Research Article Effect of Surface Charge and Hydrophobicity Modulation on the Antibacterial and ...

16 Pages·2017·3.53 MB·English

Hindawi
Journal of Nanomaterials
Volume 2017, Article ID 3528295, 15 pages
https://doi.org/10.1155/2017/3528295
Research Article
Effect of Surface Charge and Hydrophobicity
Modulation on the Antibacterial and Antibiofilm
Potential of Magnetic Iron Nanoparticles
RaniaIbrahimShebl,1FatenFarouk,1andHassanMohamedEl-SaidAzzazy2
1MicrobiologyandImmunologyDepartmentandPharmaceuticalChemistryDepartment,FacultyofPharmacy,
AhramCanadianUniversity,Cairo,Egypt
2DepartmentofChemistry,SchoolofSciences&Engineering,AmericanUniversityinCairo,NewCairo,Egypt
CorrespondenceshouldbeaddressedtoHassanMohamedEl-SaidAzzazy;[email protected]
Received 18 January 2017; Accepted 24 May 2017; Published 12 September 2017
AcademicEditor:JeanM.Greneche
Copyright©2017RaniaIbrahimShebletal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Unmodifiedmagneticnanoparticles(MNPs)lackantibacterialpotential.WeinvestigatedMNPssurfacemodificationsthatcan
impartantibacterialactivity.SixMNPsspecieswerepreparedandcharacterized.Theirantibacterialandantibiofilmpotentials,
surfaceaffinity,andcytotoxicitywereevaluated.PreparedMNPswerefunctionalizedwithcitricacid,aminegroup,amino-propyl
trimethoxysilane(APTMS),arginine,oroleicacid(OA)togivehydrophilicandhydrophobicMNPswithsurfacechargeranging
from−30to+30mV.PreparedMNPsweresphericalinshapewithanaveragesizeof6–15nm.Hydrophobic(OA-MNPs)and
positivelychargedMNPs(APTMS-MNPs)hadsignificantconcentrationdependentantibacterialeffect.OA-MNPsshowedhigher
inhibitory potential against S. aureus and E. coli (80%) than APTMS-MNPs (70%). Both particles exhibited surface affinity to
S.aureusandE.coli.DifferentconcentrationsofOA-MNPsdecreasedS.aureusandE.colibiofilmformationby50–90%,while
APTMS-MNPsreduceditby30–90%,respectively.Upto90%ofpreformedbiofilmsofS.aureusandE.coliweredestroyedbyOA-
MNPsandAPTMS-MNPs.Inconclusion,surfacepositivityandhydrophobicityenhanceantibacterialandantibiofilmproperties
ofMNPs.
1.Introduction biocompatibility,aswellastheirmanipulationbyamagnetic
field[3].MNPstypesincludehematite(𝛼-Fe2O3),magnetite
Nanoparticles (NPs) are gaining attention as a new antimi- (Fe3O4),wu¨stite(FeO),andmaghemite(𝛾-Fe2O3).Themag-
crobialandantibiofilmapproach.Theymaydirectlyexhibit netite and maghemite types have proven biocompatibility.
antibacterialeffectorbeusedasantibioticcarriers[1].Metal Bothtypesareproducedbythermalcoprecipitationmethod
oxides NPs can be prepared easily and stand harsh condi- [4, 5]. Magnetite Fe3O4 (black) is produced under anaero-
tions such as high temperature during sterilization. Metal bic conditions while maghemite 𝛾-Fe2O3 is the magnetite
oxide NPs are expected to overcome the organic molecule oxidation product (brown). MNPs range in size from few
drawbackssuchaspollution,residenceintissues,highcost, to hundreds of nanometers [6]. They have many bioappli-
toxicity,andlowstability.Theirmechanismofactionincludes cations, such as magnetic bioseparation and detection of
productionofreactiveoxygenspecieswhichdamagescellular biologicalentities,diagnosticapplicationsasmagneticreso-
structures,alterationofmembranepermeability,interruption nanceimaging,andtherapeuticapplicationsastargeteddrug
ofenergytransduction,alterationofenzymaticactivity,and deliveryandbiologicallabels[7].
DNAreplication[2]. MNPs are generally very reactive and tend to aggregate
Ironoxidemagneticnanoparticles(MNPs)haveseveral quicklytodecreasetheirsurfaceenergywhichleadstoalter-
advantages due to their stability, low preparation cost, and ationintheirsizeandmagneticproperties.Surfacecoatingof
2 JournalofNanomaterials
Table1:AntibacterialeffectsofMNPswithdifferentsurfacegroups.
Ironcoatingorfunctional Surfacecharge
Hydrophobicity/hydrophilicity Bacterialstraintested Effectonbacteria Ref.
group (mV)
Ironfunctionalizedwith
E.coli,S.enterica,P.
HennaExtract“2-hydroxy-
— — mirabilis,andS. Inhibitedgrowth [34]
1,4-naphthoquinone”and
aureus
GardeniaLeaveExtract
(i)Inhibitedgrowth
∗OA-MNP −40 Hydrophobic S.aureusandP. [35]
aeruginosa (ii)Showed
antibiofilmactivity
P.aeruginosa,E.
Glycerol,MNPs — Hydrophilic Stimulatedgrowth [36]
faecalis
ExtractofA.Mexicana,
— Hydrophilic Inhibitedgrowth
MNPs E.coli,P.mirabilis,
[37]
MNPs — Hydrophilic andB.subtilis Noeffect
Chitosan-MNPs 61.8 Hydrophilic E.Coli Inhibitedgrowth [38]
MNPs 43.7 Hydrophilic Inhibitedgrowth
CES-MNPs −15.4 Hydrophilic S.aureusandS. Inhibitedgrowth
APTES-MNPs 32.6 Hydrophilic epidermidis Inhibitedgrowth [39]
PEG-MNPs −7.7 Amphiphilic Noeffect
MNP −32.2 Hydrophilic Noeffect
B.subtilisandE.coli [8]
Chitosan-MNP +36.2 Hydrophilic Inhibitedgrowth
Streptomycin-chitosan- Enhanced
— Hydrophilic S.aureus [40]
MNPs antibacterialactivity
Enhanced
Ampicillin-chitosan-MNPs +14.4 Hydrophilic M.tuberculosis [41]
antibacterialactivity
PVA-MNPs −19 Amphiphilic S.aureus Inhibitedgrowth [42]
Citricacid-MNPs −30 Hydrophilic M.smegmatis Noeffect [43]
Polyacrylicacid-MNPs −30 Hydrophilic M.smegmatis Noeffect [7]
∗
TheOA-IONPsshowedmorepotentantibiofilminhibitoryactivityagainstGram-positivebacteriaS.aureusascomparedtoGram-negativebacteriaP.
aeruginosa.
MNPsmodulatestheiraggregation,stability,anddispersion antibiofilm ability will greatly reduce the antibiotic use.
ability[4,6]. Antibacterial NPs having ability to reduce bacterial biofilm
Owingtotheiruniqueproperties,severalattemptswere formation can exert their function by preventing bacterial
performed to investigate the antibacterial and antibiofilm adhesion to surfaces and increasing the bacterial cell expo-
potentials of MNPs. Previous studies showed that MNPs suretosurroundingenvironment[9].
exhibitedinsignificantornoantibacterialactivity,whilesome Thepresentstudyaimedtocomparetheeffectofhydro-
surfacemodificationsweresuccessfultoimpartantibacterial phobicityandsurfacechargemodulationontheantibacterial
potential(Table1).Understandingtheeffectofsurfacemodi- and antibiofilm potentials of MNPs. MNPs with different
ficationofMNPswillenabletheoptimumselectionofsurface surfacefictionalizationsthatresultindifferentsurfacecharge
coating materials offering the greatest antibacterial activity andhydrophobicityweresynthesizedandcomparedinterms
withtheleasttoxicity. of antibacterial activity, inhibition of bacterial biofilm for-
Many bacterial strains produce slime which serves as a mation, destruction of preformed biofilm, and their safety
matrixinwhichbacteriaareembeddedleadingtotheforma- to human cells. Comparison was performed against Gram-
tion of bacterial biofilm. Bacterial adhesion is mediated by positive S. aureus and Gram-negative E. coli. To the best of
electrostatic, dipole-dipole, H-bond, hydrophobic, and van our knowledge, this is the first attempt to compare the
der Waals interactions [8]. Biofilms promote antibiotic tol- effectofhydrophobicityandchargemodificationonMNPs’
erance by reducing antibiotic entry into the bacterial cells. antibacterialpotentialandantibiofilmformationability.
Bacteria in the biofilm can also grow slowly to adapt to
depletionofnutrientandaccumulationofwaste[9].Bacterial
biofilmcanadheretosurfacesorexistinflowingsystemlike 2.MaterialsandMethods
watercolumns[10].Bacterialinfectionwhichformsabiofilm
will be transformed from an acute to a chronic infection 2.1. Material and Instrumentation. FeCl3, FeSO4, ammonia
which is difficult to eradicate. Eradicating bacteria in a solution(25%),citricacid,andoleicacidwereobtainedfrom
biofilmwillrequireeithermechanicalremovalorlongtime Al-GomhoreyaforChemicalIndustriesinCairo,Egypt.Argi-
combination of high doses of antibiotics [11]. NPs with nine (Arg) and amino-propyl trimethoxy silane (APTMS)
JournalofNanomaterials 3
wereobtainedfromSigmaAldrich(St.Louis,MO).Deion- washedwithacidifiedDIwaterandthenwithcoldDIwater
ized(DI)waterwasproducedinhousebyaMilli-Qsystem toremoveexcessammoniaandotherinorganiccomponents.
(Milford, Connecticut, USA). Mueller Hinton broth was The particles were charge measured and FTIR scanned. A-
obtained from Sigma Aldrich. E. coli (ATCC-8739) and S. MNPswerethenreconstitutedinDIwater(2mg/mL)[16].
aureus (ATCC-6538) strains and normal human epithelial
(WISH) cells (ATCC-CCL25) were generously provided by
2.2.4.PreparationofCitrateCoatedMNPs(CA-MNPs). CA-
VACSERA(Cairo,Egypt).Phosphatebuffersaline(PBS)was
MNPswerepreparedasdescribedin[17,18]withsomemod-
purchasedfromBiowhittaker-Belgium.DynatechMicroplate
ifications.Solutionofcitricacidwaspreparedbydissolving
Reader (MR 5000Er, West Sussex, UK) and a Jenway 6850
38.4mg of citric acid in 1L of DI water. The solution was
Spectrometer(Staffordshire,UK)wereused. ∘
heatedat80 C(Solution1).FeCl3 (5.5g)andFeSO4 (2.75g)
were weighed and dissolved in DI water (1L). The solution
2.2.MethodsofMNPsSynthesis washeatedat70∘Cfor30min.FivemLofammoniasolution
wasaddeduntilblackprecipitateisformed.Thereactionwas
2.2.1.SynthesisofMagneticIronNanoparticles(MNPs). Ther-
continuedfor10min(Solution2).Solution2wasaddedon
mal coprecipitation method was adopted for the prepa-
solution 1 dropwise with stirring. The mixture was heated
ration of MNPs using FeCl3, FeSO4, and 35% ammonia for 20min and washed with DI water. The resulting CA-
solution (Figure 1). Briefly, 5.5g of FeCl3 and 2.75g of MNPs were airdried, grinded,andexaminedforFTIRand
FeSO4 were weighed and d∘issolved in DI water (1L). The zetapotentialchange.Finally,asuspensionof2mg/mLwas
solution was heated at 70 C for 30min [12]. Five mL of
preparedforapplication[17].
ammonia solution was added until a black precipitate was
formed (Magnetite Fe3O4). The reaction was continued for
2.2.5.PreparationofMNPsCoatedwithArginine(Arg-MNPs).
additional 10min. Solution was evaporated to dryness in
Unfunctionalized MNPs (100mg) were transferred into a
rotavap to remove adsorbed ammonia. Residue was then
glassmortarand20mgofarginine(Arg)wasaddedfollowed
washed six times with DI water and surface charge was
measureduntilshiftedtonegative(−20mV)indicatingcom- by 1mL HCl. The mixture was mixed using a pestle and
diluted with DI water (100mL) and then transferred into
plete desorptionofsurface ammonia.The MNPs were then
∘ a stoppered conical flask for 6h reflux. Arg-MNPs were
driedinovenat180 Candgrindedbeforeuse.Thepowder
turned into the brown maghemite (𝛾-Fe2O3) solid (supple- separatedusinganexternalmagnetandwashedwithDIwater
sixtimes.Arg-MNPswereairdriedfollowedbygrinding.A
mentary information 1, in Supplementary Material avail-
2mg/mLsolutionwasprepared[19].
ableonlineathttps://doi.org/10.1155/2017/3528295).Prepared
MNPs(200mg)wereweighedandsuspendedin100mLDI
water.TheX-raydiffraction(XRD)analysis(supplementary 2.2.6. Preparation of Silane Coated MNPs (APTMS-MNPs).
materials 2) and transmission electron microscope (TEM) Amino-propyl trimethoxy silane (APTMS) was used as a
imaging(Figure2)ofthepreparedparticleswereperformed. functionalizing agent for MNPs. Two protocols were fol-
lowed.Inthefirstprotocol(coldsynthesismethod),200mg
of MNPs was weighed and suspended in 3mL of DI water.
2.2.2. Preparation of Oleic Acid (OA) Functionalized MNPs
The suspension was sonicated for 30min for hydration and
(OA-MNPs). OA-MNPs were synthesized according to the
ensuring homogeneity (I). The MNPs suspension was then
methods of [13, 14], with some modifications. Excess oleic
poured (100𝜇L portions) on a glass vial containing 800𝜇L
acid (OA) was added (3mL) to black magnetite particles
∘ of APTMS placed on an ice path. The mixture was left
with stirring for 1h at 70 C. Two layers were formed: the
in ice bath for 3h and then washed with DI water for
upper OA layer was separated in a separating funnel and
6 successive times. In the second method (hot synthesis
washed three times with DI water. MNPs were collected by
method),200mgMNPsweresuspendedin10mLofacetone
a magnet and then washed 6 times with ethanol to remove
(I).APTMS(400𝜇L)weremixedwith10mLofacetone(II).
excess OA. OA-MNPs were then tested for removal of
SolutionsIandIIweremixedbysonicationfor90min.The
uncoated OA by FTIR in terms of absence of C=O peak of
contents were then transferred to a glass stoppered conical
OAindicatingchemosorptionofOAonMNPs(Figure3(a))
flask and refluxed for 3h followed by washing. From both
[14, 15]. Chemosorption of OA was further verified by the
methods,2mg/mLsuspensionsofMNPswerepreparedinDI
shiftinzetapotential(Figure3(b))andthelackofdispersion
water. Zeta potential scanning was used to compare higher
abilityofparticlesinwater(Supplementarymaterial3aand
potential change induced by APTMS using both methods
b).OA-MNPswereairdriedtoremoveexcessalcoholgrinded
(supplementaryinformation4).
and weighed (200mg). OA-MNPs were then mixed with
40𝜇LofTween80inaglassmortarandDIwaterwasadded
dropwise for the first 10mL and then portionwise until the 2.3.AntibacterialandAntibiofilmActivityof
volumewascompletedto100mL[13]. SynthesizedMNPs
2.2.3. Preparation of Amine Coated MNPs (A-MNPs). The 2.3.1.AntibacterialActivityScreening. Antibacterialeffectof
blackmagnetiteparticleswereextractedfromtheirreaction MNPs was examined against E. coli (ATCC-8739) and S.
mixtureafterbeingleftforagingatroomtemperaturefor3h aureus(ATCC-6538).MNPsweredilutedinMuellerHinton
withtheexcessammoniainclosedcondition.A-MNPswere broth to reach a final concentration of 100𝜇g/mL and
4 JournalofNanomaterials
NH4OH
FeCl3
NH3NH3NH3
NH3
NH3 NH3NH3NH3
FeSO4 NH3 FeSO4 NH3
NH3 NH3
NH4OH NH3 NH3 Wash with NH3 NH3
external
NH3 NH3 magnet 3 NH3 NH3
NH3
times on
2Fe3++Fe2−+8OH− Heat at80∘C Fe3O4+4H2O+remaining salts cold NH3 NH3 NH3
MNP with surface
adsorbed amine
A-MNP
Wash with
external
magnet 3
times at80∘C
NH3
− −
OH − − − −
O + − − − −−
−
− − − −
− − Dry at180∘C − −
− − −
− −
Oleic acid − − O2 − − − −
O OH O
O
Si MNP
HO
HO O
+
−− −−+−− −O HO O OH H2N HN NH2
− − O APTMS
− − − O CH3OH NH
− − −− −− O− OH HCl
−
− − + OH
−−−−−− −O−−− O −−−−−−−−−−O−−−O− − OHOHO−−−−−H−O−−O−−−−−−− OHOO OHOH O H2N H2NHOHNO O OH NH2
OO H −−−−−− − −−−−− HHOO OCA-MNP H O O O−S−−−i−−−−−−−S−−−−i O H+3N−−−−−−+N−−H−−2+−−NH+3
− O H2N NH
OA-MNP
Arg-MNP
NH2
APTMS-MNP
NH2 O OH
Figure1:Synthesisofmagneticnanoparticles(MNPs).
JournalofNanomaterials 5
measuredat1,2,4,6,and24hintervalsusinganELISAplate
readerat600nm.
2.3.4. Surface Affinity of MNPs to Bacteria. OA-MNPs and
APTMS-MNPsweremixedwithconcentratedbacterialsus-
pensioninDIwater(ODofsuspension=1.2)toreachafinal
concentrationof500𝜇g/mL.Thesuspensionwaspreparedin
DI rather than broth to reduce the bacterial growth during
theexperiment.However,allmeasurementswereperformed
against suspension control of the same age. The mixture of
MNPsandbacteriawereallowedtointeracttogetherthrough
incubationonashaker(200rpm)forapredeterminedperiod
oftime.MNPswerecollectedusinganexternalmagnet.The
bacterial suspension was mixed to resuspend the bacterial
cells and samples were collected to measure their OD at
600nmusingspectrometer.Theaffinitywascalculatedfrom
Figure2:TEMimageofMNPspreparedbythermalcoprecipitation
method.PreparedMNPshadanaveragesizeof6–16nmandwere the decrease in OD which occurred upon treating bacterial
sphericalinshape. suspension with MNPs relative to positive control bacteria
without MNPs. Optical densities obtained after collecting
MNPs alone with a magnet were considered as negative
incubatedwith18–20hsubculturedbacterialstrainsat37∘C control[23].ThecollectedMNPswerescannedbyFTIRand
onashaker(200rpm)for24h.Attheendoftheincubation comparedtocontrolMNPs[24].
period, samples were obtained from each flask and 10-fold
serially diluted in sterile saline. One hundred 𝜇L of each 2.4.AntibiofilmActivity
dilution as well as control was spread on the surface of 3
∘
nutrient agar plates, incubated at 37 C for 24h, and the 2.4.1. Effect on Biofilm Formation. The effect of MNPs on
average numbers of colony forming units (CFU/mL) were biofilm formation was carried out by allowing bacterial
counted[20].MNPsspeciesshowingpromisingantibacterial strains to grow in presence of double fold serially diluted
screening were imaged by scanning electron microscope MNPs (OA-MNPs and APTMS-MNPs) in 96 multiwell
∘
(SEM)tocomparetheirsurfacemorphology. plates.Plateswereincubatedat37 Cfor24h.Afterincubation
period,contentsoftheplateswerediscardedandplateswere
washed3timeswithPBStoremoveunboundbacteria.Plates
2.3.2. Effect of Concentration on the Antibacterial Activity of
were inoculated with 100𝜇L/well crystal violet (0.15%) and
MNPs. The OA-MNPs and APTMS-MNPs were prepared
in different dilutions (25–400𝜇g/mL) and incubated with incubated at room temperature for 30min. Crystal violet
(CV) was discarded; plates were washed again 3 times with
subcultured bacteria. CFU/mL was counted on surface of
phosphate buffer saline (PBS) and allowed to air dry com-
agarplatesafter24hincubationperiods.
pletely.Biofilmformedineachwellwasresuspendedin97%
ethanol(200𝜇L/well)andincubatedatroomtemperaturefor
2.3.3. MNPs Inhibitory Potentials and Growth Kinetics. To
10min. The solubilized biomass (150𝜇L) was transferred to
evaluate the inhibitory activity of MNPs as well as growth
sterile96wellplatestobemeasuredspectrophotometrically
kineticsontestedbacterialstrains,cultureturbiditywasused
at 590nm. Data were presented as percentage inhibition in
as a measure of bacterial growth. OA-MNPs and APTMS-
biofilmgrowthinpresenceaswellasinabsenceofMNPs[25].
MNPs were double fold serially diluted in 96-well plate.
PositivecontrolwellsweredoublefoldseriallydilutedwithDI
water. All plates were inoculated with test bacteria as 2.4.2. Effect on Preformed Biofilm. In 96 multiwell plates,
10𝜇L/well (105CFU/mL) except for negative control wells 100𝜇Lbacterialsuspension(105CFU/mL)wasinoculatedin
∘
(blank);thiswascarriedouttoavoidinterferencecausedby each well and incubated at 37 C for 24h to initiate biofilm
light-scatteringpropertiesofNP[21].Plateswereincubatedat formation. After incubation period, plates were washed 3
37∘Cfor24hwithcontinuousshaking.Percentageinhibition timeswithsterilePBStoremoveanyunattachedcells.Double
was calculated according to Sachidananda et al. as follows foldseriallydilutedMNPs(OA-MNPsandAPTMS-MNPs)
[22]: wereaddedinallwellsexcept innegativecontrolwellsand
∘
incubatedat37 Cfor24htoevaluatetheeffectofMNPson
𝑎
Percentageinhibition=[1−( )×100]. (1) preformedbiofilms.MNPswerediscardedandtheremaining
𝑏
biofilmswerestainedwithCVaspreviouslydescribed[26].
𝑎 is optical density of test bacteria with MNPs; 𝑏 is optical
densityofpositivecontrolwellscontainingDIwater,bacteria, 2.5. Cytotoxicity Assay. Ninety-six well-plates were inocu-
andmedia. lated with normal human epithelial (WISH) cells (ATCC-
4
MIC50 is the lowest MNPs concentration that reduces CCL25)at10 cells/well.Onconfluency,culturemediawere
the bacterial growth by >50%. Optical density (OD) was discardedandplateswereinoculatedwithdoublefoldserially
6 JournalofNanomaterials
87.0
86.5
86.0
85.5
85.0
nce 8844..05 APTMS-MNPs ×105 Zeta potential distribution
mitta 8833..05 OA-MNPs 67
% trans 88881122....0505 MNPs ounts 45 OA-MNPs
788900...505 Total c 23 MNPsAPTMS-MNPs
79.0 1
78.5
0
4000 3500 3000 2500 2000 1500 1000 500 −100 0 100 200
Wavenumbers (cm−1) Apparent zeta potential (mV)
(a) (b)
MNPs
OA-MNPs
APTMS-MNPs
(c)
Figure3:(a)FT-IRspectraofAPTMS-MNP,OA-MNP,andMNPs.MNPs(10mg)weremixedandgrindedwithKBrtogiveafinalweightof
−1
0.12g.Themixturewasthenpressedintoadiscforanalysis.SampleswereFTIRscannedintherangefrom400to4000cm ataresolution
−1
of4cm .Eachspectrumisanaverageof32scans.Dataispresentedas%transmittance.(b)SurfacechargeofAPTMS-MNP,OA-MNP,and
MNP.(c)ScanningelectronmicrographofMNP,OA-MNP,andAPTMS-MNP.
JournalofNanomaterials 7
dilutedMNPs(exceptnegativecontrolwells)andincubated Table2:AveragezetapotentialofpreparedMNPs(𝑛=3).
at37∘Cfor24h.Residuallivingcellsweretreatedwith20𝜇L
Typeofparticles Zetapotential(mV)
sterile 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) dye (5mg/mL) at 37∘C for 4h. MTT was MNPs −18±3
discarded and plates were washed with PBS three times. CA-MNPs −31±4
Aliquots of 50𝜇L DMSO were added to each well. Plates OA-MNPs −29±4
were left on plate shaker for 30min to allow dissolution of Arg-MNPs −6±2
the intracellular formed blue formazan complex. OD was A-MNPs −9±3
measured at 570nm using an ELISA plate reader [27]. ATPMS-MNPs 24±5
Viabilitypercentagewascalculatedasfollows[27]:
Note.CA-MNP:citricacidmodifiedMNP;OA-MNP:oleicacidmodified
MNP;Arg-MNP:argininemodifiedMNP;A-MNP:aminemodifiedMNP;
APTMS-MNP:amino-propyltrimethoxysilaneMNP.
Cellviabilitypercentage
(2)
ODoftreatedcells
=( )×100.
ODofuntreatedcells )L 90
m 80
U/ 70
3.ResultsandDiscussion CF
( 60
80
1 50
3.1.Results ×
nt 40
u
3.1.1. Synthesis and Characterization of MNPs. MNPs were co 30
prepared using thermal coprecipitation method [12, 28]. erial 20
Results of XRD analysis shown in supplementary materials ct 10
a
(2)areinagreementwiththetypicalpatternfor𝛾-Fe2O3 as B 0 −31 −18 −6 −9.6 24.5 −29.2
initiallypredictedfromthechangeoftheircolorfromblack
troevberaolwednt[h2a9t,t3h0e]s.iTzreaonfsMmiNssPiownaesle6c–t1r5onnmmiacnrdostchoaptethimeyawgienrge Control A-MNPs MNPs g-MNPs A-MNPs S-MNPs A-MNPs
sphericalinshape(Figure2).FunctionalizationoftheMNPs C Ar M O
T
was performed to prepare hydrophobic MNPs and charge P
A
modulated ones (ranging approximately from −30mV to
S. aureus 74 72 75 60.2 50.5 42.4 29.03
+30mV). E. coli 68 66.2 67 64.7 52.2 44 31
SixtypesofMNPwereprepared(Figure1).Threepositive
charge shifted MNPs (A-MNPs, Arg-MNPs, and APTMS- Figure4:InhibitorypotentialofMNPs(MNPS,OA-MNPS,CA-
MNPs, Arg-MNPs, A-MNPs, and APTMS-MNPs) prepared as
MNPs) and three negatively charged particles (MNPs, OA-
100𝜇g/mLonS.aureusandE.coliafter24htreatmentbasedonCFU
MNPs, and CA-MNPs) were prepared and their surface assaymethod.Barchartrepresentsthemeanbacterialcount×108
chargewasmeasured(Table2).Figure3comparestheFTIR
aftertreatmentbyeachMNPspecies(chargedisplayedinmV)as
spectrum (a), the surface charge (b), and the scanning
comparedtocontrol.
electronmicrographofparticles(c).
3.1.2.AntibacterialandAntibiofilmPotentialofMNPs a concentration of 400𝜇g/mL, the OA-MNPs showed inhi-
bitionof83%and79%,whilethehydrophilicMNPsshowed
(1)AntibacterialActivity inhibitionof73%and72%forS.aureusandE.coli,respec-
tively(Figures5(a)and5(b)).
(a) Antibacterial Activity Screening. Synthesized MNPs
(100𝜇g/mL) with different surface charge and functional (b) Effect of MNPs on Bacterial Growth. Inhibitory activity
groups were examined for their antibacterial activities by of MNPs was determined using microdilution method by
countingthenumberofbacterialcoloniesdevelopedoverthe calculatingMIC50after24htreatment.TreatingS.aureusand
surface of agar plates. The highest antibacterial effect was E.coliwithOA-MNPsresultedinanMIC50 valueof31and
observed for OA-MNPs and APTMS-MNPs. Hydrophobic 63𝜇g/mL,respectively.Ontheotherhand,greaterconcentra-
negatively charged OA-MNPs showed greater reduction in tion of APTMS-MNPs (125𝜇g/mL) showed 50% inhibition
the number of CFU/mL (61%, 54%) compared to that towardsbothbacterialstarins(Table3).MIC50 resultsindi-
obtained posttreatment with hydrophilic positively charged catedthatOA-MNPsandAPTMS-MNPsexhibitedagreater
APTMS-MNPs(43%,35%)forS.aureusandE.coli,respec- inhibitorypotentialonS.aureusthanthatonE.coli(Table3).
tively(Figure4). Analysis of growth kinetics reflects a significant time
Investigation of the APTMS-MNPs and OA-MNPs and concentration dependent decrease in OD after OA-
antibacterialpotentialusingdifferentconcentrations(25–400 MNPsandAPTMS-MNPstreatmentandrevealedthegrowth
𝜇g/mL)revealedaconcentrationdependentreductioninthe inhibitorypotentialsoftheseparticlesontestbacteria(Fig-
CFU24hposttreatment(supplementaryinformation5).At ure6).
8 JournalofNanomaterials
Table3:Assessmentof50%minimuminhibitoryconcentrations(MIC50)ofMNPsusingmicrodilutionmethod.
%inhibition
MNPs(𝜇g/mL) S.aureus E.coli
OA-MNPs APTMS-MNPs OA-MNPs APTMS-MNPs
15.625 46±2 13±1 31±1 9±1
31.25 58±2 22±2 47±2 20.0±2
62.5 62±2 37±1 57±2 34.0±1
125 65±2 54±4 62±2 51±3
250 68±3 58±2 68±2 60±1
500 80±4 64±1 71±3 68±2
Note.OA-MNP:oleicacidmodifiedMNPandAPTMS-MNP:amino-propyltrimethoxysilaneMNP.
100 100
90 90
%) %)
nt ( 80 nt ( 80
u 70 u 70
o o
bacterial c 5600 bacterial c 5600
n 40 n 40
n i n i
o 30 o 30
ducti 20 ducti 20
Re 10 Re 10
0 0
25 50 100 200 400 25 50 100 200 400
MNPs(g/mL) MNPs(g/mL)
OA-MNPs OA-MNPs
APTMS-MNPs APTMS-MNPs
(a) (b)
Figure5:QuantificationofbacterialcellnumberposttreatmentwithdifferentconcentrationsofOA-MNPsandAPTMS-MNPsincaseofS.
aureus(a)andE.coli(b),respectively.Datarevealedaconcentrationdependentreductioninbacterialcount.Valueswereexpressedasmean
percentagereductioninbacterialcount±standarddeviationofthreeindependentexperiments.
(c)SurfaceAffinityofMNPstoBacteria.Afterbacteriawere (2)EffectonBiofilmFormationandPreformedBiofilm.Table4
allowed to interact with MNPs, MNPs were collected using andFigure8displaythepercentagereductioninabsorbance
an external magnet at varying time intervals (3–240min). of stained biofilm by CV assay after treatment of S. aureus
The reduction in OD as compared to initial OD of treated and E. coli with different concentrations of OA-MNPs and
bacterial suspension represents the bacterial cells bound to APTMS-MNPs.
MNPs. OA-MNPs(15.6–2000𝜇g/mL)reducedbiofilmformation
MNPs bound to bacteria instantly (after 3min). The by62–94%and48–96%forS.aureusandE.coli,respectively.
period of interaction did not affect the binding capacity. APTMS-MNPshowed30–91%and34–74%reductionofthe
OA-MNPsshowedthehighestpercentagereductionranging ability of S. aureus and E. coli to form a bacterial biofilm,
between 15%–46% and 30%–83% for S. aureus and E. coli, respectively.
respectively. Positively charged hydrophilic APTMS-MNPs OA-MNPs (15.6–2000𝜇g/mL) destroyed the preformed
recordedalowerreductionpercentagebetween14%–32%and biofilm by 29–94% for S. aureus and 17–93% for E. coli.
12%–23%forS.aureusandE.coli,respectively(Figure7). APTMS-MNPs (15.6–2000𝜇g/mL) were able to destroy the
The FTIR spectral difference between OA-MNPs before preformedbiofilmby19–89%and9–77%forS.aureusandE.
andafterattractingbacteriaisshown(supplementaryinfor- coli,respectively.
mation6).Arrowsindicatethepositionofthecharacteristic The inhibitory potential of OA-MNPs and APTMS-
peaksforE.coliandS.aureus,where(a)indicatespolysaccha- MNPs on the bacterial biofilm during its development
−1
ride (900–1200cm ) and (b) indicates the band attributed increased by increasing the concentration of MNPs. OA-
−1
to primary amine (1640–1560cm ) [31] for E. coli. For S. MNPs and APTMS-MNPs were able to destroy the pre-
aureus,thepeaksdenotedby(c)and(d)mayrepresentthe formed biofilm. A higher inhibitory biofilm potential was
C=NandtheC-H,respectively[32,33]. observedforOA-MNPsoverAPTMS-MNPs(Table4).
JournalofNanomaterials 9
1
1.0 0.9
0.9 0.8
0.8 0.7
m) 0.7 nm) 0.6
600D (n 000...456 600OD ( 00..45
O 0.3 0.3
0.2 0.2
0.1 0.1
0.0 // 0 //
1 2 4 6 24 1 2 4 6 24
Time interval (h) Time interval (h)
250g/mL 31.25g/mL 250g/mL 31.25g/mL
125g/mL 15.625g/mL 125g/mL 15.625g/mL
62.5g/mL Control S. aureus 62.5g/mL Control S. aureus
(a) (b)
0.8
0.8 0.7
0.7
0.6
0.6 m) 0.5
m) 0.5 n
600D (n 00..34 600OD ( 00..34
O
0.2
0.2
0.1 0.1
0 // 0 //
1 2 4 6 24 1 2 4 6 24
Time interval (h) Time interval (h)
250g/mL 31.25g/mL 250g/mL 31.25g/mL
125g/mL 15.625g/mL 125g/mL 15.625g/mL
62.5g/mL Control E. coli 62.5g/mL Control E. coli
(c) (d)
Figure6:GrowthkineticsofS.aureus(aandb)andE.coli(candd)inabsenceandpresenceofdifferentconcentrationsofOA-MNPs(aand
c)andAPTMS-MNPs(bandd).BacterialcellswereexposedtodifferentconcentrationsoftheMNPs(15.625,31.25,62.5,125,and250𝜇g/mL)
atdifferenttimeintervalscomparedtountreatedcontrolcells.Independenttriplicateexperimentswerecarriedoutforeachreaction;error
barrepresentsthestandarddeviationofmean.
3.1.3.CytotoxicityAssay. ThedegreeofcytotoxicityofMNPs The prepared MNPs were spherical and of average size of
tonormalhuman(WISH)cellswascarriedoutusingMTT 6–16nm. The charge of the synthesized MNPs was −18mV
assay(Table5).APTMS-MNPshadnocytotoxiceffectupto [8,31].TheXRDanalysis(supplementarymaterials2)showed
250𝜇g/mL.Theviabilitydecreasedto85.2%ataconcentra- typicalpatternformaghemite(𝛾-Fe2O3).
tionof500𝜇g/mL.Cellviabilitydeceasedfrom100%to52% Surface functionalization of MNPs with citric acid was
upontreatmentwithOA-MNPsconcentrationsbetween62.5 successfultoshiftsurfacechargeintoamorenegativepoten-
and1000𝜇g/mL,respectively(Figure9). tial (−31mV) [17]. Functionalizing MNPs with OA resulted
inanincreaseinnegativepotentialto−29.2mV[35,44].To
3.2.Discussion. Thereisaneedtoreducetheuseofconven- synthesize positive charge shifted MNPs, Arg (amino acid)
tional antibiotics and find alternatives to combat bacterial inducedachargeshiftfrom−18to−6mV[45–47].Ammonia
infections and biofilms. The ability of bacteria to form a adsorbedonMNPs’surfacewasabletoshiftsurfacechargeto
biofilm reduces its vulnerability to antibiotics and compli- −9.6mV,whileAPTMSshiftedsurfacechargeofMNPsfrom
cates eradication efforts. In this study, we have investigated −18to24.5mV[23].HydrophobicitywasinducedonMNPs
theinfluenceofsurfacefunctionalizationontheantibacterial bysurfacefunctionalizationwithOA.
andantibiofilmpropertiesofMNPs. The functionalization of the synthesized MNPs was
MNPs were synthesized by the classical thermal copre- confirmed by FTIR scanning. The unfunctionalized MNPs
cipitation method using ammonia as the alkylating agent. showed Fe-O characteristic absorption band at 634.4 and
10 JournalofNanomaterials
Table4:(a)Effect(%reduction)ofOA-MNPandATMPS-MNPonpreformedbiofilmforS.aureusandE.coli.(b)Effect(%reduction)of
OA-MNPandATMPS-MNPonbiofilmformationforS.aureusandE.coli.
(a)
MNPs(𝜇g/mL) OA-MNPs/S.aureus APTMS-MNPs/S.aureus OA-MNPs/E.coli APTMS-MNPs/E.coli
15.625 29 19 17 9
31.25 53 24 33 15
62.5 70 41 45 39
125 76 52 55 45
250 79 60 62 58
500 80 72 70 64
1000 86 74 79 72
2000 94 89 93 77
(b)
MNPs(𝜇g/mL) OA-MNPs/S.aureus APTMS-MNPs/S.aureus OA-MNPs/E.coli APTMS-MNPs/E.coli
15.625 62 30 48 34
31.25 67 33 64 35
62.5 86 43 67 38
125 86 50 81 39
250 90 74 87 44
500 92 79 92 51
1000 93 86 93 69
2000 94 91 96 74
y 100 Table 5: Cytotoxicity of APTMS-MNPs and OA-MNPs against
nsit 90 WISHcells.
e
cal d 80 MNPs(𝜇g/mL) Average%cellviability
pti 70 OA-MNPs APTMS-MNPs
o
duction in 456000 12625250.5 11800000 111000000
e re 30 500 74 85
ag 20
nt 1000 52 64
ce 10
er
P 0
0 50 100 150 200 250 300
Time (min) −
between the COO and the MNPs which resulted in the
OA-MNPs/E. coli OA-MNPs/S. aureus blue shift of the original C=O peak of OA which appears
APTMS-MNPs/E. coli APTMS-MNPs/S. aureus −1 −
at 1731cm . Harris et al. also reported the COO of OA
−1
Figure 7: Surface affinity of MNPs to S. aureus and E. coli to appear as a broad band between 1541 and 1639cm and
postexposureatdifferenttimeintervals.MNPswerecollectedwith concluded that the bonding pattern of the carboxylic acids
externalmagnetandopticaldensityoftheremainingsolutionwas onthesurfaceoftheNPwasatanangletothesurface[50].
measuredspectrophotometrically.Datawasrecordedaspercentage A-MNPs showed an amine peak between 3410 and
reductioninopticaldensityattimeintervalofthreeindependent 3457cm−1. CA-MNPs showed a peak at 2958.5cm−1 which
tests.
corresponds to CH2 stretching. The C=O of citric acid
−1
appeared at 1638cm [51]. Arg-MNPs showed amine peak
557.5cm−1 [48]. Two other bands around 3422.7cm−1 and between3410and3450cm−1,aC=Opeakat1634.9cm−1,and
1634.9cm−1correspondtostretchingandbendingvibrations a CH2 stretching band at 2923.2cm−1 [52]. APTMS-MNPs
of surface hydroxyl groups on surface of MNPs [49]. OA- werecharacterizedbytheCH2 stretchingwhichappearedat
−1 −1
MNPsshowedOAcoatingcharacteristicbandsat2923.8and 2920.8and2851.3cm ,N-Hbendingappearedat1631cm ,
−1 −1
2852.9cm whichcorrespondtoCH3 stretching.TheC=O C-Nbendingappearedat1384cm ,andabsorptionbandsin
−1 −1 −1
appeared as a peak at 1638cm and at 1618.3cm which theregion1000–1227cm canbeduetoSi-O-SiandSi-OH
supports the formation of a chelating bidentate interaction [53].FTIRspectraofthesynthesizedMNPsagreewiththose