotionarethe resultsfromPVAMw85,000.Here,aggregateswereveryfrequent. AstheMwincreasessodoestheviscosityofthesolution.Asaresult, theAgNPsbecomeentrappedwithinthepolymericmatrixandare mostdifficulttoidentifyindividually.Thesamewasobservedfor chitosan800Cpsand1600Cps,alsoaveryviscouspolymer.
DifferencesinconcentrationbetweenequalMwpolymerswere neglectedfromthisanalysis,sincetheywerefoundnotsignificant. Onlythehighestconcentrationstestedweretakeninto considera-tion.
3.3. Lacactivityandstability
CharacterizingtheLackineticsasfunctionofpH,temperature andsubstrateprovidesimportantinformationabouttheenzyme stabilitywithtimeanditssaturationpoint,whicharecrucialto pre-dictitsbehavior.Lacwasdilutedinappropriatebuffersat1:30,000 forABTS,1:60,000forDMPand1:1000forcatecholsubstrates,and itsoptimalrelativeandspecificactivitywasestablished. Accord-ingtothecollecteddata(Fig.4),Lacdisplaysoptimalactivityat pH3inABTS,pH6inDMPandpH7incatechol.Itsspecific activ-itywasthehighestinABTSandthelowestincatechol[27].The
Fig.2.UV–visspectraofPEG-andPVA-AgNPswithrespectivedilutionfactor(Dnx)appliedperconcentration.
same wasobserved during temperature testing (Fig.5A). Once theoptimumpHforeachsubstratewasestablished,thekinetics oftheenzymewastestedagainstarangeoftemperatures,from 20◦Cto70◦C.Asexpected,atlowtemperatures(20◦C)the activ-itywastheslowestsincethereisabreakinthekineticenergyof themoleculesinthesystem[56].Theenzymaticactivityincreases rapidlyasthetemperaturerisesto≈50◦C.Afterthispoint,asthe
riskofdenaturationincreases,theenzymaticactivitydecreases sig-nificantly.Indeed,afteracertaintemperature,whichforLacisclose to60◦C[57],enzymeslosetheirnativestructureorconformational integrityandirreversiblelossesinfunctionarelikelytooccur[58]. Theoptimaltemperatureforthethreesubstrateswasestablished at50◦C.
ThestabilityoftheenzymeLacwasfollowedfor1hat50◦C, withdatabeingcollectedevery10min(Fig.5C).TheABTS sub-stratewastheonepromotingthehighestenzymaticactivityinthe veryfirstmoments.However,itsstabilitywascompromisedvery
quickly(10min),becominginactiveafter1h.Thishappensbecause LacoxidizesABTStoformABTS+andABTS++.Lacisquitestablein
thepresenceofABTS+;however,thedivalentcationsdestabilize
theenzymewithtimeastheygenerateconditionsunsuitedtoits performance[59].Althoughcatecholwasthesubstratewiththe higheststabilityafter1htesting,DMPwastheselectedsubstrate becauseofitssimilarlygoodstabilitywithtimeandhigherspecific activitycomparedtocatechol(Figs.4and5).
3.4. LacactivityandstabilityinDMPonpolymer-stabilized AgNPs
The processof immobilizing enzymesor proteinsonNPs is known to alter their conformation and ionization/dissociation states[60].Byusingpolymersasstabilizingagents,randomintra andintermolecularcross-linksaregeneratedwiththesemolecules providinggreatstabilityduringimmobilization[61].
Fig.3. TEMmicrographiesofthePEG-,PVA-andchitosan-AgNPsat5%concentration(scale100nmforPVAMw9000and200nmforallothersamples).
Fig.4.LacrelativeandspecificactivityasfunctionofpHinABTS,DMPandcatechol(SD,n=3).
Lacandpolymer-stabilizedAgNPswereincubatedat50◦Cand theenzymeactivitywasmeasureduntilitwasinexistentor resid-ual,using DMP assubstrate (Fig.6).The maximum incubation period was established at 13days, at which point most tested groups ceased theiractivity.Afterday 1,all polymer-stabilized AgNPsgroupsexhibitedspecificactivity,howeverlowerthanthe controls(Fig.6A).Thehighestactivitywasregisteredby1%PVA Mw9000andMw 31,000,3%PVA Mw13,000and5%PEGMw 1500. After 3days of incubations, Lac lost its activity in most polymer-stabilizedAgNPswithonly1%PVA85,000and3%PVAMw 9000registeringactivityabove100U/mL.Pastthispoint,the Lac-AgNPscontrolgroupbecameinactive,whilethefreeLacremained active untilday 9, however ata much lower level. Atthe end of6daysbothPVAMw9000and31,000losttheiractivity com-pletely,whichoccurredatday10forPEGMw10,000andchitosan 1600Cps.
In theory,it would be expectedPEG torepel Lacdue toits antifoulingnature.Yet,theinteractionwassuccessful.Recent stud-ieshaveshownthatPEGbindstoproteinsundercertainconditions [62].Protein-PEGinteractionsinsolutiondisplaya temperature-dependent behaviour, which becomes more favourable as the temperaturerisesabove37◦C.Thetemperatureusedto synthe-sizetheAgNPswas100◦CandalltheLacactivitymeasurements wereconductedat50◦C.PEGchainsegmentscanalsoadopt multi-pleconfigurationsanddifferentconformerswhileinteractingwith watermoleculesandNPs.Hence,PEGinteractionswithwater, pro-teinandNPscannotbereducedtoasimplepolymertheory,such astheexcludedvolume,sincethereisnotheoreticalmodelthat adequatelypredictsthebehaviourofPEG[63].
Aparticularkineticsofenzymaticactivitywasobservedwith PEGMw1500andMw10,000andPVAMw13,000andMw85,000: a slowdecrease in activitywasfollowedby a suddenincrease,
Fig.5.(A)Lacrelativeandspecificactivityasfunctionoftemperatureand(B)Lacstabilityasfunctionoftime,inABTS,DMPandcatechol(SD,n=3).
whichwasfinallysucceededbyanevenquickerdrop.This phe-nomenonisexplainedbythepolymerswater-solubleproperties. Asthepolymerlosesitsstabilityinwater,theenzyme immobi-lizeddetachesfromthepolymericmicelleand,consequently,an increaseinitsspecificactivityisdetectedbythesystem.However, becausetheenzymelosesitspolymericprotection,itsactivity can-notstandanddecaysveryquickly[64].Inmostgroups,atapolymer concentrationof5%,theLacactivityislost(orbecomesresidual) attheendof3days.Theamountofpolymerinteractingwiththe enzymepreventsitsaccessbytheDMPsubstrate.Lacrevealedgreat stabilityinthepresenceofPVAMw13,000,particularlyat1% con-centration,expressingactivityevenafter13days(upto18days, datanotshown).Formostpolymers,abalancebetweenthehighest andthelowestconcentration,3%,wasseentobethemosteffective inpromotingLacactivity.
Incontrastwiththeotherpolymers,chitosaninstigatedvery littleenzymaticactivity.Withtheexceptionof1%chitosanat800 Cps,therestwerebarelynoticed.Chitosanbeingapolyelectrolyte ofpositivechargeiscapableofdestabilizingtheenzymeactivity andsignificantlyinfluencetheimmobilizationprocess.Initslinear polyglucosaminechainsofhighMw,chitosanpossessesreactive aminoandhydroxylgroupsamenableofchemicalmodifications.Its singularalkalineproperties,whichconferapositivecharge,allow chitosantoefficiently interactwithpolyanioniccompoundslike proteins[65].Thus,byenablingmanyionicinteractions,chitosan restrictsthemasstransferbetweenthesubstrateandLac.Despite theclearinterferenceofchitosaninthestabilityandactivityofthe enzyme,therepulsionbetweentheAgNPsandthechitosanpositive chargesallowsforsomemobility.Thismayberesponsibleforthe residualactivitymanifestedafterday3inthepresenceofAgNPs butnotinitsabsence(Fig.6C).
In the absence of AgNPs (Fig. 6C), Lac activity only lasted 3days regardlessthe polymer in useand concentration tested.
Thekinetics ofactivitywas alsosimilar,starting highat day1 andcontinuouslydecreasinguntilday3.Theseresultsattestto theefficacyofthepolymer-stabilizedAgNPstopromoteLac activ-ityandstabilitythroughouttime.Datasuggeststhattheenzyme becomes protectedonce incontact withthepolymer-stabilized AgNPs,whichguaranteesitsstability.Lac-AgNPsorfreeLac (con-trols)showedlowstabilities [66,67].Althoughextrapolymeris presentinthesolutionandcaninteractwiththeNPsorenzyme, itsprotectionisnotaseffectiveaswhenthepolymerisconjugated withAgNPs.Thepolymersarenotcapableofstabilizingtheenzyme bynon-specificinteractions[68].Itisnecessarytheformationof astablehydrogelstructuretowhichtheenzymebindsstrongly, avoidingdiffusionintothesurroundingmedia,acquiring protec-tionfromdenaturingfactorsand,simultaneously,obtainingaccess totheactivesites,thuspotentiallyenhancingitsoperational stabil-ity[69].Underthesepremises,itislikelythePVAorPEGtoregulate notonlytheNPssizeandshapebutaswelltolimitthedeleterious effectoftheAg+releaseontheenzymestructure.Datafromthe
con-trolgroups(Fig.6A),showthatLacactivityonlylasts3daysinthe presenceofAgNPs,whileinitsabsence,freeLac,remainsinaction foranothersixdays,howeveratamuchlowerlevel(≈130U/mL), andonlyceasesatday9 (≈5.1U/mL).Theenhanced stabilityof AgNPsdispersionsprovidedbywater-solublepolymersisusually basedonthestericrepulsionof thepolymersthatare immedi-atelyadsorbedatthephaseinterphase.Thebalancebetweenthe attractiveandtherepulsiveforcesis stronglydependentonthe thicknessoftheadsorbedlayer,which,onitsturn,isfunctionof thechainlengthandadsorptionmodeoftheusedpolymer[70]. ThiscouldexplaintheincreaseinLacactivitymanifestedbythe hybridAgNPs-polymer-Lacwithtime.Thedecreaseindiameterof thepolymericcoatingsurroundingtheNPsmayfavorthe detach-mentanddiffusionoftheenzymeinsolution,untiltheprotection offeredistooweaktopreventtheAg+fromreleasingand
dena-Fig.6.LacactivityandstabilityinDMP(A)asafreeagentorassociatedwithAgNPs(controls),(B)onPEG-,PVA-andchitosan-AgNPsand(C)inthepresenceofonlythe polymersPEG,PVAandchitosan(withoutAgNPs)(SD,n=3).
Fig.6. (Continued)
turingtheenzyme[66].Thisbehaviornotonlyrevealsasynergistic effectontheLacstabilitybutalsoallowsatime-dependentactionof theantimicrobialsinwhich,firsttheLacandsecond,afterenzyme denaturation,theAg+acttoreducetheriskofresistance-associated
mutations.
AquickanalysisoftheLacactivityonpolymer-stabilizedAgNPs inthepresenceofABTS(pH3)substratewasalsoconducted(Fig.S2 intheSupportinginformation).Datacollectedrevealedverylittle activitythroughouttimeregardlessthepolymerusedasprotecting agent.ThedifferencesinoptimalpHbetweenABTSandthe pheno-licsubstrateDMPreflectthedifferenceinLacoxidationmechanism foreach substratethat mayaffectthestability of complex sys-temslikethosein study[71].Evenso,intheabsenceofAgNPs, LacdemonstratedgreateractivitythanwithDMP.Thisisexplained bythefactthattheoxidationof ABTStothestablecation
radi-caldoesnotinvolveprotons,andthuspossessesaredoxpotential independentofpH.Ontheotherhand,Lacisgreatlyaffectedby thedifferenceinpHduetothecontributionoftheOH−inhibition. AnotherexplanationcanbeattributedtotheenhancedAg+
solu-bilityandhighdegreeofaggregationoftheAgNPsinpresenceof dissolvedO2atlowpHvalues[72,73].
4. Conclusions
SynthesisofAgNPsbycitratereductionmethodusingpolymeric reducingagents,PEG,PVAandchitosan,resultedintunableAgNPs sizesandshapes.Asprotectingagents,PEGandPVApromotedthe formation ofspherical uniformly-shaped,small-sized, monodis-persedNPs.WiththeexceptionofPVAMw85,000,whichgenerated aggregates(highviscosity),highMwpolymerswereestablishedas
mosteffectiveinproducingsmall-sizedNPs.Chitosan’sviscosity impairedthisphenomenon.
Lacactivityandstability werestimulatedin thepresenceof DMP.AdecreaseintheenzymaticactivitywasdetectedonceLac wasimmobilizedonthePEG-andPVA-AgNPs.This,however,was promptlydisregardedasthepolymer-stabilizedAgNPsincreased theenzymestabilityovertimeandwerecapableofmaintainingits activityupto13days.Atthispoint,thepolymerslosttheirstability inwaterandtheenzymelostitsprotection.Athighpolymer con-centrations(5%),however,Lacactivitywaslostafter3dayssince thepolymerpreventedtheaccessbyDMP.Byenablingmanyionic interactions,chitosanrestrictedthemasstransferbetweenLacand substrateand,thus,promotedverylittleenzymaticactivity.
Generally,theefficacyofthepolymer-stabilizedAgNPsin pro-motingLacstabilitywithtimewasestablished.Byimplementing this strategy,thelimitations of usingboth AgNPsandenzymes as therapeutic agents in the fight against microbial organisms andbiofilmformationcouldbeovercome,andanew antimicro-bialsynergisticapproachwithpromisingbiomedicalapplications uncovered.Moreover,sincethereleaseoftheLacandAg+
antimi-crobialeffectsaredifferentintime,thishybridallowsasuperior antimicrobialeffectreducing theriskoftheappearanceofdrug resistance-associatedmutations.
Atthismoment,microbiologyexperimentsusinggram-positive andgram-negativebacteriaareatcoursewiththegoalof proof-ingthis concept. Differentmethods are beingusedtoestablish theantimicrobialsynergisticeffectofLac,polymerstabilizersand AgNPs, and its cytotoxic profile is being outlined recurring to humanfibroblasts.Inanearfuture,areportofthedataacquired willbepublished.
Competingfinancialinterests
Theauthorsdeclarenocompetingfinancialinterest.
Acknowledgements
Thisworkwasfunded byPortugueseFoundationforScience andTechnologyFCT/MCTES(PIDDAC)andco-financedbyEuropean funds(FEDER)throughthePT2020program,research projectM-ERA-NET/0006/2014.A.ZilleandH.P.Felgueirasalsoacknowledge fundingfromFCTwithinthescopeoftheproject POCI-01-0145-FEDER-007136andUID/CTM/00264.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb.2017.03. 023.
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