http://informahealthcare.com/ddi ISSN: 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
Ching-Yee Loo1, Paul M. Young2,3, Rosalia Cavaliere4, Cynthia B. Whitchurch4, Wing-Hin Lee1, and Ramin Rohanizadeh1 1
Advanced 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
Keywords
Objectives: Silver nanoparticles (AgNPs) with a size ranging from 7 to 70 nm were synthesized using 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 and used as seed to prepare AgNPs. Results: The presence of ascorbic acid and citrate allows the regulation of size and size distribution of the nanoparticles. The increase in free silver ion-to-seed ratio (Agþ/Ag0) resulted in 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 particles with higher polydispersity index and minor distributions of tetrahedral particles. Citrate-capped AgNPs were stable and did not agglomerate upon centrifugation. The effect of AgNPs on biofilm reduction was evaluated using static culture on 96-well microtiter plates. Results showed that AgNPs with the smallest average diameter were most effective in the reduction of Pseudomonas aeruginosa biofilm colonies, which accounted for 90% of removal. Conclusion: The biofilm removal activities of the nanoparticles were found to be concentrationindependent particularly for the concentration within the range of 80–200 mg/mL.
Antibacterial, biofilm, chemical reduction, Pseudomonas aeruginosa, silver nanoparticles
Introduction Biofilms are typically made up from communities of microorganisms which have switched from a planktonic to a sessile state after adhering to a particular surface1. Unlike their planktonic state, biofilms are highly organized, in which microorganisms communicate by chemical signals and build protective structures that enable them to survive the hosts’ natural defenses and antimicrobial agents. Once adhered, it is extremely difficult to remove biofilms from the surface; thus biofilms can cause many lifethreatening infections in patients treated using medical devices, such as intratracheal tubes for ventilation1,2. It is therefore a challenge to identify antimicrobial agents that are effective against the colonization of wide ranges of biofilms and simultaneously exhibit low toxicity levels for patients. The renewed interest in silver and its nano-sized particles may be generally related to the rapid emergence of antibioticresistance bacteria. Unlike conventional antibiotics that have narrow therapeutic targets, the likelihood of microorganism acquiring resistance against silver ions is slim, since silver ions simultaneously act on multiple sites within bacterial cells that are critical to physiological function (e.g. cell wall, DNA/RNA
Address for correspondence: Dr Ramin Rohanizadeh, Faculty of Pharmacy (A15), University of Sydney, Sydney, New South Wales, Australia. E-mail:
[email protected]
History Received 22 November 2012 Revised 14 February 2013 Accepted 20 February 2013 Published online 17 April 2013
syntheses and electron transport)3. Most importantly, silver ions are generally reported to be compatible with human tissues4. According to WHO standards, 0.1 mg/L of soluble silver (ions) in drinking water is considered safe for humans5. Interestingly, however, the soluble silver ions are only effective for short periods of time. In comparison, nano-sized silver particles can be prevalent for as long as 200 d due to longer residence time at the site of infection1. Silver nanoparticles (AgNPs), clusters of silver atoms ranging in diameter from 1 to 100 nm are potent antimicrobial agents used in many fields including the medical, food, electrical and textile industries6,7. It is believed that antimicrobial efficiency of AgNPs is influenced by their size in which smaller particles confers higher surface area-to-volume ratio, and thus greater bactericidal effect8. Literature on the bactericidal effect of AgNPs with respect to their size and synthesis methods is extensive9–12. Martı´nez-Castan˜o´n et al. reported a size-dependent bactericidal effect of AgNPs in which minimum inhibitory concentration (MIC) of 7 nm and 29 nm AgNPs towards Escherichia coli were 6.25 and 13.02 mg/mL, respectively13. The MIC of 40 nm AgNPs prepared via irradiation in the presence of sodium dodecyl sulfate (SDS) toward Pseudomonas aeruginosa was 2 mg/mL14. In another study, the MIC of 26-nm AgNPs against P. aeruginosa CCM 3955, S. auereus CCM 3953, E. coli CCM 3954, Enterococcus faecalis CCM 4224 and various isolated clinical strains were in the range of 1.69–13.5 mg/mL15.
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Silver nanoparticles enhance Pseudomonas aeruginosa PAO1 biofilm detachment
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A remarkable range of novel techniques have been employed to synthesize AgNPs with varying degrees of sizes, shapes and stability. The most common approach is through the reduction of silver nitrate (via a reducing agent). When AgNPs are prepared using strong reducing agents, such as sodium borohydride16 and gallic acid13 the resulting particles tend to be monodispersed and spherical. While this process produces highly reproducible particles, the size range is somewhat limited to 510 nm. The use of weaker reducing agents, results in broader particle size ranges; however, poly-dispersed systems are often produced with a combination of two or more particle shapes17. In addition, methods such as photoreduction via UV-light18, irradiation19, sol-gel process20, microemulsions21,22 and manipulation of the microenvironment (e.g. ionic strength, pH, temperature and the use of capping agents) have successfully produced AgNPs with controllable sizes and shapes. Seeding-mediated growth is an another relatively easy approach to regulate the shapes and sizes of nanoparticles such as gold23. Here, we report a simple and relatively fast synthesis of un-agglomerated AgNPs of 7–70 nm in diameter through seedmediated growth assisted by ascorbic acid and citrate. This approach involves a step-wise procedure for the preparation of larger AgNPs using a weaker reducing agent from monodispersed small AgNPs (Figure 1). Specifically, we report on the preparation of AgNPs with various sizes using the seeding growth approach by manipulating synthesis conditions such as ion-to-seed ratio (Agþ/Ag0), ascorbic acid addition rate and concentration. While, the antimicrobial effect of AgNPs against planktonic microorganisms has been previously documented, studies on their effect on biofilm are relatively few. Therefore, this study aims to investigate the efficacy of different AgNPs sizes on the detachment of P. aeruginosa PAO1 biofilms.
Materials and methods Materials Silver nitrate AgNO3 (MP Biomedicals, Santa Ana, CA), sodium citrate (Sigma Aldrich, St. Louis, MO), gallic acid (MP Biomedicals) and ascorbic acid (Sigma Aldrich) were used as received. Deionized water (H2O) was purified by reverse osmosis (milliQ, Millipore, Billerica, MA).
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Preparation of AgNPs seeds AgNPs with average diameter of 8 nm were prepared according to the gallic acid reduction method as described by MartinezCastan˜o´n et al13. All glassware were ultrasonically cleaned with absolute ethanol before use. Briefly, 100 mL of aqueous AgNO3 (103 M) was prepared in a 250 mL flask. While stirring, 10 mL of deionized H2O containing 10 mg gallic acid was added into the solution, followed immediately by adjusting the pH solution to 11 using 1 M NaOH. The stirring continued for 10 min. The prepared AgNPs (sample A) were used as seed particles for subsequent experiments (Figure 1). Preparation of differently sized AgNPs ranging from 10 to 60 nm in the presence of 8 nm seeds Five sets of solutions labeled B to F containing sodium citrate (5 104 M) and AgNO3 (Table 1) were prepared. Different amounts of the 8 nm seeds (sample A) were added into the solutions so that the final silver atom concentration in the solutions was 1 103 M and the mixture was brought up to 150 mL using deionized H2O. While stirring, 100 mL of 2 104 M ascorbic acid was added at a rate of 7.5 mL/min followed by continuous stirring for 1 h. Preparation of AgNPs in the presence of sample B as seeds A set of six solutions labeled as B1 to B6 was prepared containing different ratios of Agþ/Ag0 (Table 2). The final silver atom concentration in the solutions was 1 104 M and the mixtures were diluted to 150 mL using deionized H2O. Immediately, while stirring, 100 ml of 4 104 M ascorbic acid was added at the rate of 10 mL/min. Recovery and purification of AgNPs using dialysis and centrifugation Two methods were applied and compared to purify prepared AgNPs: centrifugation (20 min, 15 000 g, 15 C) and dialysis using a Cellu-Sep T2 (Fisher Biotec, Perth, Australia) membrane in a buffer 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.
Antibiofilm of citrate- and ascorbic acid-capped AgNPs
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Table 1. Summary of AgNPs synthesis in the presence of 8 nm AgNPs as seeds (sample A). For samples B to F, AgNO3 was added so that final silver atom concentration was 103 M.
Sample
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A B C D E F
Seeds added (mL)
Volume of 0.1 M AgNO3 added to the reaction (mL)
Agþ/Ag0
None 15.00 5.00 2.50 0.50 0.25
None 2.35 2.45 2.48 2.50 2.50
None 1.6 5 10 50 100
Table 2. Summary of AgNPs synthesis using predetermined amount of sample B as seeds. AgNO3 was added so that final silver atom concentration was 104 M.
Sample B1 B2 B3 B4 B5 B6
Seeds added (mL)
Volume of 0.01 M AgNO3 added to the reaction (mL)
Agþ/Ag0
15.00 7.50 2.50 1.00 0.75 0.50
1.00 1.75 2.25 2.40 2.43 2.45
0.7 2.35 10 25 35 50
centrifugation method, the pellet was washed with deionized H2O to remove the excess of Agþ ions and the pellet was subsequently lyophilized. For the dialysis of AgNPs, the ratio of AgNP to the buffer was 500 and buffer solutions were changed three times. Both lyophilized AgNPs powders (from centrifugation) and AgNPs suspensions (from dialysis) were analyzed using transmission electron microscope (TEM) and UV-vis spectrophotometry. Characterization of AgNPs Absorption spectra of particle suspensions were recorded using a Hitachi U-200 (Tokyo, Japan) spectrophotometer at a wavelength from 280 to 600 nm. Particle size distribution was determined using dynamic light scattering (DLS, Malvern, UK). DLS was performed using a Malvern Zetasizer Nano ZS with the following settings: the refractive index for silver was 1.35 and the viscosity of water was 1.0002 mPas. The samples were measured in a quartz cuvette at 25 C. Particle size distributions were measured in triplicate (n ¼ 3). Transmission electron microscopy (TEM) was carried out using a JEOL1400 (Tokyo, Japan) electron microscope operating at 200 kV. Approximately 1 mL of particle suspension were added dropwise onto 200-mesh carbon coated Holey copper grids and dried at room temperature. TEM images were 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 in which P. aeruginosa PAO1 was first allowed to attach to the surface of 96-well polystyrene plate. Three independent replicates of experiments were conducted on separate days with five technical replicates performed during each assay. Briefly, P. aeruginosa was grown overnight in cation adjusted Mueller– Hinton broth (CAMHB, Oxoid, Cambridge, UK) medium at 37 C, shaken at 250 rpm and diluted 100-fold to 106 CFU/mL (determined by OD and plate count assay) with CAMHB or
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modified M9 media. A 100 mL aliquot of the culture was placed into each well and the plates were incubated without shaking at 37 C. After 24 h, the liquid was gently removed and the wells were rinsed twice with phosphate buffer saline (PBS) before being subjected to AgNPs treatment. The effect of AgNPs on the biofilm was studied at a range of different concentrations for each particle size as well as for free Ag in solution. The freeze-dried AgNPs powders were re-suspended in water and homogenized thoroughly using a water bath-sonicator for 15 min, prior to the study. A 100 mL aliquot of re-suspended AgNPs was added to the wells containing biofilm and incubated for 2 h at 37 C. Five wells were treated for each particle size and concentration. The wells were washed extensively with PBS and the remaining attached biofilm was stained with crystal violet (CV), left for 1 h before being re-washed and de-stained with ethanol-acetone. The optical density (OD) was measured using a Beckman microtiter plate reader at 595 nm. The biomass of biofilm corresponded to the absorbance at 595 nm. The percentage of removal biofilm was calculated using the following equation: Initial biomass remaining attached biomass 100% Initial biomass
ð1Þ
Qualitative imaging of P. aeruginosa PAO1 biofilms using confocal laser scanning microscopy The morphologies of the biofilm were analyzed using confocal laser scanning microscopy (CLSM) (Nikon A1, Tokyo, Japan). Samples were treated with different AgNPs sizes containing 600 mg/mL equivalent of Ag content. Both control and treated biofilm samples were stained using SYTO9 (Invitrogen, Grand Island, NY) and fixed with 4% paraformaldehyde. After washing, the stained biofilm samples were imaged using CLSM using oil immersion lens (100 objective lens and numerical aperture of 1.4). Recorded images were reconstructed by Imaris and presented as three-dimensional structures. In addition, enumerations of remaining biofilm were performed using viable plate counting and data were expressed as colony forming units per well (CFU/well). Statistical analysis Statistical analysis of data was performed using the SPSS Statistics 19 software package (IBM, Armonk, NY). All data were collected (n ¼ 5) and the mean values and standard deviations (SD) were calculated. The statistical differences between the groups were determined by analysis of variance (ANOVA). The pairwise comparisons of individual group means were performed using the Tukey post-hoc analysis. Values of p50.05 were considered statistically significant.
Results and discussions Optimization of AgNPs fabrication method and particle characterization In this study, the seeding growth approach was applied with 8-nm AgNPs as seeds and ascorbic acid as reducing agent in order to produce a wider range of AgNPs sizes. Small, spherical AgNPs seeds were synthesized using a chemical reduction method with a strong reducing agent. In this case, gallic acid was used as both reducing agent and stabilizer13. In acidic condition, the solutions were colorless and turned immediately dark yellow as the pH was adjusted to 11. The reduction of metal ions into metal particles was achieved through the oxidation of phenolic groups and subsequently stabilized the formed AgNPs24.
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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.
It is suggested that the oxidation of phenolic groups is pH-dependent and the rate is higher in alkaline conditions, thus in turn enhancing the metal ion reduction rate forming small spherical particles24. The TEM micrographs (Figure 2A) showed that the particles prepared via this method were relatively monodispersed and primarily of spherical shape. Figure 3(A) shows the corresponding histogram for the particle size distribution (n ¼ 500 measurements) determined from TEM micrographs (Figure 2A) whereby the average diameter of AgNPs was 8 nm. Although the majority 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 narrow distribution, a seeding growth approach was used. The 8 nm AgNPs were employed as seeds with ascorbic acid as reducing agent. The addition of ascorbic acid into the solutions containing both AgNPs seeds, metal salts (AgNO3) and sodium citrate as stabilizer was conducted drop-wise using a burette at 7.5 mL/min
rate and the resulting particles were stirred continuously for more than 1 h. As a control, the same procedure conducted without the addition of AgNPs seeds, resulted in the Agþ solutions that remained clear, without the Ag plasmon band (410 to 460 nm), indicating that AgNPs were not formed. Such observations demonstrate that ascorbic acid was too weak to reduce Agþ into Ag0 at room temperature without seed material (results not shown) possibly related to a coordination effect with citric acid, as reported previously25. In the presence of seed AgNPs, the solutions changed immediately from yellowish to red color as ascorbic acid was added. Five sets of AgNPs corresponding to the samples B to F were prepared according to the synthesis conditions outlined in Table 1 and Figure 1. The respective shapes and sizes of samples B to F are shown in Figure 2(B) to (F). By varying the ratio between free silver ion to silver seed (Agþ/Ag0), different particle sizes were achieved. When the Agþ/Ag0 ratio was 1.6 and 5, respectively,
Antibiofilm of citrate- and ascorbic acid-capped AgNPs
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Figure 3. (A to F) Histograms showing the size distribution of AgNPs determined from TEM. (G) Extinction spectra of AgNPs prepared via the seeding 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.
the produced nanoparticles were spherical and enlargement of particles were not significant (Figure 2B and C) with sizes determined from TEM images being 7 and 11 nm, respectively (Figure 3B and C). As the amount of nanoparticle seed material decreased to 2.5 mL (sample D), 0.5 mL (sample E) and 0.25 mL (sample F), the resultant nanoparticles became pseudo-spherical with average diameters of 20, 35 and 50 nm, respectively (Figures 2
and 3). Sample F was stable only for a few hours and turned into dark purplish color with black precipitates. The particle plasmon resonance bands for both sample A and B were sharp and intense at 410 nm and red-shifted as the particle sizes increased (Figure 3G). In particular, the full half-width maximum (FHWM) for sample F was broader and a weak band at 500–550 nm was visible; indicating particle aggregation.
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In our study, the use of sodium citrate as capping agents confers several advantages: (i) regulating size distributions with respect to the concentrations used17,26; (ii) limiting the aggregation process during early particle formations via electrostatic mechanisms27; and (iii) acting solely as stabilizer in reaction temperature without competing against the reducing agent (ascorbic acid). Strong capping agents has been known to inhibit particle growth which in turn generates high monodispersity26. In addition, we observed that AgNPs (e.g. sample E in Figure 2) produced in similar conditions, without the presence of sodium citrate, were approximately two orders of magnitude larger than the AgNPs prepared in the presence of sodium citrate; with broader size distributions and less stable (results not shown). The stabilization effect of AgNPs by citrate relied on the electrostatic interactions between capping ligands and the core of nanoparticle28 and it is influenced to a larger extent by the citrate concentration26. The effective range of concentration26 was between 1 and 5 104 M as coalescence/agglomeration was observed at the concentrations of 51 104 M or 15 104 M. As reported by Pillai and Kamat17, anionic citrate bonded strongly on the surface of Ag0 seeds and grew slowly to reach an optimal point where strong repelling layers of citrate is enough to prevent the aggregation. Generally seed-mediated nanoparticle growth is considered to be due to growth along nanocrystal lattice of silver ions at the seed surface29, suggesting that size should increase in tandem with increasing Agþ/Ag0 ratio. However, in sample B, the average diameter was approximately similar to that of the initial seed material with a narrower distribution. The size-dependent concentration relationship was further assessed using a simple theoretical calculation based on the assumption that all spherical particles grow with no nucleation and without apparent changes in particle shape30. On the basis of spherical model for AgNPs, the expected 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 seeds and metal salts, respectively. The Equation (2) assumes the conversion efficiency of Agþ into Ag0 is 100% and all seeds grew. In Figure 3(H), the estimated theoretical particle size was compared to the experimental value. From Figure 3(H), it is clear that the particle diameters determined from TEM images were smaller than the theoretical values. Although values for the seed radius, additional metal ion concentration and the amount of added AgNPs as seeds could be theoretically calculated, the actual concentration of AgNPs and Agþ involved in seeding growth may not be 100%. In general, the variation of Agþ/Ag0 ratio should directly influence the particle size whereby as the amount of seeds was decreased, the particles size increases. However, in our study, it was observed that the addition of a high volume of seeds led to the formation of AgNPs which were smaller or of similar size to that of the seed (e.g. Figure 3B). The discrepancy in theoretical values and experimental size, shown in Figure 3(H), reflects the over-simplification of the spherical model for AgNP formation. Additional factors including particle dissolution, the presence/ types/mode of actions of stabilizers, concentration and type of reducing agent and other microenvironment conditions including pH, temperature and ionic strength may contribute to such a variation in reduction rate of Agþ into Ag0. In an earlier study by Jana et al., it was observed that the size of gold nanoparticles differed 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 of reducing agent was effective in suppressing further nucleation, subsequently improving the monodispersity of particle size distributions31. As mentioned above, the presence of citrate inhibited or slowed the growth of particles that possibly explained the relatively smaller particle size showed in Figure 3(H) compared to that of the theoretical data. We also prepared AgNPs in different concentrations of ascorbic acid and sodium citrate. Preliminary data obtained by the TEM analysis showed that the increase of ascorbic acid concentration significantly increased the final particle size at the expense of the particle stability, which further demonstrated that the external factors are important in regulation of AgNP particle size. Sample B resulted in narrowly distributed mono-dispersed AgNPs (7 2 nm) that could be used as initial seeds for further ascorbic acid-citrate mediated growth, using a final concentration of silver atoms fixed at 104 M. The TEM micrographs of resultant particles were shown in Figure 2 (B1–B6) and their corresponding distribution histograms in Figure 4. A typical UVvis spectrum monitoring the formation of AgNPs in the presence of 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 was evident as the ratio of Agþ/Ag0 was increased. In addition, max increases with an increase in nanoparticle size. The estimated particle sizes were relatively higher than the experimental data at Agþ/Ag0 below 25 (Figure 4B8). At ratios above 25, the measured sizes fitted closely to estimated values, indicating that growth only occurred under these experimental conditions. This indicated that the AgNPs were dominantly spherical or roughly spherical in shape32, which was further supported by TEM images (Figure 2). Meanwhile, the max of B4, B5 and B6 were shifted to 420, 430 and 440 nm, respectively, which could be correlated to the presence of polyhedral (e.g. decahedral) shaped particles as demonstrated 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. The appearance of minor percentages of faceted polyhedral or truncated shaped nanoparticles as a result of repetitive seeding is possibly attributed to the variation in relative growth rates at different faces of particles and the interference of reduction rate due to the competition between the reduction process and the capping action of stabilizers33. After AgNPs preparation, two different methods for purification (dialysis and centrifugation), were assessed, prior to biofilm assay. However, in the dialysis approach, irreversible agglomeration 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 no visible aggregation was observed in TEM images. Pseudomonas aeruginosa biofilm detachment assays P. aeruginosa is an opportunistic pathogen to immuno-suppressed patients which causes urinary tract infections, respiratory infections and several systemic infections through the establishment of biofilm, exopolysaccharides secretion and resistance to antibiotics34. In this study, P. aeruginosa was employed to evaluate the effect of AgNPs on the removal of established biofilms. The biofilm assay was performed in two different growth media of P. aeruginosa, M9 (defined minimal media) and CAMHB (nutrient rich complex media). In view of the differences of nutrient composition between M9 and CAMHB, both media were evaluated for their effect on the pre-formation of P. aeruginosa
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Figure 4. Size distribution of AgNPs determined from TEM (n ¼ 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-Vis absorption spectra of synthesized AgNPs. (B8) Comparison of the theoretical and experimental particles sizes showing the correlation between particles sizes and concentration of seeds and ions.
biofilm prior to the treatment with AgNPs. The density of P. aeruginosa biofilm biomass pre-attached to the wall surface was almost two-fold higher when grown in CAMHB (OD595nm ¼ 4.0) compared to M9 (OD595nm ¼ 2.6). Figure 5 shows the detachment (expressed in terms of volume reduction) of pre-formed biofilm colonies grown in CAMHB and
M9 after the treatment with different sizes of AgNPs. The OD at 595 nm shown in the figures corresponded to the amount of remaining attached biofilm biomass after 24 h treatment. The OD for control samples (without Ag treatment) in CAMHB and M9 was 4.0 and 2.6, respectively. As shown in Figure 6(A), the increase of 8-nm AgNPs concentrations from 32 to 200 mg/mL did
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Figure 5. Volume reduction of preformed biofilm colonies of P. aerugionosa PAO1 grown for 24 h in the wells of 96-microtiter plates supplemented with (A) CAMHB or (B) M9. The biofilm colonies were treated with different concentrations of 8-nm AgNPs (white bar), 20-nm AgNPs (black bar), 35-nm AgNPs (gray bar) and AgNO3 (white striped bar) and the remaining attached biofilms were stained with CV. The OD at 595 nm corresponds 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 to control groups (p50.05).
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not result in any significant differences in the detachment of P. aeruginosa biofilm (p50.05). At this range, 30% (OD595 nm ¼ 1.2) of biofilm remained adhered on the surface of microtiter plates. However, as nanoparticle concentrations exceeded 200 mg/mL; it was demonstrated that the detachment activity was significantly higher (p50.05). Approximately 90% (OD595 nm ¼ 0.4) of biofilm was successfully detached when treated with 600 mg/mL of 8-nm AgNPs (Figure 5A white bar). Similar trends were observed for biofilm volume reduction treated with 20-nm and 35-nm AgNPs (Figure 5A black and gray bar). At low nanoparticles concentrations ranging from 4 to 40 mg/mL, only 40% (OD595 nm ¼ 2.5) of P. aeruginosa biofilm was removed. However at concentration exceeding 120 mg/mL, the removal of biofilm was more effective in which 70% of the biofilm was detached (Figure 5A gray bar). Free Agþ ions were also studied to compare the differences in the biofilm detachment assay to AgNPs. From Figure 5(A) (white striped bar), the remaining biofilm colonies attached to the surface was as high as 3.70 (93%) at an OD 595 nm when treated with high concentration of Agþ (600 mg/mL). The removal of biofilms was influenced by the concentration of free Agþ ions, whereby a gradual increase in biofilm detachment was seen with an increase in Agþ concentration from 4 to 80 mg/mL. However, the detachment was ineffective at high Agþ concentration. A sharp decline in the biofilm removal was observed (from 55% to 7.4%) when Agþ concentration was increased from 400 to 600 mg/mL. The detachment of pre-formed P. aeruginosa biofilms grown in M9 media using different sizes of AgNPs was illustrated in Figure 5(B). In contrast to the results exhibited in CAMHB, it was found that biofilm reductions by 8-nm and 20-nm AgNPs were dose independent. Visual confirmation on the effect of NP size on the biofilm detachment was performed using CLSM (Figure 6A–E). As observed in Figure 6, significant removal of P. aeruginosa PAO1 biofilm is clearly seen after AgNP treatment which correlated with results from CV assay. After treatment with 8-nm AgNPs, only small clusters of cells were remained attached while significant reduction in the microcolonies in both 20-nm and 35-nm AgNPs treatment were observed compared to control. The biofilm removal effect is size-dependent with the decreasing trend: 8-nm AgNPs420-nm AgNPs435-nm AgNPs4Agþ. In addition, the corresponding CFU of attached wells for control cells, and biofilm cells treated with 8-nm AgNPs, are 5 106 and 0.18 106, respectively (Figure 6F). Figure 7 demonstrates the relationship between the size of nanoparticles and the detachment efficiency of pre-formed biofilms grown in either CAMHB or M9 media. It is seen that irrespective of growth media the effectiveness of biofilm removal corresponded to the size of nanoparticles: the smaller the nanoparticles, the higher the amount of biofilm reduction. For instance, when pre-formed biofilm grown in M9 medium was treated with 600 mg/mL AgNPs, the order of biofilm removal efficiency was as follows: 8-nm AgNPs (90%)420-nm (69%)435-nm (52%). In comparison with CAMHB medium, 89% of biofilm was removed using 600 mg/mL of 8-nm AgNPs. However, no significant differences in terms of effectiveness of biofilm removal were observed between 20-nm and 35-nm AgNPs. Both sets of nanoparticles showed the removal of approximately 75%. It is well known that the antibacterial activity of AgNPs is activated with the release of Agþ from the particle surface when exposed to a liquid media1. A particle with smaller size has a higher surface area to volume that translates to a higher availability of surface area for oxidation and therefore Agþ release. For example, the surface area1 for a gram of 10 nm AgNPs is 0.6 million per cm2 which is about 55 000 times higher than a gram of pure solid spherical silver with the surface area of 10.6 cm2.
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Figure 7. The effect of nanoparticle sizes on the reduction of pre-formed P. aeruginosa biofilm grown in respective media (CAMHB and M9). The biomass of biofilm corresponded to the absorbance at 595 nm. Data was expressed in precentage of removal activities (n ¼ 5).
Extensive studies have been previously conducted on the inhibitory effect of AgNPs concentration on growth of planktonic P. aeruginosa. In a recent report by Guzman et al., it was demonstrated that the MIC of 9-nm and 14-nm AgNPs for achieving an antibacterial effect was between 14 and 28 mg/mL, respectively35. In another study, a MIC ranging from 1.69 to 13.5 mg/mL was used to kill various bacterial and clinical isolates of P. aeruginosa15. A rule of thumb suggests that 1000 to 1500 times higher concentrations of AgNPs are required to eradicate biofilm when compared to killing a planktonic bacterium1. Here, we have demonstrated that the complete detachment of P. aeruginosa biofilm colonies was not reached even at AgNPs concentration of 600 mg/mL. For 8-nm AgNPs, the removal of biofilm was 90% while only 75% detachment was demonstrated by both 20-nm and 35-nm particles (Figure 6). On the contrary, a recent study demonstrated that complete eradication of P. aeruginosa biofilm was achieved using approximately 45-nm AgNPs in concentrations as low as 1 mg/mL14. In another study, the treatment of P. aeruginosa with 100 nM of AgNPs resulted in rapid decrease of biofilm amounting to 95% detachment36. One probable reasons contributing to the lower anti-biofilm activity of AgNPs in our study might be due to the capping effect of citrate, which potentially reduced the silver particle effectiveness (e.g. via retention of the silver ions or inhibition of local oxidation37,38. Studies on the inhibitory mechanisms of free Agþ ion on planktonic bacteria have shown that the microorganisms treated with Agþ lost the ability to replicate DNA and the function of some essential enzymes and cellular proteins was impaired3. It is also shown that Agþ binds to functional groups of proteins forming salt bridges, resulting in protein denaturation. Importantly, in this study, we have demonstrated that the addition of high concentration of Agþ directly onto pre-formed biofilm has an opposite effect on detachment activity. It was also observed
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that upon addition of Ag , white precipitate immediately became visible that remained attached to the wall of the microtiter plate throughout the treatment. The surface of P. aeruginosa PAO1 cells is negatively charged, thus the addition of a cationic Agþ ion may cause coagulation of matrix proteins in biofilm via charge neutralization39. Experimental conditions such as temperature, pH and concentrations of coagulant have been shown to affect the rate of coagulation. At low concentration of Agþ, coagulation of proteins is slow while complete charge neutralization is not obtained. At higher concentrations, the coagulation rate is fast and immediate39. Therefore, this suggests that direct contact of Agþ with the outermost layers of biofilm colonies may cause rapid protein coagulation, which indirectly triggers additional shielding effect of the inner biofilm colonies from being affected by Agþ. The fact that the biofilm removal efficiency of AgNPs differed significantly to that of its ionized form indicated that other routes are involved in the removing of biofilms using silver. As mentioned above, Ag in the metallic form acts as a reservoir and exhibits antibacterial behavior when oxidized to release Agþ. Therefore, the higher activity of AgNPs compared to Agþ could be due to its better dispersion and penetration into biofilm matrix. It is easier for AgNPs to reach colonies inside the biofilm compared to the ionized form that interacts easily with biofilm matrix protein. It is known that water channels or pores exist within biofilm matrix as nutrient transport channels. Furthermore, the rate of Agþ release from AgNPs is proportional to its surface area, whereby it could be speculated that the release of ions from 8-nm AgNPs is more rapid than AgNPs with bigger particle size. Another possible mechanism of AgNPs actions could be through the attachment of the nanoparticles onto the surface of microbial cell membrane leading to increased permeability, inhibition of cell wall synthesis, plasmolysis and subsequently cell death40,41. The binding of AgNPs to bacterial cell wall depends on factors such as availability of surface area42 and surface charges11. Therefore, it would be expected that smaller AgNPs, which have higher surface area might have higher binding interactions with bacterial cell wall compared to larger ones35. In addition, once adhered to the surface of membrane, AgNPs could penetrate and accumulate within the cytoplasm of bacteria11. It is believed that accumulated AgNPs in the cytoplasm of bacterial cell inactivate DNA and enzymes through coagulation with sulfur- and phosphorus-containing compounds43.
Conclusions Seeding growth approach was used to produce mono-dispersed and homogenous AgNPs with different sizes ranging from 8 to 70 nm. The plasmon absorbance bands of synthesized nanoparticles were approximately 400 nm for smaller nanoparticles and shifted to green part of the spectrum as the particle grew larger. In addition, max for all nanoparticles fell in the range of 400– 450 nm, indicating that the majority of particles were spherical and pseudo-spherical shaped. The appearance of polyhedral, tetrahedral and truncated particles occurred after repetitive seeding. The use of AgNPs was more effective to remove P. aeruginosa biofilm than applying silver in its ionic form. AgNPs could acts as reservoir to release smaller amount of Agþ ions, thus resulting in higher activity. In addition, the effect of AgNPs on biofilm removal was size-dependent, i.e. smaller nanoparticles had the higher efficacy to remove biofilm.
Acknowledgements The authors would like to thank Mr Shaun Bulcock from Australian Centre for Microscopy and Microanalysis (ACMM) for his valuable advice and help for TEM observations.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Dr Cynthia Whitchurch was funded by a NHMRC Senior Research Fellowship.
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