Enhanced Antibacterial Activity of Silver Doped Titanium Dioxide-Chitosan Composites under ...

17 Pages·2017·7.17 MB·English

materials
Article
Enhanced Antibacterial Activity of Silver Doped
Titanium Dioxide-Chitosan Composites under
Visible Light
JieLi1,*,† ID,BingXie1,†,KaiXia1,YingchunLi1,JingHan2 ID andChunmaoZhao1
1 SchoolofMaterialsScienceandEngineering,NorthUniversityofChina,Taiyuan030051,China;
[email protected](B.X.);[email protected](K.X.);[email protected](Y.L.);
[email protected](C.Z.)
2 SchoolofMechatronicEngineering,NorthUniversityofChina,Taiyuan030051,China;[email protected]
* Correspondence:[email protected]
† Contributedequallyandshouldbetreatedastheco-firstauthor.
(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:2May2018;Accepted:7August2018;Published:10August2018
Abstract: Nano titanium dioxide (TiO ) with photocatalytic activity was firstly modified by
2
diethanolamine, and it was then doped with broad spectrum antibacterial silver (Ag) by in situ
method. Further, both Ag doped TiO -chitosan (STC) and TiO -chitosan (TC) composites were
2 2
preparedbytheinverseemulsioncross-linkingreaction.TheantibacterialactivitiesofSTCcomposites
werestudiedandtheirantibacterialmechanismsundervisiblelightwereinvestigated. Theresults
show that in situ doping and inverse emulsion method led to good dispersion of Ag and TiO
2
nanoparticlesonthecross-linkedchitosanmicrosphere. TheSTCwithregularparticlesizeof1–10µm
exhibitedexcellentantibacterialactivityagainstE.coli,P.aeruginosaandS.aureusundervisiblelight.
ItisbelievedthatSTCwithparticlesizeof1–10µmhaslargespecificsurfaceareatocontactwith
bacterialcellwall. Theincreasedantibacterialactivitywasattributedtotheenhancementofboth
electron-holeseparationsatthesurfaceofnano-TiO bythesilverionsunderthevisiblelight,andthe
2
synergeticandsustainedreleaseofstrongoxidizinghydroxylradicalsofnano-TiO ,togetherwith
2
silverionsagainstbacteria. Thus,STCcompositeshavegreatpotentialapplicationsasantibacterial
agentsinthewatertreatmentfield.
Keywords: cross-linkedchitosan;silver;titaniumdioxide;photocatalyticantibacterial
1. Introduction
Antibacterial compounds, including inorganic compounds and organic polymers, have been
appliedasthenewfunctionalmaterialinthefieldofwaterpurification.Amongthem,chitosan,becauseof
its good antibacterial activity, strong absorbing, sustained-release, environmental friendliness,
andbiocompatibility,appearstobeoneofthemostpromisingantibacterialagents[1,2].WhenthepHis
lowerthanitspK (6.5),theprotonatedaminogroups(NH +)attheC2positionintheglucosemonomer
a 3
ofchitosanchainsallowfortheformationofapolycationicstructure,whichcaninteractwithanionic
compoundsofbacteria[3].Theaminoandhydroxylgroupsonthemacromolecularbackbonemake
chitosanphysicallyandchemicallyreactwithvariouscompounds[4–6]. Forexample,chitosancan
reactwithterephthalaldehyde,formingacross-linkedchitosanhydrogeltomakecore-shellwallof
themicrocapsulesappliedindrugdeliverysystem. Themicrocapsuleiscomposedofacross-linked
chitosanhydrogelshellandanoilycorecontainingdrugmolecules[7,8].
Thecompositesofchitosanwithinorganicmaterials(suchastitaniumdioxide)[9,10]ormetallic
materials(suchasgoldandsilvernanoparticles)[11,12]canofferspecificproperties,suchasbroad
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Materials2018,11,1403 2of17
spectrum antibacterial activity [13] and photocatalytic activity [14]. For example, titania-chitosan
compositesshowedaphotocatalyticeffectonitsantibacterialperformance[15]. Thehydroxylradicals
produced by titania under UV light hindered the growth of bacteria [16]. However, TiO with a
2
wide band-gap can only exert its antibacterial properties under UV light [17–19]. Many methods
havebeeninvestigatedtoachievethevisiblelightactiveTiO photocatalyst. Themethodsfollowed
2
made visible light activity by the doping of TiO with transition metals [20]; doping of TiO with
2 2
non-metals [21]; and coupling of TiO with grapheme et al. [22]. It could extend the absorption
2
propertyinthevisibleregion.
Amongthem,transitionmetals,suchassilver(Ag)nanoparticles(NPs)dopedtitaniumdioxide
nanomaterialscanofferenhancedvisiblelightphotocatalysis[23–25]. Also,Aghasstrongantibacterial
activities against both Gram positive and Gram negative bacteria, such as Staphylococcus aureus
and Escherichia coli, which are common pathogens in water [26,27]. Moreover, the high specific
surface area of Ag NPs makes it easier to release silver ions (Ag+) and then kill the bacteria [28].
Therefore,itisexpectedthatsilverdopedtitaniumdioxide-chitosancompositeswouldobtainenhanced
antibacterialactivity.
However,agglomerationofnanoTiO andAgNPsduringthetraditionaldopingprocessresults
2
inlargerparticlesanddecreasedphotocatalyticantibacterialproperties[29]. Thus,itisnecessaryto
modifythesurfaceofnanotitaniumdioxidebeforeuse. Meanwhile,thepreparationofsilverdoped
titaniumdioxide-chitosancompositesbytheinverseemulsioncross-linkingreactionisalsocrucialfor
optimizingtheantibacterialactivities.
Inthisstudy,thesurfaceofnanoTiO wasmodifiedusingdiethanolamineanddopedwithAg
2
NPsbyinsitumethod. Ourgoaloftheintroducingbiocompatiblechitosanasacarrierwastomakeit
swellandreleasetheAg-TiO NPswithsynergisticantibacterialactivityinthewater. Theeffectsof
2
glutaraldehydeconcentrationandinverseemulsioncross-linkingreactiontimeontheparticlesizeof
theproductswerestudied. Theantibacterialactivityofthecompositeswithdifferentparticlesizes
was examined by plate colony-counting methods. Moreover, antibacterial properties of chitosan,
titania-chitosan (TC), and Ag doped TiO -chitosan (STC) composites were compared under both
2
visible light and dark conditions. Furthermore, the possible antibacterial mechanisms of the STC
compositeswereproposed. WhencomparedtoTC,enhancedantibacterialactivityofSTCmakesit
possibletobeusedasanantibacterialagentinthefieldofwatertreatment.
2. MaterialsandMethods
2.1. Materials
Chitosan polymer (deacetylation degree of 95%, viscosity is 100–200 mPa·s), titanium oxide
(TiO , particle size < 40 nm), Span-80 (Sorbitan Fatty Acid Ester), phosphate buffer saline
2
(PBS),andnutrientAgarwerepurchasedfromAladdinIndustrialCorporation(Shanghai, China).
Paraffin liquid, ether, and acetic acid glacial were obtained from Fangzheng chemical reagent
factory (Tianjin, China). Glutaraldehyde (50%) was obtained from Damao chemical reagent
factory (Tianjin, China). Silver nitrate was obtained from Tianjin Fengchuan Chemical Reagent
Technologies Co., Ltd (Tianjin, China). Sodium borohydride was obtained from Tianjin Kemiou
ChemicalReagentCo.,Ltd(Tianjin,China). GrampositivebacteriaS.aureus(Staphylococcusaureus,
CGMCC1.2465),GramnegativebacteriaE.coli(Escherichiacoli,CGMCC1.3373),andP.aeruginosa
(Pseudomonas aeruginosa, CGMCC 1.2620) were obtained from China General Microbiological
Culture Collection Center (Beijing, China). All of the reagents were used as received without any
furtherpurification.
2.2. SurfaceModificationofNanoTiO
2
There are a large number of active hydroxyl groups on the surface of nano TiO , resulting in
2
theeasyagglomerationofnano-scaleTiO inthesolventduetothestronginteractionofhydrogen
2
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bonds. Therefore,thenanoTiO surfacewasmodifiedtoreducetheinteractionofmolecules. Firstly,
Materials 2018, 11, x FOR PEER REVIEW2 3 of 16
2.5gnanoTiO wasdispersedin80mLethylalcoholtoobtainhomogeneoussuspensionthrough
2
fomre 1c0h amniicna, lasntidr rtihnegn, 0u.0lt9ramsLondicieatlhlya ndoilsapmerisneedw faosr a2d0d medint oatt hreoosmus pteemnspioenra,tsutirrer.e Fdinatalalyc,o tnhseta mntixsptuereed
sfuosrp1e0nsmioinn ,wanasd stuhcetniounl tfrilatseorendic aalnlyd dwisapsheersde dwfitohr e2t0hmyl ianlcaothroolo. mThtee mrepseurlatitnugr es.oFliidnsa lwlye,rteh edrmieidx tfuorre
ussues.p ensionwassuctionfilteredandwashedwithethylalcohol. Theresultingsolidsweredriedforuse.
2.3. PreparationofAgDopedTitania-Chitosan(STC)
2.3. Preparation of Ag Doped Titania-Chitosan (STC)
Briefly,the3mLglacialaceticacidwasaddedtothe197mLdeionizedwatertoprepare1.5vol%
Briefly, the 3 mL glacial acetic acid was added to the 197 mL deionized water to prepare 1.5 vol%
glacial acetic acid solution. The chitosan solution was prepared by dissolving 0.3 g chitosan in
glacial acetic acid solution. The chitosan solution was prepared by dissolving 0.3 g chitosan in 20 mL
20mLglacialaceticacidsolution. Ittookonehourunderultrasonicdispersionatroomtemperature.
glacial acetic acid solution. It took one hour under ultrasonic dispersion at room temperature. Then,
Then, 0.1 g titania and 0.2 mL silver nitrate deionized water solution (1 mol/L) were added into
0.1 g titania and 0.2 mL silver nitrate deionized water solution (1 mol/L) were added into chitosan
chitosan stock solution. Next, this mixture was dispersed by ultrasonication for 30 min and the
stock solution. Next, this mixture was dispersed by ultrasonication for 30 min and the homogeneous
homogeneoussuspensionwasobtained. Afterthat,0.4mLsodiumborohydridewasaddeddropwise
suspension was obtained. After that, 0.4 mL sodium borohydride was added dropwise to the
tothesuspensionandstirredfor30minatroomtemperature,andthenbrownsuspensionwasobtained.
suspension and stirred for 30 min at room temperature, and then brown suspension was obtained.
Finally,200mLliquidparaffin,certainamount(2,3,4,5mL)ofglutaraldehyde,and4mLspan-80
Finally, 200 mL liquid paraffin, certain amount (2, 3, 4, 5 mL) of glutaraldehyde, and 4 mL span-80
wereaddedtotheabovesuspensionandstirredat450rpmfor2,4,6,or8hat40◦C,thereaction
were added to the above suspension and stirred at 450 rpm for 2, 4, 6, or 8 h at 40 °C, the reaction was
was carried out in a nitrogen atmosphere. The mixture was filtered and the obtained solids were
carried out in a nitrogen atmosphere. The mixture was filtered and the obtained solids were then
thenwashedthreetimeswithetheranddistilledwater,respectively,toremovetheresidualreagent.
washed three times with ether and distilled water, respectively, to remove the residual reagent. Final
FinalbrownpowderswereobtainedaftervacuumdryingtheSTCcomposites. Figure1illustratesthe
brown powders were obtained after vacuum drying the STC composites. Figure 1 illustrates the
fabricationprocessforSTCcomposite. Also,typicalSEMimageofpreparedSTCmicrospherehas
fabrication process for STC composite. Also, typical SEM image of prepared STC microsphere has
beenshowninFigure1.
been shown in Figure 1.

FFigiguurree 11. .TThhee fafabbrircicaatitoionn pprroocceessss foforr AAgg ddooppeedd TTiiOO22-c-chhitiotossaann (S(STTCC) )ccoommppoossitiete. .
In order to investigate the photocatalytic antibacterial activity of STC composites, the titania-
In order to investigate the photocatalytic antibacterial activity of STC composites,
chitosan (TC) composites were prepared as well for comparison. The preparation process was the
thetitania-chitosan(TC)compositeswerepreparedaswellforcomparison. Thepreparationprocess
same, except that silver nitrate and sodium borohydride were not added.
wasthesame,exceptthatsilvernitrateandsodiumborohydridewerenotadded.
22.4.4. .CChhaarraaccteterrizizaatitoionn
TThhee mmooleleccuulalarr sstrtruucctuturree aanndd inintteerraacctitoionn ooff cchhitiotossaann, ,TTCC, ,aanndd SSTTCC ccoommppoossitietess wweerree ininvveessttigigaatteedd
bbyy FFoouurrieierr trtraannssfoforrmm ininfrfarareredd ssppeecctrtorommeeteter r(F(FTTIRIR).) .TThhee FFTTIRIR sasammppleles swweerere pprereppaareredd aas sKKBBr rppeellleletst,s ,
aanndd tthhee ssppeeccttrruumm wwaassc ocolllelcetcetdeda taat rae sroelsuotliuotnioonf 4ocf m4 −c1mw−1i twhi3t2h s3c2a nsscapnesr rpuenr bryunu sbinyg uasBinrgu kae rBTruenkseorr
T2e7nFsoTrI R2s7p eFcTtrIoRm estpeerc(tErottmlinetgeern ,(GEtetrlimngaenny,) .GTehremmanoryp).h oTlohge ymofotrhpehsoalomgpyl esofw athsec hsaarmacptelerisz ewdabsy
characterized by using a JEOL JSM-6510 scanning electron microscope (Tokyo, Japan). The dried
samples were coated with gold before the characterization for better imaging. The surface of the
samples was analyzed by X-ray photoelectron spectroscopy (XPS) and Auger electron spectra (AES)
in AXIS ULTRA DLD (Kratos Analytical Ltd., Manchester, UK) with Al Kα (1486.6 eV) radiation as
Materials2018,11,1403 4of17
usingaJEOLJSM-6510scanningelectronmicroscope(Tokyo,Japan). Thedriedsampleswerecoated
withgoldbeforethecharacterizationforbetterimaging. Thesurfaceofthesampleswasanalyzed
byX-rayphotoelectronspectroscopy(XPS)andAugerelectronspectra(AES)inAXISULTRADLD
(KratosAnalyticalLtd.,Manchester,UK)withAlKα(1486.6eV)radiationastheexcitationsource,
the analysis was done in the pressures on the order of 10−8 Pa. The charges of the samples were
correctedbysettingthebindingenergyoftheC peakat284.8eV.Thebandgapofthesampleswas
1s
characterizedwithUV/Visspectroscopy(SHIMADZUUV-2550spectrophotometer,Kyoto,Japan).
2.5. AntimicrobialAssays
Theantibacterialactivityofthesamples(chitosan,TCandSTCcomposites)wastestedagainst
three different bacterial species, i.e., two Gram negative bacteria, i.e., E. coli (Escherichia coli,
CGMCC 1.3373) and P. aeruginosa (Pseudomonas aeruginosa, CGMCC 1.2620), and a Gram positive
bacteria S. aureus (Staphylococcus aureus, CGMCC 1.2465). The ASTM E2180-07 (standard method
for determining the antimicrobial effectiveness of agents that are incorporated into the polymeric
surfaces)wasadoptedtoevaluatetheantibacterialactivityofthesamples. Briefly,thesamplesandall
oftheequipmentandmediawerepreviouslysterilizedbeforeantimicrobialtesting. Firstly,astandard
0.5McFarlandsuspension(1×108CFU/mL)wasprepared. Thissuspensionwasfurtherdilutedto
achieveafinaltestconcentrationof1×104CFU/mL.Secondly,foreachtest,thesamples(1g)were
madeinto2cm×2cmsquares(theSTCpowderwasslowlypouredintotheself-made2cm×2cm
squarehollowplasticmold,thenpressedthepowderintoasheetshapewithsquaremass,andfinally
removedthemoldtogetthesample)andplacedintoasterilePetridish. Then,0.5–1.0mLinoculated
agarslurrywaspipettedontothesquaretestandcontrolsamples. Thesampleswereplacedinan
incubatorat37◦Cfor24hundervisiblelightanddarkcondition,respectively. Thirdly,theincubation
samples were transferred to sterile beakers filled with 10 mL MH (Mueller-Hinton) broth. Then,
thebeakersweretreatedunderultrasonicationfor2mininordertofacilitatethecompletereleaseof
theagarslurryfromthesamples. Atlast,thedilutedsolutionswerespreadintonutrientagarand
incubatedat37 ◦Cfor48hunderthedarkcondition. Afterincubation, thebacteriacolonieswere
countedandrecordedbynakedeyes. Visiblelightsourcesweresuppliedwith150W(840lm)halogen
lamps (Philips, Shanghai, China). The distance between the light source and the sample is 20 cm.
Eachsamplewasperformedintriplicateandtested,beingrepeatedthreetimes.
3. ResultsandDiscussion
3.1. StructureandMorphologyofSTCComposites
Figure2ashowstheopticalimagesofthechitosan,TCandSTCcomposites.Allofthematerials
were powder solid. Pure chitosan was white, whereas TC and STC composites were yellow,
which might result from the cross-linking reaction between glutaraldehyde and chitosan in the
context of the Schiff mechanism (The –NH of chitosan reacted with –CHO of glutaraldehyde to
2
form C=N) due to the photochromic properties of Schiff base that led to the yellowing of TC and
STCunderillumination[30,31]. Themolecularstructureofthethreesampleswasinvestigatedby
FTIR.InFigure2b, forpurechitosan, thebroadbandlocatedat3435cm−1 canbeattributedtothe
hydrogenbondsoftheO–HandN–Hstretchingvibrations, thepeakat2874cm−1 correspondsto
the vibration of C–H, the peak at 1596 cm−1 is related to the N–H deformation vibration, and the
peakat1075cm−1 isduetotheC–Ostretchingvibration[32]. Whencomparedwithpurechitosan,
theabsorptionbandofNH (1596cm−1)groupsforchitosanshiftedslightlytoalowerwavenumber
2
(1588cm−1)forTCandSTC,asevidencedbythehydrogenbondingbetweentheaminogroupof
chitosanandthehydroxylgroupsofTiO [31]. Moreover,theintensityofthesuperimposedpeaks
2
ofhydroxylandaminogroupswassignificantlyreducedinTCandSTC,whichcouldbeattributed
tothereductionoftheprimaryaminegroupsbythereactionoftheaminogroupsonthechitosan
molecularchainswithglutaraldehyde[33]. Whencomparedwithpurechitosan,thenewbandsat
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1664 and1657cm−1inTCandSTCemerge,respectively,canbeattributedtotheformationoftheC=N
bond,furtherdemonstratingtheformationoftheSchiffbasebythereactionbetweenchitosanand
glutaraldehyde[31,33].
Materials 2018, 11, x FOR PEER REVIEW 5 of 16

Figure 2. (a) Digital photos and (b) Fourier transform infrared spectrometer (FTIR) spectra of
Figure2.(a)Digitalphotosand(b)Fouriertransforminfraredspectrometer(FTIR)spectraofchitosan,
TiO -chitosan(TC),andSTC. chitosan, TiO2-chitosan (TC), and STC.
2
The X-ray photoelectron spectroscopy (XPS) was carried out to investigate the elementary
The X-ray photoelectron spectroscopy (XPS) was carried out to investigate the elementary
composition on the surface of STC. In Figure 3a, for the wide scan spectra of the three specimens, the
composition on the surface of STC. In Figure 3a, for the wide scan spectra of the three specimens,
peaks at 285, 398 and 533 eV are corresponding to C 1 s, N 1 s, and O 1 s, respectively. The Ti 2p peak
thepeaksat285,398and533eVarecorrespondingtoC1s,N1s,andO1s,respectively. TheTi2p
is found in the spectra of STC and TC (459 eV), and the Ag 3d peak is observed in the spectrum of
peakisfoundinthespectraofSTCandTC(459eV),andtheAg3dpeakisobservedinthespectrumof
STC (369 eV). It is demonstrated the presence of Ag and Ti elements on the surface of STC. The Ag
STC(369eV).ItisdemonstratedthepresenceofAgandTielementsonthesurfaceofSTC.TheAg3d
3d spectrum of STC composite was fitted with an asymmetric Gaussian-Lorentzian sum function
spectrumofSTCcompositewasfittedwithanasymmetricGaussian-Lorentziansumfunctionwhile
while using the fitting program XPSPEAK41 (Figure 3b). It is clearly seen that the strong peaks at
usingthefittingprogramXPSPEAK41(Figure3b). Itisclearlyseenthatthestrongpeaksat368.1and
368.1 and 374.1 eV are attributed to 3d5/2 and 3d3/2 of metallic silver (Ag0). While, the weak peaks at
374.1eVareattributedto3d and3d ofmetallicsilver(Ag0). While,theweakpeaksat367.7and
367.7 and 373.7 eV are attrib5u/t2ed to 3d35//22 and 3d3/2 of silver ion (Ag+) in silver oxides. It may be the
373.7eVareattributedto3d and3d ofsilverion(Ag+)insilveroxides. Itmaybetheresultthata
result that a small part of Ag5 /N2Ps on th3/e2 surface of STC are oxidized during the storage process. The
smallpartofAgNPsonthesurfaceofSTCareoxidizedduringthestorageprocess. TheAg3dAuger
Ag 3d Auger analyses of STC composites were carried out to further confirm the above results (Figure
analysesofSTCcompositeswerecarriedouttofurtherconfirmtheaboveresults(Figure3c). Besides,
3c). Besides, the Ag MNN Auger curve agrees with that of the Ag+ in silver oxides with two peaks
theAgMNNAugercurveagreeswiththatoftheAg+insilveroxideswithtwopeaks(M N N and
(M5N45N45 and M4N45N45) that are located around 350.9 and 356.6 eV, respectively [34]. O5n 4t5he 4o5ther
M N N )thatarelocatedaround350.9and356.6eV,respectively[34]. Ontheotherhand,twopeaks
han4d,4 5two45 peaks appear at higher kinetic energies (351.7 and 357.3 eV) are assigned to metallic silver,
appearathigherkineticenergies(351.7and357.3eV)areassignedtometallicsilver,thisfurtherproves
this further proves that Ag0 and Ag+ exist simultaneously on the surface of the STC [34,35]. Figure 3d
thatAg0 andAg+ existsimultaneouslyonthesurfaceoftheSTC[34,35]. Figure3dshowsthehigh
shows the high resolution XPS spectrum of Ti 2p. Two peaks that were observed at 458.5 and 464.2
resolutionXPSspectrumofTi2p. Twopeaksthatwereobservedat458.5and464.2eVareassociated
eV are associated with the splitting of Ti 2p3/2 and Ti 2p1/2, and the doublet separation of the peaks is
withthesplittingofTi2p andTi2p ,andthedoubletseparationofthepeaksis5.7eV,provingthe
5.7 eV, proving the existe3n/c2e of TiO2 o1n/2 the surface of STC [36].
existenceofTiO onthesurfaceofSTC[36].
Figure 4 sho2ws the microstructures of chitosan, TC, and STC composites. Chitosan presents an
irregular sheet-like structure and dimensional inhomogeneity. After the reaction, similar
morphologies were observed for the TC and STC composites. Both showed spherical micron
particles, differing greatly from chitosan. It is probably because that with the addition of Span-80
surfactant during the preparation process, micelles were generated in paraffin, and then chitosan
reacted with glutaraldehyde in the micelle structure, forming a spherical cross-linked product.
Materials2018,11,1403 6of17
Materials 2018, 11, x FOR PEER REVIEW 6 of 16

Figure 3. X-ray photoelectron spectroscopy (XPS) of chitosan, TC, and STC. (a) Wide scan; (b) High
Figure3. X-rayphotoelectronspectroscopy(XPS)ofchitosan,TC,andSTC.(a)Widescan;(b)High
resolution XPS Ag 3d spectra on the surface of STC; (c) Ag MNN Auger electron spectra of STC; and,
resolutionXPSAg3dspectraonthesurfaceofSTC;(c)AgMNNAugerelectronspectraofSTC;and,
(d) High resolution XPS Ti 2p spectra on the surface of STC.
(d)HighresolutionXPSTi2pspectraonthesurfaceofSTC.
Figure4showsthemicrostructuresofchitosan,TC,andSTCcomposites. Chitosanpresentsan
irregularsheet-likestructureanddimensionalinhomogeneity. Afterthereaction,similarmorphologies
wereobservedfortheTCandSTCcomposites.Bothshowedsphericalmicronparticles,differinggreatly
from chitosan. It is probably because that with the addition of Span-80 surfactant during the
preparationprocess,micellesweregeneratedinparaffin,andthenchitosanreactedwithglutaraldehyde
inthemicellestructure,formingasphericalcross-linkedproduct.
3.2. EffectofParticleSizeonAntibacterialActivity
In order to study the influence of particle size on the antibacterial activity, a series of STC
compositemicrosphereswerepreparedunderdifferentreactionparameters.
Asweallknow,thetwoaldehydegroupsonglutaraldehydemoleculecanreactwiththeamino
groupsontwochitosanmolecules,respectively,tomakechitosanformacrosslinkednetworkstructure.
Thus,theinfluenceofglutaraldehydedosageonthemorphologyofSTCisobvious. Fourgroupsof
sampleswithconcentrationof1.0,1.5,2.0,and2.5%(v/v)ofglutaraldehydewerepreparedandthe
resultsareshowninFigure5. Whentheconcentrationofglutaraldehydeis1.0%(v/v),themorphology
of STC is extremely irregular. This is due to the concentration of being glutaraldehyde too low to
have a full cross-linking reaction to form spherical particles. With increasing the concentration of
glutaraldehydeto2.0%(v/v),theparticlemorphologybecomesmoreregular. Whentheconcentrati on
ofglutaraldehydeincreasescontinuouslyto2.5%(v/v),theparticlemorphologyissimilartotheresult
Figure 4. SEM images of (a) chitosan, (b) TC and (c) STC.
3.2. Effect of Particle Size on Antibacterial Activity
Materials 2018, 11, x FOR PEER REVIEW 6 of 16
Materials2018,11,1403 7of17

Figure 3. X-ray photoelectron spectroscopy (XPS) of chitosan, TC, and STC. (a) Wide scan; (b) High
of2.0%(v/v),soitcanbespeculatedthatthe2.0%(v/v)concentrationofglutaraldehydecanmakethe
resolution XPS Ag 3d spectra on the surface of STC; (c) Ag MNN Auger electron spectra of STC; and,
cross(dli)n Hkiignhg rreesaoclutitoionnb XePcSo mTie 2spa stpuercattread o.n the surface of STC.
Materials 2018, 11, x FOR PEER REVIEW 7 of 16
In order to study the influence of particle size on the antibacterial activity, a series of STC
composite microspheres were prepared under different reaction parameters.
As we all know, the two aldehyde groups on glutaraldehyde molecule can react with the amino
groups on two chitosan molecules, respectively, to make chitosan form a cross linked network
structure. Thus, the influence of glutaraldehyde dosage on the morphology of STC is obvious. Four
groups of samples with concentration of 1.0, 1.5, 2.0, and 2.5% (v/v) of glutaraldehyde were prepared
and the results are shown in Figure 5. When the concentration of glutaraldehyde is 1.0% (v/v), the
morphology of STC is extremely irregular. This is due to the concentration of being glutaraldehyde
too low to have a full cross-linking reaction to form spherical particles. With increasing the
concentration of glutaraldehyde to 2.0% (v/v), the particle morphology becomes more regular. When
the concentration of glutaraldehyde increases continuously to 2.5% (v/v), the particle morphology is
similar to the result ofF i2g.u0r%e 4(.v S/EvM), ismo aigte sc aonf ( ab) ec hsiptoescaunl, a(tbe)d T Cth aantd t(hc)e S T2.C0.% (v/v) concentration of
Figure4.SEMimagesof(a)chitosan,(b)TCand(c)STC.
glutaraldehyde can make the cross linking reaction become saturated.
3.2. Effect of Particle Size on Antibacterial Activity

Figure 5. SEM images and particle size of STC composites with different glutaraldehyde dosage (a)
Figure 5. SEM images and particle size of STC composites with different glutaraldehyde dosage
1.0%, (b) 1.5%, (c) 2.0%, and (d) 2.5% (v/v).
(a)1.0%,(b)1.5%,(c)2.0%,and(d)2.5%(v/v).
In addition, SEM images of four samples with inverse emulsion cross-linking reaction time of 2,
4, 6, aInndad 8d hit aiorne ,sShEoMwnim ina gthees Foifgfuoruer 6s.a Tmhpel emsowrpithhoilnovgeyr soef eSmTCul psiaornticcrleoss sw-liitnhk 2in-hg rreeaaccttiioonn itsim irereogfu2l,a4r,,
6i.,ea.,n ad lo8th ofa rneosnh-sopwhneriincatlh peaFritgiculeres a6n.dT hweidmeo prparhtoiclloeg dyiaomfSeTteCr dpiasrttriicbluestiowni.t hIt 2is- hprroeabcatbiolyn biesciarruesgeu tlhaar,t
it.he.e, carloosts olifnnkoinng-s rpehaecrtiicoanl tpimaret iicsl etsooa nsdhowrti dtoe fpoarmrti cal ecodmiapmleettee rspdhisetrrei.b Wuthioenn. tIhtei sreparcotiboanb ltyimbee cisa u4s he,
tthhaet mthoercprhooslsolginyk oinf gthree apcatirotinclteism ies imstooroe srheogrutlator. fFourmrthaercmomorpel,e wtehsepnh ethree. rWeahcetinonth teimreea cisti o6n ht,i mthee
ims4orhp,htohleogmyo orpf hthoel opgayrtoicfleths eisp tahret imcloessti sremguolraer rieng tuhlea rs.aFmuprltehse. rNmeovreer,twhehleesns,t hwehreena cthtieo nretaicmtieonis t6imhe,
tihs einmcroerapsheodl otog y8 ohf, tah peaprat rotifc pleasrtisictlhese amreo sbtrroekgeunl aarnidn tthhee msaomrpphleosl.oNgye vise rirthreegleuslsa,rw. Iht eisn pthroebraebalcyt itohne
triemsuelits oifn tchreea csreodsst olin8khe,da cphairtotsoafnp maritcicrolesspahreerebsr owkietnh alanrdget hdeiammoertperh obleoinggy eisasirirlye gburloakre.nIt uisnpdreor bsahbelayr
tshterersess aunltdo tfhteh leoncrgo-stismlien mkeudtucahli tcooslalinsimoni cbreotswpeheenre msiwcriothsplahregreesd diaumrientge rthbee isntigrreinagsi lpyrobcreoskse. nunder
shearFsutrrethssera,n dsitzhee ldoinsgtr-itbimuteiomn utoufa lScToCll iscioonmbpeotswiteeesn municdreors pdhiefrfeersednut rirnegacthtieonst irprainragmperotecress s.was
summarized in Table 1. As shown in Table 1, with the increase of glutaraldehyde dosage, the quantity
of STC particles with diameter between 1 and 10 μm increases from 25.00% to 54.34%. Besides, the
particle diameter distribution of product becomes more concentrated and the regularity of particle is
also improved. When the concentration of glutaraldehyde is 2.5% v/v, the particle size has no obvious
change, which can be speculated that the optimal concentration of glutaraldehyde is 2.0% v/v. With
increasing the reaction time, the percentage of STC particles with diameter between 1 and 10 μm
increases from 21.74% to 68.75%, whereas the percentage of STC particles with diameter between 10
and 20 μm decreases from 32.61% to 16.67%, which means that the particle diameter distribution of
product is more concentrated. However, the regularity of particle morphology is deteriorated when
the reaction time is 8 h, thus it can be speculated that the optimal reaction time is between 4 and 6 h.
The above results are agreed with SEM images of samples in Figures 5 and 6.
Materials2018,11,1403 8of17
Materials 2018, 11, x FOR PEER REVIEW 8 of 16

Figure 6. SEM images and particle size of STC composites with different cross-linking reaction time
Figure6.SEMimagesandparticlesizeofSTCcompositeswithdifferentcross-linkingreactiontime
(a) 2 h, (b) 4 h, (c) 6 h, and (d) 8h.
(a)2h,(b)4h,(c)6h,and(d)8h.
Table 1. Size distribution of STC composites under different reaction parameters.
Further,sizedistributionofSTCcompositesunderdifferentreactionparameterswassummarized
Reaction Parameters Frequency Frequency Frequency Frequency
in Table 1. As shown in Table 1, with theFirneqcureenacsye of glutaraldehyde dosage, the quantity of STC
Dosage of Reaction Stirring of STC of STC of STC of STC
paNrot.i cleswithdiameterbetween1and10µofm STinCc (r1e–asesfrom25.00%to54.34%. Besides,theparticle
Glutaraldehyde Time Rate (10–20 μm) (20–30 μm) (30–40 μm) (40–50 μm)
10 μm) (%)
diameter d(i%st vr/ivb)u tion of p(hr)o duct(rb/mecino)m es more concent(r%at) ed and th(%e)r egularity(%o) f particle(%is) also
im1p roved. W1.h0 entheconc4e ntration45o0 fglutar2a5.l0d0e hydeis392..750% v/v,t2h6e.48p articles8i.z8e2 hasnoob0v ious
2 1.5 4 450 39.13 26.09 17.39 8.70 8.70
change, which can be speculated that the optimal concentration of glutaraldehyde is 2.0% v/v.
3 2.0 4 450 54.34 20.23 9.83 8.09 5.20
Withincreasingthereactiontime,thepercentageofSTCparticleswithdiameterbetween1and10µm
4 2.5 4 450 52.82 28.87 14.09 3.52 0.70
inc5r easesfro2m.0 21.74%to628 .75%,w4h50e reasthe21p.7e4r centage32o.f61S TCpart3i4c.l7e8s withdi8a.7m0 eterbetw2.e1e7 n10
an6d 20µmd2e.c0 reasesfrom6 32.61%t4o501 6.67%,5w8.6h4i chmean17s.2t8h atthep1a0r.4t7ic lediam8e.t9e0 rdistribu4t.1io9 nof
7 2.0 8 450 68.75 16.67 7.29 4.17 1.04
productismoreconcentrated. However,theregularityofparticlemorphologyisdeterioratedwhen
8 2.0 4 300 0 9.09 4.55 13.64 24.24
thereactiontimeis8h,thusitcanbespeculatedthattheoptimalreactiontimeisbetween4and6h.
TheaboveresultsareagreedwithSEMimagesofsamplesinFigures5and6.
Based on the above analysis, STC-3, STC-5, and STC-8 with typical particle diameter ranges
being chosen to study the effect of particle size on antibacterial activity. It can be seen from Figure 7
Table1.SizedistributionofSTCcompositesunderdifferentreactionparameters.
that the particle diameter ranges STC-3, STC-5 and STC-8 is centered in 1–10 μm, 20–30 μm, and 40–
50 μm, respectively.
ReactionParameters Frequency Frequency Frequency Frequency Frequency
No. Dosageof Reaction Stirring ofSTC ofSTC ofSTC ofSTC ofSTC
Glutaraldehyde Time Rate (1–10µm) (10–20µm) (20–30µm) (30–40µm) (40–50µm)
(%v/v) (h) (r/min) (%) (%) (%) (%) (%)
1 1.0 4 450 25.00 39.70 26.48 8.82 0
2 1.5 4 450 39.13 26.09 17.39 8.70 8.70
3 2.0 4 450 54.34 20.23 9.83 8.09 5.20
4 2.5 4 450 52.82 28.87 14.09 3.52 0.70
5 2.0 2 450 21.74 32.61 34.78 8.70 2.17
6 2.0 6 450 58.64 17.28 10.47 8.90 4.19
7 2.0 8 450 68.75 16.67 7.29 4.17 1.04
8 2.0 4 300 0 9.09 4.55 13.64 24.24
Based on the above analysis, STC-3, STC-5, and STC-8 with typical particle diameter ranges
being chosen to study the effect of particle size on antibacterial activity. It can be seen from
Figure7thattheparticlediameterrangesSTC-3,STC-5andSTC-8iscenteredin1–10µm,20–30µm,
and40–50µm,respectively.

Figure 7. SEM images and particle size distribution of (a) STC-3, (b) STC-5, and (c) STC-8.
Materials 2018, 11, x FOR PEER REVIEW 8 of 16

Figure 6. SEM images and particle size of STC composites with different cross-linking reaction time
(a) 2 h, (b) 4 h, (c) 6 h, and (d) 8h.
Table 1. Size distribution of STC composites under different reaction parameters.
Reaction Parameters Frequency Frequency Frequency Frequency
Frequency
Dosage of Reaction Stirring of STC of STC of STC of STC
No. of STC (1–
Glutaraldehyde Time Rate (10–20 μm) (20–30 μm) (30–40 μm) (40–50 μm)
10 μm) (%)
(% v/v) (h) (r/min) (%) (%) (%) (%)
1 1.0 4 450 25.00 39.70 26.48 8.82 0
2 1.5 4 450 39.13 26.09 17.39 8.70 8.70
3 2.0 4 450 54.34 20.23 9.83 8.09 5.20
4 2.5 4 450 52.82 28.87 14.09 3.52 0.70
5 2.0 2 450 21.74 32.61 34.78 8.70 2.17
6 2.0 6 450 58.64 17.28 10.47 8.90 4.19
7 2.0 8 450 68.75 16.67 7.29 4.17 1.04
8 2.0 4 300 0 9.09 4.55 13.64 24.24
Based on the above analysis, STC-3, STC-5, and STC-8 with typical particle diameter ranges
being chosen to study the effect of particle size on antibacterial activity. It can be seen from Figure 7
tMhaatte rtiahlse 2p01a8r,t1ic1,le1 4d03iameter ranges STC-3, STC-5 and STC-8 is centered in 1–10 μm, 20–30 μm, and9 o4f01–7
50 μm, respectively.

Figure 7. SEM images and particle size distribution of (a) STC-3, (b) STC-5, and (c) STC-8.
Figure7.SEMimagesandparticlesizedistributionof(a)STC-3,(b)STC-5,and(c)STC-8.
Next,thebacteriasuspensionwithaconcentrationof1×104Colony-FormingUnits(CFU)/mL
wasinoculatedonanagarplateasthereferencegrouptocomparetheantibacterialabilitiesofSTC-3,
STC-5,andSTC-8withtypicalparticlediameterranges. Allofthetestswereconductedunderthe
visiblelightconditionandtheresultsareshowninFigure8andTable2. Accordingtothebacterial
colonypicture(Figure8a),itcanbefoundvisuallythattheSTC-3whoseparticlediameterarecentered
in1–10µmpresentsabestantibacterialpropertyagainstE.coli,S.aureus,andP.aeruginosa. Whenthe
particle diameter of product are in the range of 20–30 and 40–50 µm, respectively, the number of
bacterialcolonyincreasessuccessivelyasshowninFigure8aandTable2,whichprovesthatwhenthe
particlediameterdecreases,thenumberofbacterialcolonydecreases. Thisisprobablybecausethatthe
STC-3withasmallerparticlediameterhasbiggerspecificsurfaceareaandhighersurfaceenergy[37].
ThusmoreSTCparticlescanbeindirectcontactwiththebacterialcells,sothatsilverionsandhydroxyl
radicals,whichworkonthebacteriaandthenpreventthegrowthofbacterial,canbereleasedfrom
thesilverandtitaniaNPsstabilizedonthesurfaceofcrosslinkedchitosan[38]. Inaddition,fromthe
resultsthatareshowninFigure8b,theantimicrobialeffectofSTCsmightberelatedtothespeciesof
bacteria. Itcanbefoundthattheaveragecolony-formingunitofE.coli(8CFU)isthelowestamong
thethreekinds,provingthattheSTCshaveabestantibacterialabilityonE.coli. GroupofP.aeruginosa
hadthelargestnumberofcolonies(21CFU),whichindicatedthattheantibacterialactivityofSTC
againstP.aeruginosawaslowerthanthatofE.coli. Inaddition,thecolony-formingunitofS.aureus
(10CFU)wasbetweenthatofE.coliandP.aeruginosa. Amongthethreebacteriastrains,therewere
distinguishingcharacteristicsinthecompositionsoftheircellwalls. Specificallyspeaking,thecell
wallofGrampositivebacteriaS.aureusisthickerandmorerigidthanthatofGramnegativebacteria
E.colis,whichpreventsthesilverandtitaniaNPsfromenteringbacterialcells[28]. FortheP.aeruginosa,
thebacterialbiofilmwillformasolidlayerofenvelopeduringthegrowthprocessofP.aeruginosa,
whichweakenstheinfluenceofSTCsonthecellwall,thisresultisingoodagreementwiththatalready
reportedthattheantimicrobialeffectofantibacterialagentisrelatedtobacterialspecies[28,39].
Materials 2018, 11, x FOR PEER REVIEW 9 of 16
Next, the bacteria suspension with a concentration of 1 × 104 Colony-Forming Units (CFU)/mL
was inoculated on an agar plate as the reference group to compare the antibacterial abilities of STC-
3, STC-5, and STC-8 with typical particle diameter ranges. All of the tests were conducted under the
visible light condition and the results are shown in Figure 8 and Table 2. According to the bacterial
colony picture (Figure 8a), it can be found visually that the STC-3 whose particle diameter are
centered in 1–10 μm presents a best antibacterial property against E. coli, S. aureus, and P. aeruginosa.
When the particle diameter of product are in the range of 20–30 and 40–50 μm, respectively, the
number of bacterial colony increases successively as shown in Figure 8a and Table 2, which proves
that when the particle diameter decreases, the number of bacterial colony decreases. This is probably
because that the STC-3 with a smaller particle diameter has bigger specific surface area and higher
surface energy [37]. Thus more STC particles can be in direct contact with the bacterial cells, so that
silver ions and hydroxyl radicals, which work on the bacteria and then prevent the growth of
bacterial, can be released from the silver and titania NPs stabilized on the surface of cross linked
chitosan [38]. In addition, from the results that are shown in Figure 8b, the antimicrobial effect of
STCs might be related to the species of bacteria. It can be found that the average colony-forming unit
of E. coli (8 CFU) is the lowest among the three kinds, proving that the STCs have a best antibacterial
ability on E. coli. Group of P. aeruginosa had the largest number of colonies (21 CFU), which indicated
that the antibacterial activity of STC against P. aeruginosa was lower than that of E. coli. In addition,
the colony-forming unit of S. aureus (10 CFU) was between that of E. coli and P. aeruginosa. Among
the three bacteria strains, there were distinguishing characteristics in the compositions of their cell
walls. Specifically speaking, the cell wall of Gram positive bacteria S. aureus is thicker and more rigid
than that of Gram negative bacteria E. colis, which prevents the silver and titania NPs from entering
bacterial cells [28]. For the P. aeruginosa, the bacterial biofilm will form a solid layer of envelope
during the growth process of P. aeruginosa, which weakens the influence of STCs on the cell wall, this
result is in good agreement with that already reported that the antimicrobial effect of antibacterial
Materials2018,11,1403 10of17
agent is related to bacterial species [28,39].

Figure8.AntibacterialactivitiesofSTC-3,STC-5,andSTC-8withtypicalparticlesize.(a)Flatcolony
countingmethod(StandardNormASTME2180–07)forSTCcompositeswithtypicalparticlesize
(1–10µm,20–30µmand40–50µm),againstE.coli(CGMCC1.3373),S.aureus(CGMCC1.2465)and
P.aeruginosa(CGMCC1.2620);and,(b)Antibacterialtestquantitativeevaluationresults.Theagarplates
(bacterialconcentrationof1×104CFU/mL)withoutSTCcompositesweretreatedascontrolgroup.
Table2.Thecolonyformingunits(CFU)andantibacterialrate(AR)ofSTCwithdifferentsizerange.
Control STC40–50µm STC20–30µm STC1–10µm
Bacteria
CFU* CFU* CFU*
CFU
(AR/%) (AR/%) (AR/%)
30 12 8
E.coli 1×104
(99.70) (99.88) (99.92)
73 41 10
S.aureus 1×104
(99.27) (99.59) (99.90)
107 46 21
P.aeruginosa 1×104
(98.93) (99.54) (99.79)
*Themeannumberofthecolonyformingunitsinthethreerepeatedexperiments.
3.3. EffectofVisibleLightPhotocatalysisonAntibacterialActivity
InordertostudythephotocatalyticantibacterialactivityofSTC,theantibacterialabilityofSTC-3
againstseveralbacteria(GramnegativeE.coliandP.aeruginosa,andGrampositiveS.aureus)under
visible-lightanddarkconditionswascompared. Inaddition, testsofchitosanandTCcomposites