2013

http://informahealthcare.com/ddiISSN: 0363-9045 (print), 1520-5762 (electronic)

Drug Dev Ind Pharm, Early Online: 1–11! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.780182

RESEARCH ARTICLE

Silver nanoparticles enhance Pseudomonas aeruginosa PAO1 biofilmdetachment

Ching-Yee Loo1, Paul M. Young2,3, Rosalia Cavaliere4, Cynthia B. Whitchurch4, Wing-Hin Lee1, andRamin Rohanizadeh1

1Advanced Drug Delivery Group, Faculty of Pharmacy, University of Sydney, Sydney, Australia, 2Woolcock Institute of Medical Research, University

of Sydney, Glebe, Australia, 3Discipline of Pharmacology, Sydney Medical School, University of Sydney, Australia, and 4The ithree Institute, University

of Technology Sydney, Ultimo, Australia

Abstract

Objectives: Silver nanoparticles (AgNPs) with a size ranging from 7 to 70 nm were synthesizedusing the ascorbic acid-citrate seed-mediated growth approach at room temperature.Methods: The 8 nm silver particles were prepared using gallic acid in alkaline conditions andused as seed to prepare AgNPs.Results: The presence of ascorbic acid and citrate allows the regulation of size and sizedistribution of the nanoparticles. The increase in free silver ion-to-seed ratio (Agþ/Ag0) resultedin changes of particle shape from spherical to pseudo-spherical and minor cylindrical shape.Further, a repetitive seeding approach resulted in the formation of pseudo-spherical particleswith higher polydispersity index and minor distributions of tetrahedral particles. Citrate-cappedAgNPs were stable and did not agglomerate upon centrifugation. The effect of AgNPs onbiofilm reduction was evaluated using static culture on 96-well microtiter plates. Resultsshowed that AgNPs with the smallest average diameter were most effective in the reduction ofPseudomonas aeruginosa biofilm colonies, which accounted for 90% of removal.Conclusion: The biofilm removal activities of the nanoparticles were found to be concentration-independent particularly for the concentration within the range of 80–200 mg/mL.

Keywords

Antibacterial, biofilm, chemical reduction,Pseudomonas aeruginosa, silvernanoparticles

History

Received 22 November 2012Revised 14 February 2013Accepted 20 February 2013Published online 17 April 2013

Introduction

Biofilms are typically made up from communities of microorgan-isms which have switched from a planktonic to a sessile state afteradhering to a particular surface1. Unlike their planktonic state,biofilms are highly organized, in which microorganisms commu-nicate by chemical signals and build protective structures thatenable them to survive the hosts’ natural defenses and antimicro-bial agents. Once adhered, it is extremely difficult to removebiofilms from the surface; thus biofilms can cause many life-threatening infections in patients treated using medical devices,such as intratracheal tubes for ventilation1,2. It is therefore achallenge to identify antimicrobial agents that are effectiveagainst the colonization of wide ranges of biofilms and simul-taneously exhibit low toxicity levels for patients.

The renewed interest in silver and its nano-sized particles maybe generally related to the rapid emergence of antibiotic-resistance bacteria. Unlike conventional antibiotics that havenarrow therapeutic targets, the likelihood of microorganismacquiring resistance against silver ions is slim, since silver ionssimultaneously act on multiple sites within bacterial cells that arecritical to physiological function (e.g. cell wall, DNA/RNA

syntheses and electron transport)3. Most importantly, silver ionsare generally reported to be compatible with human tissues4.According to WHO standards, 0.1 mg/L of soluble silver (ions) indrinking water is considered safe for humans5. Interestingly,however, the soluble silver ions are only effective for short periodsof time. In comparison, nano-sized silver particles can beprevalent for as long as 200 d due to longer residence time atthe site of infection1. Silver nanoparticles (AgNPs), clusters ofsilver atoms ranging in diameter from 1 to 100 nm are potentantimicrobial agents used in many fields including the medical,food, electrical and textile industries6,7. It is believed thatantimicrobial efficiency of AgNPs is influenced by their size inwhich smaller particles confers higher surface area-to-volumeratio, and thus greater bactericidal effect8. Literature on thebactericidal effect of AgNPs with respect to their size andsynthesis methods is extensive9–12. Martınez-Castanon et al.reported a size-dependent bactericidal effect of AgNPs in whichminimum inhibitory concentration (MIC) of 7 nm and 29 nmAgNPs towards Escherichia coli were 6.25 and 13.02 mg/mL,respectively13. The MIC of �40 nm AgNPs prepared via irradi-ation in the presence of sodium dodecyl sulfate (SDS) towardPseudomonas aeruginosa was 2mg/mL14. In another study, theMIC of 26-nm AgNPs against P. aeruginosa CCM 3955,S. auereus CCM 3953, E. coli CCM 3954, Enterococcus faecalisCCM 4224 and various isolated clinical strains were in the rangeof 1.69–13.5mg/mL15.

Address for correspondence: Dr Ramin Rohanizadeh, Faculty ofPharmacy (A15), University of Sydney, Sydney, New South Wales,Australia. E-mail: ramin.rohanizadeh@sydney.edu.au

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A remarkable range of novel techniques have been employed tosynthesize AgNPs with varying degrees of sizes, shapes and stabil-ity. The most common approach is through the reduction of silvernitrate (via a reducing agent). When AgNPs are prepared usingstrong reducing agents, such as sodium borohydride16 and gallicacid13 the resulting particles tend to be monodispersed and spher-ical. While this process produces highly reproducible particles, thesize range is somewhat limited to 510 nm. The use of weakerreducing agents, results in broader particle size ranges; however,poly-dispersed systems are often produced with a combination oftwo or more particle shapes17. In addition, methods such as photo-reduction via UV-light18, irradiation19, sol-gel process20, micro-emulsions21,22 and manipulation of the microenvironment (e.g.ionic strength, pH, temperature and the use of capping agents) havesuccessfully produced AgNPs with controllable sizes and shapes.Seeding-mediated growth is an another relatively easy approach toregulate the shapes and sizes of nanoparticles such as gold23.

Here, we report a simple and relatively fast synthesis ofun-agglomerated AgNPs of 7–70 nm in diameter through seed-mediated growth assisted by ascorbic acid and citrate. Thisapproach involves a step-wise procedure for the preparation oflarger AgNPs using a weaker reducing agent from mono-dispersed small AgNPs (Figure 1). Specifically, we report onthe preparation of AgNPs with various sizes using the seedinggrowth approach by manipulating synthesis conditions such asion-to-seed ratio (Agþ/Ag0), ascorbic acid addition rate andconcentration. While, the antimicrobial effect of AgNPs againstplanktonic microorganisms has been previously documented,studies on their effect on biofilm are relatively few. Therefore, thisstudy aims to investigate the efficacy of different AgNPs sizes onthe detachment of P. aeruginosa PAO1 biofilms.

Materials and methods

Materials

Silver nitrate AgNO3 (MP Biomedicals, Santa Ana, CA), sodiumcitrate (Sigma Aldrich, St. Louis, MO), gallic acid (MPBiomedicals) and ascorbic acid (Sigma Aldrich) were used asreceived. Deionized water (H2O) was purified by reverse osmosis(milliQ, Millipore, Billerica, MA).

Preparation of AgNPs seeds

AgNPs with average diameter of 8 nm were prepared according tothe gallic acid reduction method as described by Martinez-Castanon et al13. All glassware were ultrasonically cleaned withabsolute ethanol before use. Briefly, 100 mL of aqueous AgNO3

(10�3 M) was prepared in a 250 mL flask. While stirring, 10 mLof deionized H2O containing 10 mg gallic acid was added into thesolution, followed immediately by adjusting the pH solution to 11using 1 M NaOH. The stirring continued for 10 min. The preparedAgNPs (sample A) were used as seed particles for subsequentexperiments (Figure 1).

Preparation of differently sized AgNPs ranging from 10 to60 nm in the presence of 8 nm seeds

Five sets of solutions labeled B to F containing sodium citrate(5� 10�4 M) and AgNO3 (Table 1) were prepared. Differentamounts of the 8 nm seeds (sample A) were added into thesolutions so that the final silver atom concentration in thesolutions was 1� 10�3 M and the mixture was brought up to150 mL using deionized H2O. While stirring, 100 mL of2� 10�4 M ascorbic acid was added at a rate of 7.5 mL/minfollowed by continuous stirring for 1 h.

Preparation of AgNPs in the presence of sample B asseeds

A set of six solutions labeled as B1 to B6 was prepared containingdifferent ratios of Agþ/Ag0 (Table 2). The final silver atomconcentration in the solutions was 1� 10�4 M and the mixtureswere diluted to 150 mL using deionized H2O. Immediately, whilestirring, 100 ml of 4� 10�4 M ascorbic acid was added at the rateof 10 mL/min.

Recovery and purification of AgNPs using dialysis andcentrifugation

Two methods were applied and compared to purify preparedAgNPs: centrifugation (20 min, 15 000 g, 15 �C) and dialysis usinga Cellu-Sep T2 (Fisher Biotec, Perth, Australia) membrane in abuffer containing 70% (v/v) ethanol and 30% (v/v) water. For the

Figure 1. Schematic diagram outlining the preparation of different sizes of AgNPs via seeding growth approach, recovery and biofilm removal assay.

2 C.-Y. Loo et al. Drug Dev Ind Pharm, Early Online: 1–11

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centrifugation method, the pellet was washed with deionized H2Oto remove the excess of Agþ ions and the pellet was subsequentlylyophilized. For the dialysis of AgNPs, the ratio of AgNP to thebuffer was 500 and buffer solutions were changed three times.Both lyophilized AgNPs powders (from centrifugation) andAgNPs suspensions (from dialysis) were analyzed using trans-mission electron microscope (TEM) and UV-visspectrophotometry.

Characterization of AgNPs

Absorption spectra of particle suspensions were recorded using aHitachi U-200 (Tokyo, Japan) spectrophotometer at a wavelengthfrom 280 to 600 nm. Particle size distribution was determinedusing dynamic light scattering (DLS, Malvern, UK). DLS wasperformed using a Malvern Zetasizer Nano ZS with the followingsettings: the refractive index for silver was 1.35 and the viscosityof water was 1.0002 mPas. The samples were measured in aquartz cuvette at 25 �C. Particle size distributions were measuredin triplicate (n¼ 3). Transmission electron microscopy (TEM)was carried out using a JEOL1400 (Tokyo, Japan) electronmicroscope operating at 200 kV. Approximately 1 mL of particlesuspension were added dropwise onto 200-mesh carbon coatedHoley copper grids and dried at room temperature. TEM imageswere taken at random areas of the grid and approximately 500–1000 nanoparticles were measured for size distribution.

In vitro biofilm formation assay and quantification

The biofilm formation assay was performed in microtiter plates inwhich P. aeruginosa PAO1 was first allowed to attach to thesurface of 96-well polystyrene plate. Three independent replicatesof experiments were conducted on separate days with fivetechnical replicates performed during each assay. Briefly, P.aeruginosa was grown overnight in cation adjusted Mueller–Hinton broth (CAMHB, Oxoid, Cambridge, UK) medium at37 �C, shaken at 250 rpm and diluted 100-fold to 106 CFU/mL(determined by OD and plate count assay) with CAMHB or

modified M9 media. A 100 mL aliquot of the culture was placedinto each well and the plates were incubated without shaking at37 �C. After 24 h, the liquid was gently removed and the wellswere rinsed twice with phosphate buffer saline (PBS) before beingsubjected to AgNPs treatment.

The effect of AgNPs on the biofilm was studied at a rangeof different concentrations for each particle size as well as forfree Ag in solution. The freeze-dried AgNPs powders werere-suspended in water and homogenized thoroughly using a waterbath-sonicator for 15 min, prior to the study. A 100 mL aliquot ofre-suspended AgNPs was added to the wells containing biofilm andincubated for 2 h at 37 �C. Five wells were treated for each particlesize and concentration. The wells were washed extensively withPBS and the remaining attached biofilm was stained with crystalviolet (CV), left for 1 h before being re-washed and de-stained withethanol-acetone. The optical density (OD) was measured using aBeckman microtiter plate reader at 595 nm. The biomass of biofilmcorresponded to the absorbance at 595 nm. The percentage ofremoval biofilm was calculated using the following equation:

Initial biomass� remaining attached biomass

Initial biomass� 100% ð1Þ

Qualitative imaging of P. aeruginosa PAO1 biofilms usingconfocal laser scanning microscopy

The morphologies of the biofilm were analyzed using confocallaser scanning microscopy (CLSM) (Nikon A1, Tokyo, Japan).Samples were treated with different AgNPs sizes containing600mg/mL equivalent of Ag content. Both control and treatedbiofilm samples were stained using SYTO9 (Invitrogen, GrandIsland, NY) and fixed with 4% paraformaldehyde. After washing,the stained biofilm samples were imaged using CLSM using oilimmersion lens (100� objective lens and numerical aperture of1.4). Recorded images were reconstructed by Imaris andpresented as three-dimensional structures. In addition, enumer-ations of remaining biofilm were performed using viable platecounting and data were expressed as colony forming units per well(CFU/well).

Statistical analysis

Statistical analysis of data was performed using the SPSSStatistics 19 software package (IBM, Armonk, NY). All datawere collected (n¼ 5) and the mean values and standarddeviations (SD) were calculated. The statistical differencesbetween the groups were determined by analysis of variance(ANOVA). The pairwise comparisons of individual group meanswere performed using the Tukey post-hoc analysis. Values ofp50.05 were considered statistically significant.

Results and discussions

Optimization of AgNPs fabrication method and particlecharacterization

In this study, the seeding growth approach was applied with�8-nm AgNPs as seeds and ascorbic acid as reducing agent inorder to produce a wider range of AgNPs sizes. Small, sphericalAgNPs seeds were synthesized using a chemical reductionmethod with a strong reducing agent. In this case, gallic acidwas used as both reducing agent and stabilizer13. In acidiccondition, the solutions were colorless and turned immediatelydark yellow as the pH was adjusted to 11. The reduction of metalions into metal particles was achieved through the oxidation ofphenolic groups and subsequently stabilized the formed AgNPs24.

Table 2. Summary of AgNPs synthesis using predetermined amount ofsample B as seeds. AgNO3 was added so that final silver atomconcentration was 10�4 M.

SampleSeeds added

(mL)

Volume of0.01 M AgNO3 addedto the reaction (mL) Agþ/Ag0

B1 15.00 1.00 0.7B2 7.50 1.75 2.35B3 2.50 2.25 10B4 1.00 2.40 25B5 0.75 2.43 35B6 0.50 2.45 50

Table 1. Summary of AgNPs synthesis in the presence of 8 nm AgNPs asseeds (sample A). For samples B to F, AgNO3 was added so that finalsilver atom concentration was 10�3 M.

SampleSeeds added

(mL)

Volume of0.1 M AgNO3 addedto the reaction (mL) Agþ/Ag0

A None None NoneB 15.00 2.35 1.6C 5.00 2.45 5D 2.50 2.48 10E 0.50 2.50 50F 0.25 2.50 100

DOI: 10.3109/03639045.2013.780182 Antibiofilm of citrate- and ascorbic acid-capped AgNPs 3

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It is suggested that the oxidation of phenolic groups ispH-dependent and the rate is higher in alkaline conditions, thusin turn enhancing the metal ion reduction rate forming smallspherical particles24. The TEM micrographs (Figure 2A) showedthat the particles prepared via this method were relatively mono-dispersed and primarily of spherical shape. Figure 3(A) shows thecorresponding histogram for the particle size distribution (n¼ 500measurements) determined from TEM micrographs (Figure 2A)whereby the average diameter of AgNPs was 8 nm. Although themajority of particles (490%) were less than 10 nm, larger particles(e.g. 15 nm) amounting to 10% were also present.

In order to produce a wider range of AgNPs sizes with narrowdistribution, a seeding growth approach was used. The 8 nmAgNPs were employed as seeds with ascorbic acid as reducingagent. The addition of ascorbic acid into the solutions containingboth AgNPs seeds, metal salts (AgNO3) and sodium citrate asstabilizer was conducted drop-wise using a burette at 7.5 mL/min

rate and the resulting particles were stirred continuously for morethan 1 h. As a control, the same procedure conducted without theaddition of AgNPs seeds, resulted in the Agþ solutions thatremained clear, without the Ag plasmon band (410 to 460 nm),indicating that AgNPs were not formed. Such observationsdemonstrate that ascorbic acid was too weak to reduce Agþ intoAg0 at room temperature without seed material (results notshown) possibly related to a coordination effect with citric acid,as reported previously25. In the presence of seed AgNPs, thesolutions changed immediately from yellowish to red color asascorbic acid was added.

Five sets of AgNPs corresponding to the samples B to F wereprepared according to the synthesis conditions outlined in Table 1and Figure 1. The respective shapes and sizes of samples B to Fare shown in Figure 2(B) to (F). By varying the ratio between freesilver ion to silver seed (Agþ/Ag0), different particle sizes wereachieved. When the Agþ/Ag0 ratio was 1.6 and 5, respectively,

Figure 2. TEM micrographs showing an increase in AgNPs particle size, formed by slow addition of ascorbic acid as Agþ/Ag0 was increased. (A to F)correspond to histograms A to F in Figure 3 and Table 1, while B1 to B6 corresponds to Figure 4 and Table 2.

4 C.-Y. Loo et al. Drug Dev Ind Pharm, Early Online: 1–11

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the produced nanoparticles were spherical and enlargement ofparticles were not significant (Figure 2B and C) with sizesdetermined from TEM images being 7 and 11 nm, respectively(Figure 3B and C).

As the amount of nanoparticle seed material decreased to2.5 mL (sample D), 0.5 mL (sample E) and 0.25 mL (sample F),the resultant nanoparticles became pseudo-spherical with aver-age diameters of 20, 35 and 50 nm, respectively (Figures 2

and 3). Sample F was stable only for a few hours and turnedinto dark purplish color with black precipitates. The particleplasmon resonance bands for both sample A and B were sharpand intense at 410 nm and red-shifted as the particle sizesincreased (Figure 3G). In particular, the full half-widthmaximum (FHWM) for sample F was broader and a weakband at 500–550 nm was visible; indicating particleaggregation.

Figure 3. (A to F) Histograms showing the size distribution of AgNPs determined from TEM. (G) Extinction spectra of AgNPs prepared via theseeding growth approach with varying ratio of Agþ/Ag0. (H) Comparison between estimated (from Equation (1)) and experimental particle size(expressed as diameter, nm) as a function of Agþ/Ag0 ratio for ascorbic acid-seeded growth from seeds with an average diameter of 8 nm.

DOI: 10.3109/03639045.2013.780182 Antibiofilm of citrate- and ascorbic acid-capped AgNPs 5

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In our study, the use of sodium citrate as capping agentsconfers several advantages: (i) regulating size distributions withrespect to the concentrations used17,26; (ii) limiting the aggrega-tion process during early particle formations via electrostaticmechanisms27; and (iii) acting solely as stabilizer in reactiontemperature without competing against the reducing agent(ascorbic acid). Strong capping agents has been known to inhibitparticle growth which in turn generates high monodispersity26. Inaddition, we observed that AgNPs (e.g. sample E in Figure 2)produced in similar conditions, without the presence of sodiumcitrate, were approximately two orders of magnitude larger thanthe AgNPs prepared in the presence of sodium citrate; withbroader size distributions and less stable (results not shown). Thestabilization effect of AgNPs by citrate relied on the electrostaticinteractions between capping ligands and the core of nanoparti-cle28 and it is influenced to a larger extent by the citrateconcentration26. The effective range of concentration26 wasbetween 1 and 5� 10�4 M as coalescence/agglomeration wasobserved at the concentrations of51� 10�4 M or �15� 10�4 M.As reported by Pillai and Kamat17, anionic citrate bonded stronglyon the surface of Ag0 seeds and grew slowly to reach an optimalpoint where strong repelling layers of citrate is enough to preventthe aggregation.

Generally seed-mediated nanoparticle growth is considered tobe due to growth along nanocrystal lattice of silver ions at theseed surface29, suggesting that size should increase in tandemwith increasing Agþ/Ag0 ratio. However, in sample B, the averagediameter was approximately similar to that of the initial seedmaterial with a narrower distribution. The size-dependentconcentration relationship was further assessed using a simpletheoretical calculation based on the assumption that all sphericalparticles grow with no nucleation and without apparent changes inparticle shape30. On the basis of spherical model for AgNPs, theexpected diameter, d, of particles was summarized in Figure 3(H)and 4(B8) using the following equation30:

r ¼ rs �Cseeds þ Cions

Cseeds

� �1=3

ð2Þ

where r and rs indicate radius of grown particles and seeds,respectively, Cseeds and Cions refer to concentration of added seedsand metal salts, respectively. The Equation (2) assumes theconversion efficiency of Agþ into Ag0 is 100% and all seeds grew.

In Figure 3(H), the estimated theoretical particle size wascompared to the experimental value. From Figure 3(H), it is clearthat the particle diameters determined from TEM images weresmaller than the theoretical values. Although values for the seedradius, additional metal ion concentration and the amount ofadded AgNPs as seeds could be theoretically calculated, the actualconcentration of AgNPs and Agþ involved in seeding growth maynot be 100%. In general, the variation of Agþ/Ag0 ratio shoulddirectly influence the particle size whereby as the amount of seedswas decreased, the particles size increases. However, in our study,it was observed that the addition of a high volume of seeds led tothe formation of AgNPs which were smaller or of similar size tothat of the seed (e.g. Figure 3B). The discrepancy in theoreticalvalues and experimental size, shown in Figure 3(H), reflects theover-simplification of the spherical model for AgNP formation.Additional factors including particle dissolution, the presence/types/mode of actions of stabilizers, concentration and type ofreducing agent and other microenvironment conditions includingpH, temperature and ionic strength may contribute to such avariation in reduction rate of Agþ into Ag0. In an earlier study byJana et al., it was observed that the size of gold nanoparticlesdiffered according to the rate of addition of ascorbic acid,whereby experimental data closely fitted to an estimated value

when the addition rate was controlled drop wise at 10 mL/min31.According to the authors, they argued that slow addition ofreducing agent was effective in suppressing further nucleation,subsequently improving the monodispersity of particle sizedistributions31. As mentioned above, the presence of citrateinhibited or slowed the growth of particles that possibly explainedthe relatively smaller particle size showed in Figure 3(H)compared to that of the theoretical data. We also preparedAgNPs in different concentrations of ascorbic acid and sodiumcitrate. Preliminary data obtained by the TEM analysis showedthat the increase of ascorbic acid concentration significantlyincreased the final particle size at the expense of the particlestability, which further demonstrated that the external factors areimportant in regulation of AgNP particle size.

Sample B resulted in narrowly distributed mono-dispersedAgNPs (7� 2 nm) that could be used as initial seeds for furtherascorbic acid-citrate mediated growth, using a final concentrationof silver atoms fixed at 10�4 M. The TEM micrographs ofresultant particles were shown in Figure 2 (B1–B6) and theircorresponding distribution histograms in Figure 4. A typical UV-vis spectrum monitoring the formation of AgNPs in the presenceof varying amount of seeds and metal salts was shown in Figure 4(B7). As expected, a red-shift of �max from 400 to 440 nm wasevident as the ratio of Agþ/Ag0 was increased. In addition, �max

increases with an increase in nanoparticle size. The estimatedparticle sizes were relatively higher than the experimental data atAgþ/Ag0 below 25 (Figure 4B8). At ratios above 25, the measuredsizes fitted closely to estimated values, indicating that growthonly occurred under these experimental conditions. This indicatedthat the AgNPs were dominantly spherical or roughly spherical inshape32, which was further supported by TEM images (Figure 2).Meanwhile, the �max of B4, B5 and B6 were shifted to 420, 430and 440 nm, respectively, which could be correlated to thepresence of polyhedral (e.g. decahedral) shaped particles asdemonstrated by TEM (Figure 2). As observed previously,spherical or pseudo-spherical AgNPs appeared in blue parts(�400 to 450 nm) of the spectrum while the �max for pentagonal/decahedral and truncated particles tend to shift towards red(�500 nm) and green (�650 nm) regions of the spectrum32. Theappearance of minor percentages of faceted polyhedral ortruncated shaped nanoparticles as a result of repetitive seedingis possibly attributed to the variation in relative growth rates atdifferent faces of particles and the interference of reduction ratedue to the competition between the reduction process and thecapping action of stabilizers33.

After AgNPs preparation, two different methods for purifica-tion (dialysis and centrifugation), were assessed, prior to biofilmassay. However, in the dialysis approach, irreversible agglomer-ation and black precipitates were visible at the end of the dialysis.Particles purified using centrifugation (20 min, 15 000 g, 15 �C)had similar average size diameter to un-purified particles while novisible aggregation was observed in TEM images.

Pseudomonas aeruginosa biofilm detachment assays

P. aeruginosa is an opportunistic pathogen to immuno-suppressedpatients which causes urinary tract infections, respiratory infec-tions and several systemic infections through the establishment ofbiofilm, exopolysaccharides secretion and resistance to anti-biotics34. In this study, P. aeruginosa was employed to evaluatethe effect of AgNPs on the removal of established biofilms. Thebiofilm assay was performed in two different growth media ofP. aeruginosa, M9 (defined minimal media) and CAMHB(nutrient rich complex media). In view of the differences ofnutrient composition between M9 and CAMHB, both media wereevaluated for their effect on the pre-formation of P. aeruginosa

6 C.-Y. Loo et al. Drug Dev Ind Pharm, Early Online: 1–11

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biofilm prior to the treatment with AgNPs. The density ofP. aeruginosa biofilm biomass pre-attached to the wall surfacewas almost two-fold higher when grown in CAMHB(OD595nm¼ 4.0) compared to M9 (OD595nm¼ 2.6).

Figure 5 shows the detachment (expressed in terms of volumereduction) of pre-formed biofilm colonies grown in CAMHB and

M9 after the treatment with different sizes of AgNPs. The OD at595 nm shown in the figures corresponded to the amount ofremaining attached biofilm biomass after 24 h treatment. The ODfor control samples (without Ag treatment) in CAMHB and M9was 4.0 and 2.6, respectively. As shown in Figure 6(A), theincrease of 8-nm AgNPs concentrations from 32 to 200 mg/mL did

Figure 4. Size distribution of AgNPs determined from TEM (n¼ 500) synthesized in different Agþ/Ag0 ratio at (B1) 0.7, (B2) 2.35, (B3) 10, (B4) 25,(B5) 35 and (B6) 50. All particles were synthesized using ascorbic acid seeding growth using sample B as seeds. (B7) shows the respective UV-Visabsorption spectra of synthesized AgNPs. (B8) Comparison of the theoretical and experimental particles sizes showing the correlation betweenparticles sizes and concentration of seeds and ions.

DOI: 10.3109/03639045.2013.780182 Antibiofilm of citrate- and ascorbic acid-capped AgNPs 7

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Figure 5. Volume reduction of preformedbiofilm colonies of P. aerugionosa PAO1grown for 24 h in the wells of 96-microtiterplates supplemented with (A) CAMHB or (B)M9. The biofilm colonies were treated withdifferent concentrations of 8-nm AgNPs(white bar), 20-nm AgNPs (black bar), 35-nmAgNPs (gray bar) and AgNO3 (white stripedbar) and the remaining attached biofilmswere stained with CV. The OD at 595 nmcorresponds to the amount of attached cells.

Figure 6. CLSM images of (A) control biofilm pre-formed using CAMHB and samples treated with 600 mg/ml (B) 8-nm AgNPs (C) 20-nm AgNPs (D)35-nm AgNPs and (E) AgNO3. Corresponding viable attached cells were presented in (F) expressed in CFU/well. *Statistically significant compared tocontrol groups (p50.05).

8 C.-Y. Loo et al. Drug Dev Ind Pharm, Early Online: 1–11

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not result in any significant differences in the detachment ofP. aeruginosa biofilm (p50.05). At this range, �30% (OD595

nm¼ 1.2) of biofilm remained adhered on the surface of microtiterplates. However, as nanoparticle concentrations exceeded200mg/mL; it was demonstrated that the detachment activitywas significantly higher (p50.05). Approximately 90% (OD595

nm¼ 0.4) of biofilm was successfully detached when treated with600mg/mL of 8-nm AgNPs (Figure 5A white bar). Similar trendswere observed for biofilm volume reduction treated with 20-nmand 35-nm AgNPs (Figure 5A black and gray bar). At lownanoparticles concentrations ranging from 4 to 40 mg/mL, only40% (OD595 nm¼ 2.5) of P. aeruginosa biofilm was removed.However at concentration exceeding 120mg/mL, the removal ofbiofilm was more effective in which �70% of the biofilm wasdetached (Figure 5A gray bar).

Free Agþ ions were also studied to compare the differences inthe biofilm detachment assay to AgNPs. From Figure 5(A) (whitestriped bar), the remaining biofilm colonies attached to thesurface was as high as 3.70 (93%) at an OD 595 nm when treatedwith high concentration of Agþ (600 mg/mL). The removal ofbiofilms was influenced by the concentration of free Agþ ions,whereby a gradual increase in biofilm detachment was seenwith an increase in Agþ concentration from 4 to 80 mg/mL.However, the detachment was ineffective at high Agþ concentra-tion. A sharp decline in the biofilm removal was observed (from55% to 7.4%) when Agþ concentration was increased from 400to 600mg/mL. The detachment of pre-formed P. aeruginosabiofilms grown in M9 media using different sizes of AgNPs wasillustrated in Figure 5(B). In contrast to the results exhibited inCAMHB, it was found that biofilm reductions by 8-nm and 20-nmAgNPs were dose independent.

Visual confirmation on the effect of NP size on the biofilmdetachment was performed using CLSM (Figure 6A–E). Asobserved in Figure 6, significant removal of P. aeruginosa PAO1biofilm is clearly seen after AgNP treatment which correlatedwith results from CV assay. After treatment with 8-nm AgNPs,only small clusters of cells were remained attached whilesignificant reduction in the microcolonies in both 20-nm and35-nm AgNPs treatment were observed compared to control. Thebiofilm removal effect is size-dependent with the decreasingtrend: 8-nm AgNPs420-nm AgNPs435-nm AgNPs4Agþ.In addition, the corresponding CFU of attached wells for controlcells, and biofilm cells treated with 8-nm AgNPs, are 5� 106 and0.18� 106, respectively (Figure 6F).

Figure 7 demonstrates the relationship between the size ofnanoparticles and the detachment efficiency of pre-formedbiofilms grown in either CAMHB or M9 media. It is seen thatirrespective of growth media the effectiveness of biofilm removalcorresponded to the size of nanoparticles: the smaller thenanoparticles, the higher the amount of biofilm reduction. Forinstance, when pre-formed biofilm grown in M9 medium wastreated with 600mg/mL AgNPs, the order of biofilm removalefficiency was as follows: 8-nm AgNPs (90%)420-nm(69%)435-nm (52%). In comparison with CAMHB medium,89% of biofilm was removed using 600mg/mL of 8-nm AgNPs.However, no significant differences in terms of effectiveness ofbiofilm removal were observed between 20-nm and 35-nm AgNPs.Both sets of nanoparticles showed the removal of approximately75%. It is well known that the antibacterial activity of AgNPs isactivated with the release of Agþ from the particle surface whenexposed to a liquid media1. A particle with smaller size has ahigher surface area to volume that translates to a higher availabilityof surface area for oxidation and therefore Agþ release. Forexample, the surface area1 for a gram of 10 nm AgNPs is 0.6million per cm2 which is about 55 000 times higher than a gram ofpure solid spherical silver with the surface area of 10.6 cm2.

Extensive studies have been previously conducted on theinhibitory effect of AgNPs concentration on growth of planktonicP. aeruginosa. In a recent report by Guzman et al., it wasdemonstrated that the MIC of 9-nm and 14-nm AgNPs forachieving an antibacterial effect was between 14 and 28 mg/mL,respectively35. In another study, a MIC ranging from 1.69 to13.5 mg/mL was used to kill various bacterial and clinical isolatesof P. aeruginosa15. A rule of thumb suggests that 1000 to 1500times higher concentrations of AgNPs are required to eradicatebiofilm when compared to killing a planktonic bacterium1. Here,we have demonstrated that the complete detachment ofP. aeruginosa biofilm colonies was not reached even at AgNPsconcentration of 600mg/mL. For 8-nm AgNPs, the removal ofbiofilm was 90% while only 75% detachment was demonstratedby both 20-nm and 35-nm particles (Figure 6). On the contrary, arecent study demonstrated that complete eradication ofP. aeruginosa biofilm was achieved using approximately 45-nmAgNPs in concentrations as low as 1 mg/mL14. In another study,the treatment of P. aeruginosa with 100 nM of AgNPs resulted inrapid decrease of biofilm amounting to 95% detachment36. Oneprobable reasons contributing to the lower anti-biofilm activity ofAgNPs in our study might be due to the capping effect of citrate,which potentially reduced the silver particle effectiveness (e.g. viaretention of the silver ions or inhibition of local oxidation37,38.Studies on the inhibitory mechanisms of free Agþ ion onplanktonic bacteria have shown that the microorganisms treatedwith Agþ lost the ability to replicate DNA and the functionof some essential enzymes and cellular proteins was impaired3.It is also shown that Agþ binds to functional groups of proteinsforming salt bridges, resulting in protein denaturation.Importantly, in this study, we have demonstrated that the additionof high concentration of Agþ directly onto pre-formed biofilm hasan opposite effect on detachment activity. It was also observed

Figure 7. The effect of nanoparticle sizes on the reduction of pre-formedP. aeruginosa biofilm grown in respective media (CAMHB and M9). Thebiomass of biofilm corresponded to the absorbance at 595 nm. Data wasexpressed in precentage of removal activities (n¼ 5).

DOI: 10.3109/03639045.2013.780182 Antibiofilm of citrate- and ascorbic acid-capped AgNPs 9

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that upon addition of Agþ, white precipitate immediately becamevisible that remained attached to the wall of the microtiter platethroughout the treatment. The surface of P. aeruginosa PAO1cells is negatively charged, thus the addition of a cationic Agþ ionmay cause coagulation of matrix proteins in biofilm via chargeneutralization39. Experimental conditions such as temperature, pHand concentrations of coagulant have been shown to affect the rateof coagulation. At low concentration of Agþ, coagulation ofproteins is slow while complete charge neutralization is notobtained. At higher concentrations, the coagulation rate is fast andimmediate39. Therefore, this suggests that direct contact of Agþ

with the outermost layers of biofilm colonies may cause rapidprotein coagulation, which indirectly triggers additional shieldingeffect of the inner biofilm colonies from being affected by Agþ.

The fact that the biofilm removal efficiency of AgNPs differedsignificantly to that of its ionized form indicated that other routesare involved in the removing of biofilms using silver. Asmentioned above, Ag in the metallic form acts as a reservoirand exhibits antibacterial behavior when oxidized to release Agþ.Therefore, the higher activity of AgNPs compared to Agþ couldbe due to its better dispersion and penetration into biofilm matrix.It is easier for AgNPs to reach colonies inside the biofilmcompared to the ionized form that interacts easily with biofilmmatrix protein. It is known that water channels or pores existwithin biofilm matrix as nutrient transport channels. Furthermore,the rate of Agþ release from AgNPs is proportional to its surfacearea, whereby it could be speculated that the release of ions from8-nm AgNPs is more rapid than AgNPs with bigger particle size.

Another possible mechanism of AgNPs actions could bethrough the attachment of the nanoparticles onto the surface ofmicrobial cell membrane leading to increased permeability,inhibition of cell wall synthesis, plasmolysis and subsequentlycell death40,41. The binding of AgNPs to bacterial cell walldepends on factors such as availability of surface area42 andsurface charges11. Therefore, it would be expected that smallerAgNPs, which have higher surface area might have higher bindinginteractions with bacterial cell wall compared to larger ones35. Inaddition, once adhered to the surface of membrane, AgNPs couldpenetrate and accumulate within the cytoplasm of bacteria11. It isbelieved that accumulated AgNPs in the cytoplasm of bacterialcell inactivate DNA and enzymes through coagulation withsulfur- and phosphorus-containing compounds43.

Conclusions

Seeding growth approach was used to produce mono-dispersedand homogenous AgNPs with different sizes ranging from 8 to70 nm. The plasmon absorbance bands of synthesized nanopar-ticles were approximately 400 nm for smaller nanoparticles andshifted to green part of the spectrum as the particle grew larger. Inaddition, �max for all nanoparticles fell in the range of 400–450 nm, indicating that the majority of particles were sphericaland pseudo-spherical shaped. The appearance of polyhedral,tetrahedral and truncated particles occurred after repetitiveseeding. The use of AgNPs was more effective to remove P.aeruginosa biofilm than applying silver in its ionic form. AgNPscould acts as reservoir to release smaller amount of Agþ ions, thusresulting in higher activity. In addition, the effect of AgNPs onbiofilm removal was size-dependent, i.e. smaller nanoparticleshad the higher efficacy to remove biofilm.

Acknowledgements

The authors would like to thank Mr Shaun Bulcock from AustralianCentre for Microscopy and Microanalysis (ACMM) for his valuableadvice and help for TEM observations.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of this article.

Dr Cynthia Whitchurch was funded by a NHMRC Senior ResearchFellowship.

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