Research Article Enhancement of the Antibacterial Activity of Silver Nanoparticles against ...

16 Pages·2016·3.2 MB·English

Hindawi Publishing Corporation
Journal of Nanomaterials
Volume 2016, Article ID 7135852, 15 pages
http://dx.doi.org/10.1155/2016/7135852
Research Article
Enhancement of the Antibacterial Activity of
Silver Nanoparticles against Phytopathogenic Bacterium
Ralstonia solanacearum
by Stabilization
JuanniChen,ShiliLi,JinxiangLuo,RongshengWang,andWeiDing
LaboratoryofNaturalProductPesticide,CollegeofPlantProtection,SouthwestUniversity,Chongqing400715,China
CorrespondenceshouldbeaddressedtoWeiDing;[email protected]
Received16December2015;Revised5April2016;Accepted5May2016
AcademicEditor:PiersandroPallavicini
Copyright©2016JuanniChenetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Inthispaper,theenhancedantibacterialactivityofsilvernanoparticles(AgNPs)againstthephytopathogenicbacteriumRalstonia
solanacearumafterstabilizationusingselectedsurfactants(SDS,SDBS,TX-100,andTween80)wasexamined,incomparisonwith
silverion.Tween80wasfoundtobethemostpreferablestabilizerofAgNPsduetothebeneficialsynergisticeffectsoftheAgNPsand
+
surfactant.However,allthesurfactantsnearlyhadnoeffectsontheantibacterialactivityofAg .Invitro,Tween80-stabilizedAgNPs
showedthehighestbactericidalactivityagainstR.solanacearum.FurthermeasurementsusingTEM,fluorescencemicroscopy,and
+ +
sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)revealedthatthoughAg andTween80-Ag inducedhigh
toxicity,Tween80-stabilizedAgNPsdisplayedmostseveredamagewhenindirectcontactwithcells,causingmechanisticinjury
tothecellmembraneandstronglymodifyinganddestructingthecellularproteins.Meanwhile,invivo,thepotexperimentsdata
indicatedthatthecontrolefficiencyofTween80-stabilizedAgNPsontobaccobacterialwiltwas96.71%,90.11%,and84.21%,at7
days,14days,and21days,respectively.Basedontheresultsevidencingtheiradvantageouslowdosagerequirementsandstrong
antimicrobialactivity,Tween80-stabilizedAgNPsareapromisingantibacterialagentforuseinalternativecropdiseasecontrol
approaches.
1.Introduction Today,oneofthemostpromisingstrategiesforcontrol-
lingbacterialwiltincludesthebreedingofresistanttobacco
As an important economical crop, tobacco (Nicotiana linescombinedwithimprovedculturalpractices[2].Previous
tabacumL.)hasbeengrownforthousandsofyearsandculti- studies have identified several genes governing resistance
vatedworldwide[1].However,bacterialwiltdisease,caused to bacterial wilt in tomato [5]. The Laurent group recently
by the phytopathogenic bacterium Ralstonia solanacearum identified RRS1-R, a gene conferring broad-spectrum resis-
(R. solanacearum), is prevalent in tropical regions with tancetoR.solanacearumandencodinganovelRprotein[6].
∘
temperatures above 30 C and has long caused significant Meanwhile, crop rotation using maize, soybean, sugarcane,
losses in tobacco production every year [2] and has been andricehasbeenextensivelyappliedtoreducetheincidence
one of the most widespread and disastrous agricultural ofbacterialwiltingroundnutcropsinChina[7,8].However,
diseases,whichinfectsvariousplantspecies,includingalarge theefficiencyofthisprocessishighlyvariablebecauseofthe
numberofagriculturalcropplantsandnativetrees[3].The numerousfactorsonwhichitdepends,suchastheviability
soilborne pathogen R. solanacearum initially invades crop- andpopulationoflocalbacterialstrains[8].Thus,innovative
injured roots, particularly lateral roots, and spreads along approaches are required to overcome these problems, and
theentirevascularsystemofthehostplantthroughbacterial there is an urgent need for a focus on promising new
colonizationinthecrownandstem,inducingwiltsymptoms technology,suchasnanotechnology.
[3].Studieshaveshownthatmajorvirulencefactors,suchas Nanotechnology employs nanomaterials, typically with
polysaccharides and secreted proteins, greatly contribute to particlesizeslessthan100nminatleastonedimension,and
thepathogenesisofbacterialwiltdisease[4]. has promising applications in medical science and material
2 JournalofNanomaterials
sciencebecauseofthesize-dependentqualities,highsurface- the enhancement of the antibacterial action of AgNPs.
to-volume ratio, and unique optical and physiochemical Intense efforts have been devoted to comparing the
propertiesoftheseparticles[9].Previousstudieshaveidenti- antibacterial activities of pure AgNPs with those of AgNPs
fiedpotentialusesofnanotechnologyinpracticallyeveryfield stabilized using selected surfactants agents, including SDS,
of agriculture, including the controlled release of fertilizers sodium dodecylbenzenesulfonate (SDBS), octylphenol
and micronutrients [10], pesticide degradation [11], biopes- polyethoxylate (TX-100), and polysorbate 80 (Tween 80).
+
ticidestabilization[12],nanosensingofplantpathogensand Ag wasusedtobecomparedwiththeantibacterialactivity.
pesticides[13,14],andsoilconservationandremediation[15]. The results demonstrated that the surfactant Tween 80 was
Nevertheless,nanotechnology-basedantibacterialagentsfor the most effective for improving the toxicity of AgNPs.
agriculture applications remain in the margins of academic Specifically,thehighbacteriostaticandbactericidalactivities
research. Possessing excellent antibacterial activity, silver of AgNPs against R. solanacearum in vitro were examined
nanoparticles (AgNPs) strongly inactivate a variety of bac- and the underlying toxicity mechanisms were investigated
terial pathogens (Gram-positive and Gram-negative) [16], byTEM,fluorescencemicroscopy,andSDS-PAGE.Further,
fungal pathogens [17, 18], and viruses [19] and have been this study focuses on the practical application of Tween
exploited for biomedical and environmental management 80-stabilizedAgNPsforcontrollingtobaccobacterialwiltin
+
applications [20, 21]. The release of silver ion (Ag ) from vivo.
their surface can be one of the significant reasons for their
+
high antimicrobial activity. Most of the Ag release occurs 2.MaterialsandMethods
from oxidation of metallic nanosilver by dissolved oxygen
andprotons[22].Muchlessinformationisavailableforplant 2.1. Chemicals and Apparatus. Silver nitrate (AgNO3), 1%
pathogens, but several studies have attempted to develop sodiumcitratedihydrate,andsodiumborohydride(NaBH4)
nanomaterials,suchasTiO2,asantibacterialandantifungal were obtained from Sangon Biotech (Chongqing) Co., Ltd.
tools for crop disease control [20]. Moreover, AgNPs have All the surfactants, including SDS, SDBS, TX-100, and
been utilized as a novel nanopesticide in research studies Tween 80, were purchased from Sigma-Aldrich. Additional
and have been efficiently employed to control agricultural chemicalswerepurchasedfromSangonBiotech(Chongqing)
plantdiseases[21–23].Notably,themostcommonapplication Co., Ltd. All solutions were made using sterile Milli-Q
problem involves the agglomeration and diffusion of these (mQ) water. Several instruments were used to characterize
nanoparticles,whichreduceantibacterialactivity.Thus,stud- andanalyzethephysicalandchemicalpropertiesofAgNPs
ieshaveusedvariousorganic[24]andinorganicsubstances samples,includingtransmissionelectronmicroscope(TEM,
[25] as well as powerful carriers [26] to stabilize AgNPs. JEM-2100, Japan). The ultraviolet absorption spectra were
These substances can strongly influence the antibacterial acquiredontheNicoletEvolution300UV-Visspectrometer.
activity and reduce the biological toxicity of nanoparticles. TheparticlesizedistributionandzetapotentialofallAgNPs
Except for the discharge of Ag+, the toxicity of AgNPs is dispersionswereevaluatedonMalvernZetasizerNanoSeries
highly dependent on their size [27], shape [28], surface (Malvern, UK). Cell morphology was conducted on TEM
chemistry [29], stability, and surface charge [10]. Previous (FEI,CzechRepublic).
studies have focused on the positive effects of stabilizers,
including sodium dodecyl sulfate (SDS), polyoxyethylene 2.2.SynthesisofAgNPsUsingtheChemicalReductionMethod.
sorbitan monooleate (Tween 80), cetyltrimethylammonium AgNPswerepreparedusingapreviouslydescribedprocedure
bromide (CTAB), and polyvinylpyrrolidone (PVP), on the [30]. AgNPs were obtained after reducing AgNO3 with
toxicity of AgNPs, particularly when modified by SDS, for NaBH4 in a water suspension using citrate as a stabilizing
which the minimum inhibition concentration (MIC) was agent.Briefly,5mLof10mMAgNO3wasmixedwith45mL
reduced below the “magic value” of 1𝜇g/mL [10]. This is of ultrapure water at 45∘C and rapidly heated to boiling.
because their surface charge and molecular structure are Subsequently,1mLof1%sodiumcitratedihydrateand300𝜇L
consideredtobethedominantfactorinchangingthesurface of 3mM NaBH4 were injected dropwise under vigorous
chemistry of AgNPs. Additionally, Pana´cˇek et al. showed stirring, and the resulting solution was boiled for 60min.
that SDS-capped silver nanoparticles at concentrations as After cooling under ambient conditions, the solution was
low as 0.05mg/L displayed better antifungal activity than filtered through a polycarbonate membrane (0.22𝜇m). The
silver nanoparticles alone [17]. Nonetheless, the toxicity final mixture was centrifuged at 8000rpm, precipitated,
of stabilized AgNPs also depends on the bacterial strain, and lyophilized. Alternatively, the prepared AgNPs were
and the detailed toxicity mechanisms of AgNPs against furtherstabilizedusingtheselectedsurfactants(SDS,SDBS,
phytopathogenic bacteria remain elusive. Therefore, it is TX-100, and Tween 80) at a final concentration of 1%
significanttoinvestigatetheantibacterialactivityofstabilized (w/w), and the pure and modified dispersed AgNPs were
AgNPs,therebypavingthewaytowardthedevelopmentofa then serially diluted. The morphology was observed using
broaderandnovelmeasurementtoplantmanagement. TEM (JEM-2100, Japan) and average size of the prepared
Herein, we used the phytopathogenic bacteria AgNPswasanalyzedbydynamiclightscatting(DLS)using
R. solanacearum, causing far-reaching catastrophic ZetasizerNanoZS(MalvernInstruments,England),respec-
diseases in tobacco and most other cultivated crops, tively. The UV-Vis absorption spectra were recorded using
as a platform to investigate the antibacterial activity of a Shimadzu UV-1650PC spectrometer to characterize the
silver nanoparticles and screen suitable stabilizers for AgNPs.
JournalofNanomaterials 3
2.3.SilverIonReleaseandEvaluationoftheStabilityofBare examinedinthepresentstudywasbetween39and0.04mg/L.
andStabilizedAgNPs. Thesilverionreleaseconcentrationof Namely, the AgNPs were modified with surfactants after
prepared AgNPs was monitored by the inductively coupled serially diluting the initial concentration of 1% (w/w) to
plasma mass spectrometer (ICP-MS) (Thermo Scientific). generate concentrationsrangingfrom0.5%to9.76×10−4%
Mainly, 1mL of 10mg/L AgNPs in Nutrient Broth (pH 7.0) (w/w). As a positive control, 100𝜇L R. solanacearum was
at different times (0, 5, 10, 20, 60, and 120min) was taken inoculated into the same volume of NB medium in the
to be centrifuged at 9000rpm for 40min. After that, the absence of nanoparticles, and 100𝜇L of pure broth mixed
supernatantcontinuedtobefilteredbyMicroconcentrifugal with same volume of nanoparticle served as a negative
filters(3000MWCO),madeofregeneratedcellulose.100𝜇L control. The inoculated plates were incubated at 30∘C for
ofthefinalfiltrateswasaddedto2%HNO3solutiontomakea 24h with shaking (120rpm), and the absorbance of each
totalvolumeof10mL.Afterward,thesamplesweremeasured well was determined using an automatic ELISA microplate
using inductively coupled plasma mass spectrometer (ICP- reader(ThermoMultiskanMK3,USA).TheMICvaluewas
MS)(XSeries2,ThermoScientific). defined as the lowest concentration of AgNPs at which the
To understand the stability characteristics of AgNPs, visiblegrowthofmicroorganismswassignificantlyinhibited
experiments were performed to evaluate whether the sur- comparedwiththeblank.
factants improved the particle stability by influencing the TheviabilityofR.solanacearumcellsmixedwithAgNPs
surface charge and aggregation state. The zeta potentials was determined after counting the standardized colony-
were measured by Zetasizer Nano ZS mentioned above formingunits(CFU).Briefly,afterincubatingtheinoculum
under different pH value via adjustment of the pH of the suspensions(∼105CFU/mL)andnanoparticlesfor24hwith
dispersionswithHCl(acidregion)orNaOH(basicregion). shaking (120rpm), gradient dilutions of each sample were
Wedeterminedtheparticlediametertostudytheeffectofion transferred onto LB agar plates, cultured at 30∘C for two
strengthonthestabilityofnanoparticle.NaClwasmixedwith days,andspotteduntilgrowthwasobservedonthecontrol
bare and surfactants-stabilized AgNPs (pH 7.0) to maintain plates.Subsequently,thecellcolonieswerecounted,andthe
the ion strength at various concentrations (10, 50, 100, and lowestconcentrationsshowingnogrowthorfewerthanthree
200mM). After incubation for 16min, particle diameter of colonies (approximately 99 to 99.5% killing activity) were
nanoparticlewasdeterminedbythesameinstrument. recorded as the MBC. The cell mortality (% of the control)
wasexpressedas(countsofthecontrol–countsofthetreated
2.4. Bacterial Culture. R. solanacearum strains were pro- samples)/counts of the control. All treatments were indi-
vided from the Laboratory of Natural Product Pesticide of vidually repeated at least three times. Control experiments
Southwest University (Chongqing, China). The separated were also conducted in parallel. Similarly, surfactants were
R. solanacearum strains were cultured in Nutrient Broth diluted with NB medium in a geometric progression from
(NB medium) containing 3.0g/L beef extract, 1.0g/L yeast 2to2048timestoobtainconcentrationsrangingfrom0.5%
extract, 5.0g/L peptone, and 10.0g/L glucose, pH 7.0, in to 9.76 × 10−4% (w/w). Given the major role played by the
a humidified incubator overnight at 30∘C under constant releasedAg+ ionsintheantibacterialactivityofAgNPs[31],
agitation. The bacteria cultures were harvested at the mid- asacomparisonassay,wealsomeasuredtheMICandMBC
exponential growth phase and centrifuged at 6,000rpm for ofsurfactants,Ag+,andsurfactants-modifiedAg+ usingthe
5mintocollectthecells.Thebacterialpelletwasthenwashed same methods. AgNO3 was used to supply the source of
three times with deionized water to wash off the medium Ag+.1000mg/LofAgNO3whichcontains635mg/LAg+was
constituents and other chemical macromolecules. The cells mixedwithsurfactantstoabstaintheinitialconcentrationof
wweerreedreilsuutsepdetnod1e0d5∼in10d6ecioolnoinzeyd-fowrmateinr,gaunndittsh(eCsFuUsp/menLs)iofnors a1%ge(owm/wet)r,iacnpdroagsrsetsastieodna.bove,AgNO3wasseriallydilutedin
experimentaluse.
2.6. Inhibition of Bacterial Growth. The bacteria cells were
2.5. Determination of the MIC and MBC. To investigate grown in liquid medium, and the bacterial growth was
the antibacterial activity of the synthesized nanomaterials measured using the same culture medium by comparing
against phytopathogen, stabilized AgNPs were compared the growth in the presence and absence of each type of
+
with pure AgNPs. The bacteriostatic and bactericidal activ- AgNP (pure AgNPs, Ag , and surfactant-stabilized AgNP
itiesofAgNPsagainstR.solanacearumwereexaminedusing dispersions)atdifferentconcentrations.Thebacteriagrowth
atypicalmicrodilutionmethod(ISO10932|IDF223:2010)to curvewasgeneratedaspreviouslydescribed[32].Typically,
determinetheminimalinhibitoryconcentration(MIC)and 100𝜇Lofthedilutedcellsuspensions(∼105CFU/mL)mixed
minimalbactericidalconcentration(MBC). with identical volumes of nanomaterials at different test
AgNPswereinitiallydilutedto78mg/LinaNBmedium. concentrations (39, 19.5, 9.76, 4.88, 2.44, 1.22, 0.61, 0.31,
∘
The dispersed AgNP stocks were then diluted with NB 0.15, and 0.08mg/L) was incubated at 30 C for 2h with
mediuminageometricprogressionfrom2to2048timesto gentle shaking. The control sample contained 100𝜇L of the
obtainconcentrationsrangingfrom39to0.08mg/L(39,19.5, cell suspensions added to 100𝜇L of DI water. The mixture
9.76, 4.88, 2.44, 1.22, 0.61, 0.31, 0.15, and 0.08mg/L). Next, was then transferred to 5mL tubes containing 2mL of NB
100𝜇Lofthepreparedbacterialsuspension(∼105CFU/mL) medium,andthetubeswereincubatedat30∘Cfor24hwith
was mixed with 100𝜇L of a series of diluted AgNPs in a shakingat120rpm.Thebacterialcelldensitywasmeasured
96-well ELISA plate. The final AgNP concentration range atanopticaldensity(OD)of600nmeverytwohoursusing
4 JournalofNanomaterials
aNicoletEvolution300UV-Visspectrometer.Allthesamples 2.10. A Pot Experiment. The control efficacy of Tween 80-
weretestedintriplicate,andtheaveragevaluewascalculated. stabilizedAgNPsontobaccobacterialwiltwasexaminedin
ThegrowthcurveswereplottedbytheODvalueat600nm vivo by pot experiments. Four leaves old tobacco seedlings
versusgrowthtime. growninpotswereselectedand20plantswerepreparedfor
each treatment. And 30mL of diluted Tween 80-stabilized
AgNPs suspension (1.22mg/L) was evenly irrigated into
2.7.CellImagingUsingTEM. Thebacterialcellswereexpo-
nentially collected and diluted to 4 × 108 colony-forming tobaccoroots.Ablankcontrolsamplewastreatedwithsterile
waterinthisexperiment.Thesepotswerearrangedrandomly
units(CFU)/mL.Subsequently,thecellswereincubatedwith
+ with three replicates per treatment. Twelve hours later, R.
Tween 80 (8% w/w), Ag (2.48mg/L), AgNPs (19.5mg/L),
solanacearumwasinoculatedvianoninjuredrootinoculation
and Tween 80-stabilized AgNPs (1.22mg/L) in the dark at at OD600 = 0.1 (≈107CFU/mL) per plant. The inoculated
∘
30 C for 3h with shaking at 120rpm. TEM was then used
tobaccoseedlingswerecultivatedinaplantgrowthchamber
to observe the morphological changes of the bacteria as at the temperature of 30 ± 1∘C, relative humidity of 85–
previouslydescribed[31].Aftertreatmentandcentrifugation
90%,andlightperiodof14h.FollowingtheinoculationofR.
at 6000rpm, the condensed cells were fixed with 2.5% solanacearum,thediseaseoccurrenceanddiseaseindexevery
glutaraldehyde,postfixedwith1%aqueousOsO4(Fluka),and 2dfromthefirstobservationofbacterialwiltdiseasedplants
washed with 0.1M phosphate buffer, pH 7.0. The samples were monitored until all of the control plants had almost
were then dehydrated in an ascending ethanol series (30, withered. The disease incidence, disease index, and control
50, 70, 80, 90, and 100%) for 15min and dried in a vac- efficacywerecalculatedataperiodof7d,14d,and21d.
uum oven. Thin sections containing the cells were placed
onto copper grids and observed under TEM (FEI, Czech
3.ResultsandDiscussion
Republic).
3.1. Dispersibility and Morphology Dispersibility. The mor-
2.8. Cell Imaging Using Fluorescence Microscopy. Briefly, phologyandaggregationofAgnanoparticlespreparedusing
7 8
1mL of the bacterial suspension (10 to 10 CFU per mL) the chemical reduction method were examined using TEM
+
and 100mL of 2.48mg/L Ag , AgNPs (19.5mg/L), and (JEM-2100,Japan).AsshowninFigure1(a),thesynthesized
Tween 80-stabilized AgNPs (1.22mg/L) were mixed in a silver nanoparticles were nearly spherical, and aggregates
∘
centrifuge tube and incubated at 30 C for 2h with gentle ranging from 10nm to 100nm in size were observed. After
shaking. After harvesting via centrifugation at 6000rpm, stabilizationwiththefoursurfactants,AgNPsseemtodisplay
the bacteria were resuspended in 1mL of water, and the betterdispersibility.Acomparisonwiththesizedistribution
cells were stained with 10mL propidium iodide (PI, excita- between the uncapped and stabilized AgNPs was further
tion/emissionat535nm/617nm;Sigma-Aldrich)for15min, analyzedbyDLS.Theresultsshowedthatparticleswithsmall
followed by counterstaining with 10mL 40-6-diamidino-2- size(<10nm)wereslighterinthepresenceofsurfactantsthan
phenylindole(DAPI,excitation/emissionat358nm/461nm; pristineAgNPs,butthedifferenceswerenotsignificant(Fig-
Sigma-Aldrich)for5mininthedark.Asacontrol,thecells ure1(a)).Thesesmallparticlesmaybefragmentedfromlarger
wereincubatedwithdeionizedwater.Thetestsampleswere spherical particles due to partial dissolution and recrystal-
then observed under an inverted fluorescence microscope lization [34]. It would be indicated that there is no change
(EclipseTi,Nikon).Thecelldeathpercentagewascalculated inmorphologyoraggregation.Inaddition,thenanoparticles
as the ratio of the number of cells stained with PI (dead were characterized using UV-visible spectroscopy, one of
bacteria) to the number of cells stained with DAPI plus PI the most widely used techniques for the structural char-
(totalbacteria). acterization of nanomaterials. The results revealed a max-
imum absorption peak at approximately a wavelength of
2.9. Protein Damage Assay. The protein damage in R. 410nm, consistent with the reported characteristic of the
solanacearum cells incubated with AgNPs was investi- surface plasmon absorption band of silver nanoparticles
gated using SDS-PAGE as previously described [33]. R. (see Figure S1 in Supplementary Material available online
solanacearumcellswerecollectedattheexponentialgrowth athttp://dx.doi.org/10.1155/2016/7135852).But,forstabilized
phaseanddiluted.Weselectedthefinalhighestconcentration AgNPs, there was slight red shift of the absorption band
+
to evaluate the damage of AgNPs to bacterial protein. Ag indicating the presence of an oxidized layer on the AgNPs
(2.48mg/L), Tween 80 (8% w/w), 19.5mg/L of AgNPs, and particlesurface[35,36].
1.22mg/L Tween 80-stabilized AgNPs were mixed with the
bacterial suspension for 2h, respectively, and cells treated 3.2.SilverIonReleasefromBareAgNPsandStabilizedAgNPs.
+
with sterile deionized water were used as a control. Subse- AgNPs can leach silver ion (Ag ) due to the oxidation of
quently,alltreatedsampleswereboiledinwaterfor10min, metallicnanosilverbydissolvedoxygenandprotons,which
and30𝜇Lofbacteriasuspensionwasmixedwith6𝜇Lof5x is an important factor to value their fate, transport, and
standardSDS-PAGEsampleloadingbuffer.Aftercentrifuga- biological interaction [36]. Silver ion was greatly in charge
tion,thesupernatantswereloadeddirectlyonto10%precast of the high antibacterial activity of AgNPs [37]. In this
+
polyacrylamide gels. Electrophoresis was performed on a experiment,Ag releasekineticsofbareAgNPsandstabilized
Bio-Rad vertical electrophoresis system at 100V for nearly AgNPsinNBmediumweremonitoredelectrochemicallyand
2h. analyzedusingICP-MStechnology.AccordingtoFigure2(a),
JournalofNanomaterials 5
14
12 12
12
10 10
10
ensity (%) 68 nsity (%) 68 nsity (%) 68
Int 4 nte 4 nte 4
I I
2 2 2
0 0 0
1 10 100 1000 1 10 100 1000 1 10 100 1000
Size (nm) Size (nm) Size (nm)
(a) (b) (c)
12 14
10 12
%) 8 %) 10
y ( y ( 8
nsit 6 nsit 6
nte 4 nte 4
I I
2 2
0 0
1 10 100 1000 1 10 100 1000
Size (nm) Size (nm)
(d) (e)
Figure1:TransmissionelectronimagesofbareAgNP(a),SDS-AgNPs(b),SDBS-AgNPs(c),TX-100-AgNPs(d),andTween80-AgNPs(e)
inwaterandtheircorrespondingparticlessizedistributionhistogram.NotethattheacronymsSDS,SDBS,TX-100,andTween80indicate
sodiumdodecylsulfate,sodiumdodecylbenzenesulfonate,TritonX-100,andpolysorbate80,respectively.
unstabilized AgNPs rapidly discharged high concentration 3.3. Effects of pH Value and Ionic Strength on the AgNPs
silver ions within 20min, and the content nearly reached Stability. To evaluate the stability of surfactants-modified
saturation (38.2𝜇g/mL) 20min. The reason for silver ion AgNPs,weinvestigatedthezetapotentialandparticlediam-
discharge involves the presence of an oxide layer on the eterunderdifferentpHvalueandionicstrength.Asweknow,
nanosilversurface[36,38].ForstabilizedAgNPs,theinitial AgNPspossessextremelyhighsurfacechargeddensities[35].
dissolution was more slightly prompt, as soon as, however, Stabilizer agents can usually change the surface charge and
+
the slowly ascending discharge occurs, and the Ag hardly hinder the nanoparticle aggregation process by enhancing
uncharged.Itcanbenotedthatthepresenceofthesurfactants electrostaticorstericrepulsioninteraction,therebyimprov-
induced subdued dissolution of silver ion from AgNPs and ing the stability of the nanoparticle suspension [31, 38].
+
thecontentofthereleasedAg decreasedwereconsistently Generally, zeta potentials greater than +30mV or less than
lessthanbareAgNPs,especiallyTween80-stabilizednanosil- −30mV are considered strongly to be stable. It can be seen
+
ver, Ag from which is rather minimal, reduced by 50% in from Figure 2(b) that bare AgNPs exhibited an obvious
comparisonwithbareAgNPs. declineinzetapotentialwhenthepHvalueofsolutionranged
6 JournalofNanomaterials
60 20
10
50
L) 0
𝜇g/m 40 mV) −10
elease ( 30 ential ( −20
Silver ion r 20 Zeta pot −−4300
10
−50
0 −60
0 20 40 60 80 100 120 2 3 4 5 6 7 8
Time (min) pH
AgNPs TX-100-AgNPs AgNPs TX-100-AgNPs
SDS-AgNPs Tween 80-AgNPs SDS-AgNPs Tween 80-AgNPs
SDBS-AgNPs SDBS-AgNPs
(a) (b)
140
120
m)
er (n 100
et
m 80
a
di
e
articl 60
P
40
20
0 10 50 100 200
NaCl concentration (mM)
AgNPs TX-100-AgNPs
SDS-AgNPs Tween 80-AgNPs
SDBS-AgNPs
(c)
Figure2:ReleaseofsilverionfrombareAgNPsandsurfactants-stabilizedAgNPs(a)andtheeffectofdifferentpHonthemeasuredzeta
potential(b)andtheeffectofionicstrengthontheparticlediameter(c)underpH6.8inthepresenceof100mMNaCl.
from 2 to 8, displaying small value. SDS induced a rapid chloride (NaCl) solution. It could be noted that, in the
decline in the surface charge of AgNPs, but zeta potentials presenceofelectrolytesolution(NaCl),sliverchloride(AgCl)
were moderatelydecreasedwiththeincreasingpHvaluein depositspossiblyaredevelopedonthesurfaceoforaround
the presence of SDBS and TX-100. The zeta potential was the nanoparticle, on account of the interaction between
fluctuatedat−30mV.FollowingtheDLVOtheory,thestrong chloridion (Cl−) and the Ag+ released from the dissolv-
stabilityeffectswereobservedbecausetheabsolutevalueof ing uncapped silver nanoparticle [35]. Our results in this
the surface charge reflected in the nearly doubled value of experiment showed that SDS- and SDBS-modified AgNPs
zetapotentialwassignificantlyincreased.However,changes exhibited a slighter decrease in the aggregation particle
insolutionpHhadnearlynoeffectontheelectricpotentialof diameter than that of pure AgNPs under the highest NaCl
stericallystabilizedTween80-stabilizedAgNPs,theaverage concentration (200mM), with the value equal to 83.2nm
ofwhichwas−36.7mVinthepHvariationrange,persistently and105.5nm,respectively.Differently,therewasalmostlittle
beingthestabilizingforceforAgNPs. change after stabilizing with Tween 80 under systems with
Further, we assessed the AgNPs stability by motion- different NaCl concentration and pH 7.0 within previous
ing the aggregation kinetics of nanoparticles in sodium 16min (Figure 2(b)), implying the nearly instantaneous
JournalofNanomaterials 7
+
Table1:Theminimuminhibitoryconcentrationandminimumbactericidalconcentrationofpuresurfactants,Ag ,andAgNPswithand
withoutsurfactantstowardR.solanacearum.
Dispersions MIC,mg/L(%) MBC,mg/L(%)
SDS 0.125% 0.5%
SDBS 0.0625% 0.125%
TX-100 2% 4%
Tween80 8% >16%
+
Ag 1.24mg/L 2.48mg/L
Ag++SDS 1.24(0.2×10−3%) 2.48(0.39×10−2%)
Ag++SDBS 1.24(0.2×10−3%) 2.48(0.39×10−2%)
Ag++TX-100 1.24(0.2×10−2%) 2.48(0.39×10−2%)
Ag++Tween80 1.24(0.2×10−3%) 2.48(0.39×10−2%)
AgNPs 4.88mg/L 19.5mg/L
AgNPs+SDBS 1.22(1.56×10−2%) 4.88(6.25×10−2%)
AgNPs+SDS 1.22(1.56×10−2%) 4.88(6.25×10−2%)
AgNPs+TX-100 4.88(6.25×10−2%) 19.5(0.25%)
AgNPs+Tween80 0.61(0.78×10−2%) 1.22(1.56×10−2%)
Noticethattheresultswererepeatedatleastintriplicate.
sterical stabilization for AgNPs by the protective Tween 80 concentrationvaluesof1.22mg/L.Incontrast,afterstabiliza-
layer. Further, the phenomenon greatly associated with the tionwithTX-100,nochangesinthebacteriostaticactionof
+
strongpreventionofAg dissolutionandparticleaggregates thisnanoparticlewereobserved,withthesamevalueforboth
+
(Figure 2(c)). This is because the less Ag released from MICandMBC.
AgNPsmaybesorbedontheirsurfaceandinducedtheless We also assessed the bactericidal activity of the various
formationofAgClsedimentation[38]. AgNPdispersions.Thecolony-formingunits(CFU)method
Above all, all the surfactants seem to enhance the wasused tofurtherverifythemortalityof R. solanacearum
nanoparticle’sstability.However,itisworthmentioningthat cells from the growing cells in the presence of different
Tween 80 tremendously plays a more important role than concentrations of suspensions. Notably, all the surfactants
other agents like SDS and SDBS in stability of the AgNPs testedinthepresentstudysignificantlydecreasedtheMBC
dispersion by forming low zeta potential and preventing ofAgNPs,particularlyTween80.Consistentwithaprevious
particleaggregates.Lietal.alsodemonstratedthattheSDS study, pure AgNPs, without the introduction of any surfac-
andTweencoatingcanconsistentlyreducetheinitialparticle tant, showed a high-intensity lethal effect at a high dose
sizesofcappedAgNPsacrosstheentirerangeofNaClcon- of 19.5mg/L. Surprisingly, the MBC of Tween 80-stabilized
centrationsduetoblockingparticledissolutionprocess[35]. AgNPs was 1.22mg/L, which is lower than that of pure
+
Similartothepreviousresults,thenonionicsurfactantTween AgNPs. In comparison, 2.48mg/L of Ag and 2.48mg/L
+
80performedstericallyrepulsiveinteractionsbythewayof of Tween 80-Ag induced a complete death of bacteria,
formation of thicker and denser coating layers compared respectively (Table 1). As for Tween 80-stabilized AgNPs, it
withelectrostericrepulsionofanionSDS,thusinducingthe contains0.46𝜇g/mLofAg+whenatthelethalconcentration.
improvementofnanoparticlestabilitymorethanotheragents ItmeansthatTween80-stabilizedAgNPsmakebettertoxicity
[31,39],beingconsistentwithourresults. effectsthanTween80-stabilizedAgNO3.Andweconcluded
the fact that the high antibacterial activity of Tween 80-
3.4. Bacteriostatic and Bactericidal Activity of the Pure and stabilized AgNPs is obviously associated with the released
Surfactant-Stabilized AgNPs. In this experiment, a phy- silverion,asstatedinpreviousstudy[40],but,foranother,it
topathogenicstrainofR.solanacearum,whichcausessevere isattributedtonotonlythepresenceofsilverionbutalsothe
bacterial wilt in tobacco, was used to investigate the bac- AgNPs.Thus,Tween80providedthegreatestincreaseinthe
teriostatic and bactericidal activity of pure and surfactant- antibacterial activity of silver nanoparticles, improving the
stabilized AgNPs. The MIC and MBC were determined toxicityefficiencyby16-fold.Theextraordinaryimprovement
using the microdilution method. As shown in Table 1, the ofantibacterialactivityofAgNPsbyTween80issupposedly
surfactantsaffectedtheantibacterialactivityofAgNPstoward connected with the strong steric stabilization effect, caus-
R.solanacearumtodifferentextents.Specifically,theantibac- ing the formation of weak interaction among nanoparticle
terial action of pure AgNPs was evidently enhanced upon assemblies,althoughTween80hadslighteffectonthesurface
dispersion in these surfactants. Among the four stabilizing chargeofAgNPs(Figure2)[31].
agents, Tween 80 was the most effective, yielding a MIC SDS and SDBS exhibited similar effects on the antibac-
of AgNPs (0.61mg/L, an extremely low value) that was 8- terialactivityofAgNPs,bothwithanMBCof4.88mg/L,at
foldlowerthanthevalueofpureAgNPs(upto4.88mg/L). which complete bactericidal activity was observed. Indeed,
The antibacterial activity of AgNPs was 4-fold higher in bothsurfactantsenhancedtheantibacterialactivityofAgNPs
bothSDBSandSDS,displayingidenticalminimalinhibition fourfold. The results have strong relations with the better
8 JournalofNanomaterials
stability of the silver NPs modified by anionic surfactants, R. solanacearum was evaluated in detail by measuring the
SDSand SDBS. Based on the research, the stabilizingeffect bacterial growth curves. The bacterial logarithmic growth
wasconnectedtotheelectrostaticstabilization,inducingdra- kinetics was monitored in NB medium (initial bacterial
maticincreaseofthesurfacechargeofAgNPs,thepotential concentration,∼105CFU/mL)afterincubationwithdifferent
value of which is approximately double that of unmodified concentrationsofpureandstabilizedsilvernanoparticles.
AgNPs(Figure2).Foranother,theproposedstericeffectof AsshowninFigure3,AgNPsinhibitedbacterialgrowth
the hydrophilic and hydrophobic groups of the surfactant in the presence and absence of surfactants over the wide
moleculesshouldalsobetakenintoaccount[41].Incontrast, range of concentrations assayed, and the growth inhibition
theMBCofAgNPsinthepresenceofTX-100was19.5mg/L, rateofthetestedbacteriadependedstronglyonthesurfactant
demonstratingthatTX-100hadnoeffectontheantibacterial concentration and type. To obtain clearer results from the
activityofAgNPs. bacterial growth curves, only five different representative
Ontheotherhand,toinvestigatetherelationshipbetween AgNPs concentrations were examined in the present study.
AgNPs toxicity and the use of surfactants, we conducted Theresultsshowedthatsurfactant-stabilizedAgNPsshowed
another antibacterial experiment using surfactants alone. astrongerinhibitioneffectonthegrowthofR.solanacearum
The results suggested that the application of surfactants- than did pure AgNPs. It is evident from Figure 3(a) that
stabilizedAgNPsnotonlyincreasedtheantibacterialactivity when treated with SDS and SDBS-stabilized AgNPs at a
of AgNPs against R. solanacearum, with the exception of concentration of 4.88mg/L, the R. solanacearum growth
TX-100, but also enhanced the activities of the surfactants inhibitionwasmuchfasterthanthataftertreatmentwiththe
(Table 1). Notably, a synergistic effect of the stabilizers sameconcentrationofpureAgNPs.Inparticular,Tween80-
and AgNPs on the antibacterial activity was observed. The stabilized AgNPs exhibited the strongest and most efficient
interaction efficiency differed by surfactant. As shown in growth inhibition at concentrations ranging from 0.15 to
Table1,pureSDSindicatedinhibitoryandcompletelylethal 1.22mg/L.AsshowninFigures3(a)–3(e),atarepresentative
effects of R. solanacearum at a treatment concentration of concentration of 1.22mg/L, all surfactant-stabilized AgNPs
0.125% and 0.5% (w/w), respectively, just as shown in the showedamuchstrongerinhibitoryeffectonbacterialgrowth,
growth curve (Figure S2A). However, the SDS-stabilized displayingtype-dependentinhibitioneffects.Theinhibition
AgNPs exhibited bactericidal effects at a concentration of tendencyofTween80-stabilizedAgNPsatadoseof1.22mg/L
4.88mg/L, in which the SDS content was only equal to indicated that they caused almost no bacterial growth or
0.0625% (w/w), a much lower dosage than that of pure cell death, similar to that observed with pure AgNPs at
+
SDS solution. Thus, the concentration of SDS-stabilized a concentration of 9.76mg/L. However, Ag induced an
AgNPs needed to induce a lethal effect on bacteria was obviousinhibitioneffectonthebacterialgrowthatitsMIC
4 and 2.5 times lower than that of pure SDS and pure (1.24mg/L).1.22mg/Lofsurfactants-stabilizedAgNPs,which
AgNPs, respectively. Previous studies have also confirmed only contains 0.46𝜇g/mL of Ag+, caused better bacterio-
+
thatSDS-stabilizedAgNPsexhibitedevenhigherfungicidal static activity than Ag . The phenomenon in our present
activity than pure AgNPs [17]. Similarly, SDBS and TX-100 experiment consists with previous studies, in which Tween
solution absolutely induced no growth of R. solanacearum 80-stabilizedAgNPsdisplaynotonlystrongerbacteriostatic
under the concentration of 0.125% (w/w) and 4% (w/w), activitybutalsobactericidaleffect.
respectively (Figure S2 and Table 1). In the case of bacteria
killingwithSDBS-andTX-100-stabilizednanoparticles,the 3.6.ObservationofMorphologicalChangesUsingTEM. The
concentration of SDBS decreased from 0.125% to 0.0625% important results obtained thus far suggest that the various
(w/w) and the concentration of SDBS decreased from 4% stabilizers affect the antimicrobial activity of AgNPs differ-
to 0.25% (w/w). Significantly, higher antibacterial activity ently.Amongthem,Tween80hasbeenfoundtobethemost
was observed for Tween 80-stabilized AgNPs (1.22mg/L), effective modifier for the improvement of the antibacterial
which contained only 0.0156% (w/w) Tween 80. In fact, activity of AgNPs. Therefore, in the further study, Tween
however,asshowninFigureS2DandTable1,theMICand 80-stabilizedAgNPswereselectedtoinvestigatethetoxicity
MBC of Tween 80 were 8% and above 16%. Although pure mechanism.
Tween 80 exhibited an inhibitory effect, it did not provide The initial step in AgNPs antibacterial activity involves
completebactericidalaction.Accordingtotheaboveresults, contact with the biologicalsamples [42]. The direct contact
surfactant-stabilizednanoparticlesstrengthenedthetoxicity betweenbiologicalcellsandnanomaterialsestablishesaseries
ofsurfactantsagainstR.solanacearum.However,itseemsthat ofnanoparticle/biologicalinterfaces,whichinducebiocom-
applicationofsurfactantshadnochangeontheantibacterial patible or adverse outcomes [43]. Therefore, it is necessary
+
activity of AgNO3, possibly because the Ag itself induced to study the interactions between pathogenic bacteria and
strongtoxicity,andthesurfactantconcentrationsinAgNO3 AgNPs. The underlying antibacterial mechanism induced
solution under MIC and MBC of mixture were not high throughphysicalcontactwasexaminedusingTEMtovisu-
enoughtoexertantibacterialactivity(Table1). alizethecellmorphology.AsshowninFigure4,thecontrol
samples(untreatedR.solanacearumcells)wereobservedas
3.5. Effects of Pure and Surfactant-Stabilized AgNPs on integratedcellstructureswithobviousouterenvelopes.How-
∘
Bacterial Growth. The growth inhibitory effect of AgNPs ever, after incubation with pure AgNPs (19.5mg/L) at 30 C
stabilized with four surfactant agents (SDS, SDBS, TX-100, for3h,thebacterialcellsdisplayed morphologicalchanges.
and Tween 80) against the tobacco bacterial wilt pathogen Thecellmembranebecamerough,andthecellstructurewas
JournalofNanomaterials 9
2.5 2.5 2.5
m
m 2.0 n 2.0 m 2.0
n 0 n
600D 1.5 60OD 1.5 600D 1.5
of O 1.0 e of 1.0 of O 1.0
Value 0.5 Valu 0.5 Value 0.5
0.0 0.0 0.0
0 2 4 6 8 101214161820222426 0 2 4 6 8 101214161820222426 0 2 4 6 8 101214161820222426
Time (h) Time (h) Time (h)
Control 2.44mg/L Control 1.22mg/L Control 1.22mg/L
0.61mg/L 4.88mg/L 0.31mg/L 2.44mg/L 0.31mg/L 2.44mg/L
1.22mg/L 9.76mg/L 0.61mg/L 4.88mg/L 0.61mg/L 4.88mg/L
(a) (b) (c)
2.5 2.5 2.5
m 2.0 m 2.0 m 2.0
n n n
600D 1.5 600D 1.5 600D 1.5
Value of O 01..50 Value of O 01..50 Value of O 01..50
0.0 0.0 0.0
0 2 4 6 8 101214161820222426 0 2 4 6 8 101214161820222426 0 2 4 6 8 101214161820222426
Time (h) Time (h) Time (h)
Control 4.88mg/L Control 0.31mg/L Control 1.24mg/L
1.22mg/L 9.76mg/L 0.08mg/L 0.61mg/L 0.31mg/L 2.48mg/L
2.44mg/L 19.5mg/L 0.15mg/L 1.22mg/L 0.62mg/L
(d) (e) (f)
Figure3:GrowthkineticscurveofR.solanacearuminthepresenceofdifferentconcentrationsofpuresilvernanoparticles(a)aswellasAgNPs
+
stabilizedbySDS(b),SDBS(c),TX-100(d)andTween80(e),andTween80mixedwithAg (f).Theconcentrationofsurfactantinstabilized
AgNPsisindicatedintheformof{AgNPsmg/L(surfactant%)},suchas19.5mg/L(0.25%),9.76mg/L(0.125%),4.88mg/L(6.25×10−2%),
2.44mg/L(3.125×10−2%),1.22mg/L(1.56×10−2%),0.61mg/L(0.78×10−2%),and0.31mg/L(0.39×10−2%).Similarly,theconcentrationof
surfactantinstabilized-Ag+isindicatedas0.31(0.04×10−2),0.62(0.09×10−2),1.24(0.19×10−2),and2.48(0.39×10−2).
partlyhollow,withdistortion.InthepresenceofTween80- greatly improves the ability of AgNPs to attach to bacterial
stabilizedAgNPs (1.22mg/L),muchmoredamage tothe R. cells.
solanacearumcellswasobserved.AsshowninFigure5(d),the
cellstructurewaslooser,andthecytoplasmicdensityofcells 3.7. Fluorescence Microscopy Imaging. The results of the
wasreduced,likelyindicatingthatthecellularcontentleaked present study provide strong evidence that bacteria expe-
out during the direct nanoparticle-cell contact, inducing rience structural changes and cell membrane damage in
subsequentbacterialgrowthinhibitionandcelldeath.These the presence of Tween 80-stabilized AgNPs. To determine
observationsclearlyconfirmedthatTween80couldstrongly whetherAgNPsexhibitstrongtoxicitytobacteriaandverify
enhancetheantibacterialactivityofAgNPs.Theparticle-cell the reliability of the bactericidal experiment, we conducted
interfaceplayedanessentialroleintheantibacterialactivity experiments using fluorescent dyes, namely, propidium
of AgNPs [40]. Strong interactions with the cell wall were iodide (PI) and 4-6-diamidino-2-phenylindole (DAPI). PI
observed,reflectingthehighsurfaceenergyandmobilityof and DAPI are effective imaging tools for the identification
+
nanoparticles[40].Meanwhile,thedilutionofAg fromthe of cell membrane damage. Live bacteria cells have intact
surface of AgNPs and subsequent attachment to negatively membranes and are impermeable to PI, which can only
chargedbacterialcellsisnotanegligibleantibacterialmech- enter cells with disrupted membranes. However, DAPI, a
anism[44,45],asstatedinTable 1. Basedonacomparison membrane-permeable dye, can easily cross the bacterial
+
assay, though Tween 80-Ag (2.48mg/L) also displayed a membrane and combine with DNA in the cell nucleus,
significant reduction in the bacterial growth in Figure 3, emitting blue fluorescence. After treatment with Tween 80-
some moderate destructions on the cell membrane were stabilizedAgNPsat1.22mg/L,thebacteriasuspensionswere
observed in TEM images (Figure 4(d)). Thus, the stabilizer dyedusingPIandDAPI,referringtothepreviousstudy[46].
10 JournalofNanomaterials
(a) (b)
(c) (d)
(e)
Figure4:TEMmicrographsof R. solanacearumcellsalone(a)andincubatedwith8%(w/w)Tween80(b),19.5mg/LpureAgNPs(c),
+ ∘
1.22mg/LTween80-stabilizedAgNPs(d),and2.48mg/LTween80-stabilizedAg (e)at30Cfor3hwithshakingat120rpm.
AsshowninFigure5(a),asignificantuptakeofblueDAPIby cells[47].Apreviousstudyinvestigatingthemolecularbasis
untreated R. solanacearum cells was clearly observed using of the induced toxicity of some compounds showed that
a fluorescence microscope (Eclipse Ti, Nikon). However, poly(phenyleneethynylene)(PPE)basedcationicconjugated
after counterstaining with red PI, the R. solanacearum cells polyelectrolytes (CPEs) and oligo-phenylene ethynylenes
+
incubatedwithTween80-stabilizedAgNPsandAg primar- (OPEs) can exert toxicity on E. coil cells, which is strongly
ily displayed red fluorescence as a result of the influx of associatedwiththeactionofROS,whichdirectlyorindirectly
membrane-impermeable fluorescent PI, indicating that the covalentlymodifycytoplasmicbiomolecules,suchasproteins
membraneintegrityofthebacteriawasdisturbed,consistent andDNA[48].Toourknowledge,thesestudieshaveshown
withthemorphologiesobservedintheTEMimagingstudies. that the strong toxicity of AgNPs toward bacteria reflects
physicalandchemicalinterferenceinvolvingcellmembrane
3.8.ProteinDamage. WeemployedSDS-PAGEtodetermine change or permeability induced by AgNPs and the release
whether the bacterial proteins were damaged in response of silver ions [37, 40]. However, studies have also shown
toexposuretomodifiedsilvernanoparticles.Examiningthe thatthegenerationoffreeradicals(intracellularROS)con-
cytoplasmicproteinsisimportantfordeterminingtheeffects tributestoAgNP-triggeredantibacterialactivity[49].Indeed,
ofnanomaterialexposure,particularlyexaminingthegener- there is lack of information available regarding the effect
ationoffreeradicalsandreactiveoxygenspeciesinbiological of AgNPs (or stabilized AgNPs) on the cellular protein of