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Synthesis, Characterization and Antibacterial Activity against Gram Positive and Gram Negative Bacteria of Biomimetically Coated Silver Nanoparticles Elvio Amato,† Yuri A. Diaz-Fernandez,†,* Angelo Taglietti,†,* Piersandro Pallavicini,† Luca Pasotti,† Lucia Cucca,† Chiara Milanese,‡ Pietro Grisoli,§ Cesare Dacarro,§ Jose M. Fernandez-Hechavarria,|| and Vittorio Necchi^ †
Dipartimento di Chimica, Sezione di Chimica Generale, Universita di Pavia, viale Taramelli, 1227100 Pavia, Italy C.S.G.I. & Dipartimento di Chimica, Sezione di Chimica Fisica, Universita di Pavia, viale Taramelli, 1627100 Pavia, Italy § Dipartimento di Scienze del Farmaco, Universita di Pavia, viale Taramelli, 1027100 Pavia, Italy BioInfo, Instituto Superior de Tecnologias y Ciencias Aplicadas, Ave. Salvador Allende y Luaces, Havana, Cuba ^ Centro Grandi Strumenti, Universita di Pavia, Via Bassi 2127100 Pavia, Italy
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bS Supporting Information ABSTRACT: In the present work, we describe a simple procedure to produce biomimetically coated silver nanoparticles (Ag NPs), based on the postfunctionalization and purification of colloidal silver stabilized by citrate. Two biological capping agents have been used (cysteine Cys and glutathione GSH). The composition of the capped colloids has been ascertained by different techniques and antibacterial tests on GSH-capped Ag NPs have been conducted under physiological conditions, obtaining values of Minimum Inhibitory Concentration (MIC) of 180 and 15 μg/mL for Staphylococcus aureus and Escherichia coli, respectively. The antibacterial activity of these GSH capped NPs can be ascribed to the direct action of metallic silver NPs, rather than to the bulk release of Ag+.
’ INTRODUCTION Silver is a popular material for domestic uses since ancient times,1 and silver containers have been used for centuries to purify potable water, since Ag+ is toxic to a wide range of microorganisms.2,3 In low concentrations, silver is not toxic for human cells, and hence it can be considered as an environmentally friendly antimicrobial. In recent decades, the scientific community has focused great effort on the investigation of the antibacterial activity of silver, due to the inability of bacteria to develop resistance toward silver ions.4,5 Recently, silver nanoparticles (Ag NPs) have been studied as sensors and biomarkers,6 exploiting the Localized Surface Plasmon Resonance effect,7 but the most popular application remains the antibacterial use, since nanotechnology has immense potential in biomedical applications, and has become one of the most important research fields in the scientific world. A huge amount of research describes the bactericidal activity of Ag NPs811 and its related aspects. To cite just a few: emphasis on biocompatibility and toxicity,9 possibility to bind the colloids to solid surfaces like fabrics and textiles,10 and mechanism of transport through bacterial membrane.11 The size-dependent interaction of silver nanoparticles with gram-negative bacteria and the biological activity of silver nanoparticles as a function of the particle shape have also been investigated.4 r 2011 American Chemical Society
The scientific debate concerning the mechanism of the antibacterial effect of silver is still open.8 Some authors have suggested that the effect should rely on silver ions released from metallic bulk silver or from NP surfaces, followed by the interaction of Ag+ with the thiol groups in bacterial proteins or by interfering DNA replication.12,13 It has also been reported that silver ions can affect the respiratory chain in bacteria.14 From this perspective, it sounds obvious that nanocrystalline silver has to be more active than a similar mass amount of bulk silver: the close proximity of Ag NPs to bacteria involves high concentrations of released Ag+ in cell surroundings. This has to be considered together with the high surface/mass ratio typically present in nanomaterials: the smaller the particles, the higher the metallic surface exposed, and subsequently higher microbicidal effect can be expected.15 On the contrary, other authors have suggested that Ag NPs toxicity may arise directly from physical processes caused by nano-objects, like disruption of cell membrane and penetration of NPs into the cytoplasm,8,11 with subsequent Ag+DNA binding or interaction with bacterial ribosome.16 This mechanism has been observed mainly for Gram negative bacteria, Received: April 1, 2011 Revised: June 16, 2011 Published: July 07, 2011 9165
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Langmuir and has been attributed to the low mechanical resistance of the phospholipidic cellular membranes, compared with the peptidoglycane rigid cellular walls of Gram positive bacteria. The biological applications of Ag NPs require the appropriate coating of the NPs surface, to avoid aggregation in highly saline media and to favor the solubility in water-based environments. The decoration of the surface with some biomimetic motif can also be desirable, which could favor interactions with biosystems.15,17 Glutathione (GSH) is an ideal candidate for this purpose since this biomolecule displays a thiol function, which strongly binds to the silver surface, and three pH dependent charged functional groups (carboxylates and amine), that promote water solubility and interactions toward more complex biostructures. Moreover, due to the importance of NP dimensions on antibacterial activity,18 it is strongly desirable to find a reliable procedure for coating NPs already synthesized with determined dimensions and shapes with biomimetic protecting agents. In fact, synthesis of Ag NPs in the presence of coating agents, such as amino-acids or small peptides, often produces NPs with variable dimensions, which depend on the concentration of the coating agent19 and on reaction conditions.20 In the present work, we report a coating procedure that allows us to stabilize Ag NPs with biological capping agents featuring thiols moieties (i.e., GSH and cysteine), in order to investigate their use as antibacterial agents. NPs are prepared with a wellestablished method21 that yields reproducible citrate capped Ag NPs of defined dimensions and shapes. The coating procedure with GSH and cys, which substitute citrate on the NP surface, has been investigated and optimized, and final stable products (i.e., GSH or cys coated Ag NPs) fully characterized with several techniques. Finally, it is described how these biomimetically coated NPs can be purified and redissolved in aqueous solutions under physiological conditions, in order to be used for investigation of their antimicrobial activity against bacteria.
’ EXPERIMENTAL SECTION Materials. Silver nitrate (>99.8%), sodium borohydride (g99.0%), sodium citrate (>99.0%), glutathione (99%), cysteine (99%), NaOH (99%), NaCl (99%), standard 1 M HNO3 solutions, PBS (pH = 7), HEPES (pH = 7), CAPS (pH = 10), potassium dihydrogenphosphate (99%), sodium hydrogenphosphate (99%), and Dialysis Tubing Benzoylated pore size 2000NMWCO were purchased from Sigma-Aldrich. Buffer solutions of HEPES and CAPS were prepared in order to obtain a final concentration of 0.01M. The desiderated pH was adjusted, when needed, by adding proper amounts of concentrated HNO3 or NaOH solutions. Iso-Sensitest Broth (ISB) and Tryptone Soya Broth (TSB) for bacteria culture were purchased from Oxoid, England. Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 10536 bacterial strains were used. All reagents were used as received. Water was deionized and bidistilled. Glassware was carefully cleaned with aqua regia, and then washed several times with bidistilled water under sonication before use. Citrate capped NPs were synthesized using procedures previously described.21 Stability of Citrated Capped NPs Solutions. Thermal stability tests were performed on citrate capped NPs solutions by heating inside a closed flask up to 95 C for 1 h. Stability under UV irradiation was tested by placing the citrated capped colloidal solution in a transparent quartz flask inside an irradiation chamber set to maximum power (120 W, 366 nm) for 1 h. UVvis spectra were recorded at regular time intervals, after diluting the NPs solutions 1:6 with bidistilled water. UVvis spectra were registered using an HP 8452 diode array spectrophotometer. NP dimensions were assayed using a Nano ZS90 DLS
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(Malvern) before and after UV and thermal treatments. Chemical stability of the citrated-capped colloids was probed qualitatively by adding PBS Dulbecco’s, HEPES, CAPS, potassium dihydrogenphosphate-sodium hydrogenphosphate buffer (chloride free, pH = 7), and NaCl to the NPs solutions. Stability could be determined even at necked eye, since the solutions on destabilization change visibly from yellow to black-green, evidencing the precipitation of silver.
Spectro-Photometric Titrations and Mie’s Effective Refractive Index Calculations. The spectrophotometric titrations were performed after dilution 1:6 of the synthesis batch solution of citrate capped NPs with bidistilled water and the appropriate buffer solutions (HEPES pH = 7, CAPS pH = 10). The final concentration of the buffer was 0.01 M. Aliquots of the capping agents (cysteine and glutathione) were added from stock concentrated solutions and, after equilibration, the spectra were recorded using HP 8452 diode array spectrophotometer. The variation of the total volume during the titrations was less than 1%. The spectra were analyzed using a homemade software.21 The background was interpolated and subtracted with a cubic-spline function and the parameters of the peaks were determined. The corrected spectra were interpreted applying the Mie’s formalism7 combined with the dielectric function data for silver.22 This approach allows us to calculate the effective refractive index (neff) experienced by the NPs.
Preparation, Purification and Stability of Cysteine and GSH Capped NPs. Coated NPs solutions were prepared by adding different amounts of capping agents (cysteine or GSH) to the synthesized batch solutions of citrate-capped NPs, followed by centrifugation at the appropriate pH. For GSH coated NPs, 0.03 equivalents of GSH with respect to the total silver concentration were added and the pH of the solution was manually set to 3 by adding standard HNO3 (1M) before centrifugation. For cysteine-coated NPs, 0.1 equiv of cysteine with respect to the total silver concentration were added and centrifugation was carried out at the native pH of the solution (pH = 5). Full precipitation was obtained by centrifugation at 8000 rpm for 15 min. The yield of this process was about 5 mg/100 mL of starting solution. For the stability tests of GSH capped NPs, the precipitates were redissolved in PBS Dulbecco’s and HEPES (pH = 7). Cysteine-coated NPs were prepared using CAPS (pH = 10). Thermal, photochemical, and chemical stability tests on glutathione and cysteine-capped NPs were performed as described for citrate-capped NPs. The solutions were characterized by UVvis spectra and DLS.
Bacterial Culture and Antibacterial Activity Evaluation. The antimicrobial activity of GSH capped NPs and silver ions (as silver nitrate) in disperse solution was evaluated against S. aureus and E. coli. For Minimum Inhibitory Concentrations (MICs) evaluation the microorganisms were grown overnight in TSB at 37 C. Washed cells were resuspended in Dulbecco’s PBS and optical density (OD) was adjusted to 0.1 at 655 nm wavelength, corresponding approximately to 1 108 Colony Forming Units/mL (CFU/mL). MIC values of NPs and ionic silver were determined by the standard broth macro-dilution method in ISB with an average inoculums of 107 CFU/mL as described by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS).23,24 MIC was defined as the lowest concentration that completely inhibited bacterial growth after an incubation of 24 h at the temperature of 37 C. The antibacterial activity of GSH coated NPs was also investigated against S. aureus and E. coli by suspending the bacteria in PBS and inoculating concentrations of silver colloid equal to the MIC values for each microorganism. In these experiments, the microorganisms were grown overnight in TSB at 37 C. Washed cells were resuspended in Dulbecco’s PBS and optical density (OD) was adjusted to 0.1 at 655 nm wavelength, corresponding approximately to 1 108 Colony Forming Units (CFU/ml). Then aliquots of GSH coated NPs solutions in PBS were added to reach the MIC values for each microorganism. Bacterial suspensions were incubated at room temperature and viable microbial 9166
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counts were evaluated after 0, 2, 5, and 24 h of contact. Bacterial colonies were counted after incubation at 37 C for 24 h in TSB. The microbicidal effect (ME) was calculated for the two microorganisms at each contact time according to the equation:25 ME ¼ logðN C Þ logðN D Þ
ð1Þ
where NC is the number of colony forming units (CFU) of the control microbial suspension and ND is the number of CFU of the microbial suspension in presence of the GSH capped NPs. TEM Imaging. TEM images at different magnifications were obtained from colloidal solutions of citrate capped Ag NPs, and from solutions of particles coated with GSH and cysteine. After 10-fold dilution with bidistilled water, 10 μL drops were deposited on Nickel grids (300 mesh) covered with a Parlodion membrane and observed with a Jeol JEM1200 EX II instrument.
Characterization of Purified Capped NPs and Capping Agents. The NPs samples for physicochemical characterization were prepared as described for the purification procedure, and were kept overnight under vacuum. The Ag+ complexes of cysteine and glutathione were prepared by mixing 20 mL of 0.1 M AgNO3 solution and 20 mL of 0.1 M cysteine or glutathione solutions, followed by filtration. Precipitation was observed after mixing. The wet samples were kept overnight under vacuum. Thermogravimetric analysis (TGA) was performed on the pure capping agents GSH and cysteine (as received commercial products), on the synthesized Ag+ complexes, and on capped Ag NPs. Powders were placed in alumina crucibles in variable amounts (from 3 to 5 mg) and analyzed using a SDT Q600 instrument (by TA Instruments). The TGA curves were recorded in two experimental set-ups, under static air and under nitrogen flux (15 mL/min), heating the samples from 25 to 1000 C at 1 C/min. Over this temperature, the inorganic residual mass varied less than one hundredth of a milligram. Coupled TG-DSC measurements were performed on GSH and cysteine-capped nanoparticles under nitrogen flux (15 mL/min), by Labsys Evo TGA-DSC (Setaram, France). About 10 mg of powder were placed in an Al2O3 crucible (DSC plate attachment of the instrument) and heated up to 1100 at 2 C/min. Over this temperature, the inorganic residual mass varied less than one hundredth of a milligram. The NP samples for SEM analysis were placed on a biadhesive carbon slide, fixed on the aluminum sample-holder. The typical golden-coating for SEM preparations was avoided in order to allow the quantitative determination of the elementary composition of the samples. The SEM analysis was performed using an EvoMA10 microscope from Zeiss. The experiments were carried out using a LaB6 filament and the tension of the beam and sample current were set to 20 kV and 50 pA, respectively. The composition of the samples was determined by Energy Dispersion Microanalysis (EDS), using an INCA Energy 350 X Max detector from Oxford Instruments, equipped with a Be window. Cobalt standard was used for the calibration of the quantitative elementary analysis. Several determinations were made for each sample at different points of the sample holders, and the determined values were reproducible within 20%. X-rays patterns were collected on capped NPs, pure capping agents and their silver complexes using a Bruker D5005 diffractometer with BraggBrentano geometry equipped with a Cu cathode source (1.54 Å, 30 kV, 40 mA) and a Position Sensitive Detector PSD ASA-S from MBraun. The powder samples were placed on the Si slide of a Bruker A100B36 sample holder. The patterns were collected from 10 to 120 setting 0.014 of angular step and 8.5 s of collection time for every angular value. Infrared spectra were collected on capped NPs, capping agents, and complexes with Spectrum BX FT-IR system from Perkin-Elmer, placing the sample in a sodium chloride window and using Nujol as dispersion media. Determination of Ag Release from the NPs. The experiments of determination of the Ag+ release from GSH capped NPs were
performed using Dialysis Tubing Benzoylated of nominal molecular weight cut off 2000 Da. The NPs solutions were prepared by redissolving different amounts of GSH coated NPs in PBS or Iso-Sensitest Broth in order to obtain total silver concentrations ranging from 20 to 200 μg/mL. Control experiments were performed using silver nitrate dissolved in water and Iso-Sensitest Broth in the same concentration range. The solutions were kept in contact with closed dialysis tubes, containing the same dispersion media, for 24 and 48 h. After carefully washing the external side of the tubes, the contained solutions were recovered and the concentration of silver was determined by means of Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-OES Optima 3300 DV, Perkin-Elmer). Appropriate dilutions with bidistilled water were made on the original samples to reduce the salinity before the analysis. Matrix effects were excluded, since the addition of standard silver solutions produced quantitative responses.
’ RESULTS AND DISCUSSION Citrate Nanoparticles Stability. As already demonstrated by TEM imaging,21 our synthetic procedure yields spherical citratecapped NPs with a large size distribution, i.e., ∼7 ( 4 nm diameter. Dynamic Light Scattering analysis (DLS) on the colloidal solutions gave a comparable hydrodynamic diameter of 9 nm, displaying a broad distribution of particle size. The spectra of this citrate-stabilized silver NPs colloid shows a Localized Surface Plasmon Resonance (LSPR)26,27 band centered at λ = 394 nm in aqueous solutions. LSPR is a characteristic phenomenon of colloidal suspensions of Ag NPs.28,29 The intensity, the shape, and the position of the LSPR bands depend on the size and shape of the nanoparticles,30 on the stabilizing agents coating the NPs surface, and on the dielectric constant of the surrounding environment.31 In the case of spherical silver nanoparticles with size 10 disaggregation and redissolution can be easily obtained. Conversely, for GSH-coated NPs, at pH = 5 no precipitation is observed. However, when the colloidal solution was treated with HNO3 until reaching the iso-electric point of GSH (pH = 3), the LSPR spectra of the GSH-coated NPs features a broad band with a maximum between 550 and 600 nm (data not shown), evidencing interparticle aggregation.37,40 This aggregation process leads to the slow formation of a dark red precipitate that can be separated from the solution by centrifugation. All experiments were conducted adding the minimum quantity of capping agent that completes the coating of Ag NPs surface, i.e., 0.03 equiv for GSH (Ag:GSH 100:3) and 0.1 equiv for cysteine (Ag:Cys 100:10). After centrifugation of the coated NP solution at the appropriate pH, the solid obtained could be dried under vacuum and became a dark-green metallic-like powder, that can be stored and redissolved at the appropriate pH to give stable solutions of biomimetically coated colloidal silver. We characterized the coated NPs samples by means of IR and XRD techniques, and we analyzed also the pure organic coaters, GSH and cysteine, and the silver complexes of these molecules. The IR spectra of GSH featured the characteristic sharp peak of the SH in-plain stretching at 2524 cm1 (Figure 7S, Supporting Information). This peak is absent in the complexes and in the coated NPs, and we interpreted this result as an evidence of the formation of the AgS bond to a total extent, i.e., no excess of unreacted coating agent was present in the precipitates. The unequivocal attribution of other IR peaks cannot be done due to the complexity of the samples. The peaks observed for pure GSH at 3340, 3244, and 3024 cm1 are weaker and broadened on GSH capped Ag NPs, probably reflecting hydrogen bonding between adjacent tripeptide or weak coordinative interactions with silver surface. Similar results were obtained comparing cysteine and cysteine-capped Ag NPs IR spectra (data not shown). XRPD spectra of the organic coaters featured the most intense peaks at low angles, while Ag glutathionate and cysteinate XRPD 9169
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Figure 5. UVvis spectra of redissolved GSH NPs solutions in PBS (solid line), after being heated at 95 C for 1 h (dashed line), and after being irradiated for 1 h in UV chamber (dotted line).
Figure 4. Coupled TGA-DSC profiles under inert atmosphere of (a) GSH-coated silver NPs; (b) cysteine-coated silver NPs. Dotted lines represent the TGA curves, while solid lines depict the DSC profiles.
profiles displayed only broad and poorly intense peaks, indicating low crystallinity (see Supporting Information). On the contrary, silver coated NPs showed intense and broad peaks centered at the distinctive diffraction peaks of silver-fcc (JC-PDF n004 0783, Figure 3). For coated NPs samples, the relative intensities of the peaks were similar to the theoretical values (JC-PDF n0040783), except for the 200 peak that was considerably depressed. The half widths of the peaks were very large, but Scherrer analysis gave unexpectedly large diffraction domains (about 30 nm), inconsistent with TEM and DLS results. We ascribed the discrepancy to the polydispersity of our samples, a well-known interference factor in classical XPRD data interpretation of nanomaterials.41 Additionally, the peaks ascribed to silver-fcc in our case presented peculiar shoulders, suggesting that the structure of the metallic colloid is very complex at the nanoscale. Further investigations are ongoing to clarify this fact. By means of Scanning Electron Microscopy imaging coupled with Energy Dispersion Microanalysis (EDS) it was possible to establish the elementary composition of the precipitated NPs. EDS is known to be quantitative for the relative determination of heavy elements like silver and sulfur, while for carbon and oxygen interferences from instrumental environment and the sample holder can be expected. In our case, it is reasonable to consider that the only source of sulfur in our samples are the organic coaters (GSH or cysteine) and using the minimum quantity of capping agent to reach the full coating of the NPs surface, the silver complex would not be form. Therefore, the Ag:S ratio from EDS determinations would be an indirect evidence of the proportionality between colloidal silver and coating agent in the purified NPs samples. The results showed that the ratios Ag:S for GSH and cysteine-coated NPs were 100:3 and 100:10, respectively, (full EDS data can be found in Figures 9S and 10S, Supporting Information), in perfect agreement with the results obtained from spectrophotometric titrations in solution. Considerable amounts of oxygen and carbon were present, but quantitative determination was not reliable, hence these elements were excluded from the quantitative analysis. We also analyzed the precipitated NPs samples using thermogravimetric analysis, in order to establish the quantitative relationship between sulfur and coater contents. TGA was performed on
both GSH and cysteine-capped nanoparticles, in static air and under inert atmosphere, by treating the samples at low heating rate from room temperature up to 1000 C. To attribute the observed thermal processes, we also performed the TGA of the 1:1 silver complexes for GSH and cysteine, and of the pure GSH and cysteine molecules. Coupled TG-DSC profiles on cysteine and GSH coated NPs were performed under inert atmosphere (Figure 4). We performed a correction of the TGA profiles, excluding the mass loss due to the evaporation of water, and calculating the “dry” mass at 140 C from the experimental profile. At this point, due to the uncertainty of the decomposition mechanism of the organic capping agents in presence of Ag, we applied two different models in order to obtain meaningful data concerning the molar ratio between the capping agents and Ag in our samples. The weight loss due to the thermal decomposition of the organic part allowed to calculate a ligand/silver molar fraction which is in agreement with the sulfur/silver ratio obtained from EDS experiments (see Supporting Information). Therefore, all of the surface bound organic ligands which are decomposed during the TGA measurements are molecules containing a thiol moiety and this means the coating layer is composed only by GSH (or cysteine). It is interesting to notice that the results obtained by the two models, constituting the compositional lower and upper theoretical limits, are similar between them (see Supporting Information) and close to the results obtained using complementary techniques. The quantitative agreement, within the experimental errors, for UVvis, EDS, and TGA data allow us to conclude that the samples were composed mainly by colloidal silver coated by a dense monolayer of the capping agent GSH or cysteine, which has thus completely substituited citrate on particles surface.28,34b Furthermore, only one endothermic DSC peak was observed, centered about 900 C, confirming that the residues of the samples after thermal decomposition of the organic part is composed of metallic silver. The solid obtained after centrifugation of GSH-coated NPs redissolved in a classical biological buffer solutions at pH = 7 (Dulbecco’s PBS and ISB), giving an homogeneous solution with LSPR band (Figure 5) and DLS size distribution identical to the original GSH-coated NPs (see Supporting Information for details). It must be stressed that this fact is the direct evidence of the stability of capped and purified NPs in the presence of high concentrations of electrolytes, simulating physiological conditions.The stability of the redissolved NPs solutions in PBS was monitored for several weeks at room temperature, and no change of the LSPR position or of the bandwidth was observed. Similar results were obtained with the cysteine capped NPs, using CAPS buffer (pH = 10) as dispersion media. Despite the coated NPs 9170
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Langmuir displaying long-term stability in PBS, during the measurement of the Z-potential, we observed color change and precipitation of the colloid, indicating aggregation and probable decomposition of nanoparticles in the conditions of the experiment. Therefore, unreliable data could be obtained. Redissolved samples were analyzed by TEM and DLS, and the size of the NPs was similar to the size obtained on the original citrate NPs samples. Furthermore, photochemical and thermal stability in solution of cysteine and GSH-capped nanoparticles did not change after purification (Figure 5). Combining all of these results with the qualitative and quantitative analysis of the precipitated samples, we can conclude that the adopted precipitation procedure does not modify the properties of the capped nanomaterials, and allows the purification of the NPs from the synthetic batch solution, avoiding the inconvenient effects of synthesis byproduct or contaminants. The precipitates obtained can be handled in noninert environment and be weighted as a solid. Furthermore, GSH-capped NPs solutions are stable above pH = 3 and up to pH = 10 in the presence of high electrolyte concentrations, allowing the evaluation of their antibacterial activity in vitro under physiological conditions (6.8 < pH < 7.4), while cysteine-coated colloids in this pH interval tend to aggregates, precluding any real-world biological application. Antibacterial Activity of GSH-Coated NPs. Determination of MICs values of GSH-capped NPs was carried out using two model bacteria strains: S. aureus and E. coli. These microorganisms are popular as representative of the two major classes of bacteria: Gram positive (S. aureus) and Gram negative (E. coli) bacteria. Gram positive bacteria present a relatively thick and continuous cell wall (thickness 2080 nm) consisting mainly of peptidoglycane. Conversely, Gram negative bacteria feature a thinner peptidoglycane layer (thickness 510 nm) surrounded by an outer phospholipidic membrane. This membrane contains, among other components, a group of protein-channels called porins, and allows the passage of small hydrophilic antibiotics and metabolites into the cell. The antibacterial efficacy of Ag+ against E. coli have been related to presence of these outermembrane porins proteins.1a In our case, MIC evaluation was carried out under physiological conditions, by using ISB at pH = 7 as dispersion media. The MICs for the GSH-capped NPs were 180 and 15 μg/mL for S. aureus and E. coli, respectively (exact concentration of silver after redissolution of purified precipitates in PBS was assayed by ICP-OES prior to the dilution with ISB, reported values are referred to this “real” concentration of silver). The greater resistance of Gram positive bacteria to silver ions is well-known in literature,12,42 but in our case, the difference observed between the two microbial strains was surprisingly huge. Control experiments using silver nitrate instead of colloidal silver found MICs of 15 and 10 μg/mL for S. aureus and for E. coli, respectively. It is interesting to note that for E. coli, the MIC values for colloidal and ionic silver are very similar, while for S. aureus, the MIC of the NPs is 1 order of magnitude larger than the MIC for ionic silver. We performed experiments to determine the microbicidal effect (ME) of GSH-coated NPs against E. coli and S. aureus in PBS suspensions, using the MIC values for each microorganism. The relatively logarithmic lowering of the bacterial population was calculated after 0, 2, 5, and 24 h of contact with the silver colloid using a standard procedure.25 The results (Figure 6) shown that the antimicrobial effect of the biomimetic NPs increases with time, enhancing the differentiation between Gram
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Figure 6. Microbicidal effect (ME) of GSH-coated silver NPs at the minimum inhibitory concentration for E. coli (triangle) and S. aureus (circle). Lines are depicted as guides for the eye.
positive and Gram negative bacteria. The greater effect was observed for E. coli after 24 h of contact, reaching about 3 logarithmic units lowering of the bacterial population. Nevertheless, for both bacterial strains and every contact time investigated, the values of ME are not sufficiently high to reach the full disinfectant effect at the concentration investigated. Therefore, we can conclude that at the MIC, our biomimetic coated Ag NPs interfere with the cell division of bacteria, inhibiting their replication, and are able to considerably reduce the bacterial vitality, leaving a residual population of viable bacteria about 3% for S. aureus and less than 0.05% for E. coli after 24 h of contact. Release of Silver from GSH Capped NPs under Physiological Conditions. The quantitative analysis of the release of silver from the capped NPs was performed combining equilibrium dialysis with ICP determinations. The procedure adopted allows the determination of the ionic “free” silver, if the selected dialysis membrane pores are sufficiently small to retain colloidal silver. We optimized the procedure by making several experiments in different conditions, and we found that using water as solvent in a wide range of concentration of silver nitrate (from 20 μg/mL to 200 μg/mL), the equilibrium across the dialysis membrane is reached after 24 h. When we used the culture broth as dispersion media, the dialyzed concentration of silver nitrate was much lower than the equilibrium concentration expected, even after 48 h. This result could be ascribed to the complexation of silver ions by the protein fragments present in the culture broth (the nominal composition of the culture broth declares a high concentration of hydrolyzed casein). In these experiments, when the initial concentration of silver nitrate was set to 20 μg/mL, the concentration of dialyzed silver was about 2 μg/mL after 24 h. Conversely, when GSHcapped NPs were used at the same initial concentration of silver (20 μg/mL), the dialyzed amount of ionic silver after 24 h was even less than 0.3 μg/mL. Furthermore, for silver NPs at a concentration of about 200 μg/mL, dialyzed silver was less than 0.5 μg/mL (all data set is described in detail in SI). Comparing these results, we conclude that the quantity of Ag+ released from GSH-capped NPs in the culture broth is considerably less than the MICs values of ionic silver, when the concentration of colloidal silver is within the range of the MICs for E. coli and S. aureus. Therefore, we can conclude that the antimicrobial activity of our coated colloidal silver is not a consequence of the bulk release 9171
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Langmuir of Ag+ from Ag NPs surface, but a result of the direct interaction of the metallic biomimetic coated NPs with cells, and probably the mechanism of action of coated colloidal silver is different from Gram positive to Gram negative bacteria, as expected from the different cellular structure. Further experiments to fully understand the mechanism of antibacterial action of GSH-capped Ag NPs are currently being carried on in our lab.
’ CONCLUSIONS A simple procedure to produce and purify biomimetic coated silver nanoparticles has been developed. The accurate characterization of the colloidal biomimetic-coated materials using different techniques showed that the molar composition of the capped colloids resulted Ag1.00GSH0.03 and Ag1.00 Cys0.09. GSH capped NPs were stable under physiological conditions and antibacterial tests gave MICs values of 180 and 15 μg/mL for S. aureus and E. coli, respectively. Dialysis experiments demonstrated that Ag+ release from the capped colloid is less than MIC values under physiological conditions. Hence, the antibacterial activity of dispersed GSH capped Ag NPs can be ascribed to the direct action of metallic silver NPs, rather than to the bulk release of Ag+. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional characterization data of GSH and cysteine-capped NPs in dispersed solution and solid state, discussion of TGA experiments and ICP-OES data of dialyzed solutions are available. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +39 0382987985; Fax: +39 0382 528544; E-mail:
[email protected] (A.T.),
[email protected] (Y.A.D.-F.).
’ ACKNOWLEDGMENT Authors acknowledge financial support by Fondazione Cariplo (Bandi Chiusi 2007, “Superfici vetrose a azione antimicrobica basata sul rilascio modulato e controllato di cationi metallici”). J.M.F.-H. thanks the University of Pavia for a CICOPS Scholarship grant (2009). Alessandro Girella is also acknowledged for SEM-EDS microanalysis (Pavia H2 Lab, University of Pavia, Italy). ’ REFERENCES (1) (a) Silver, S.; Phung, L. T.; Silver, G. J. Ind. Microbiol. Biotechnol. 2006, 33, 627–634. (b) Russell, A. D.; Hugo, W. B. Prog. Med. Chem. 1994, 31, 351. (2) Liau, S.; Read, D.; Pugh, W.; Furr, J.; Russell, A. Lett. Appl. Microbiol. 1997, 25, 279–283. (3) Pradeep, T. A. Thins Solid Films 2009, 6441–6478. (4) Sukdeb, P.; Yu Kyung, T.; Joon Myong, S. Appl. Environ. Microbiol. 2007, 73, 1712–1720. (5) (a) Shahverdi, A. R.; Fakhimi, A.; Shahverdi, H. R.; Minaian, S. Nanomedicine 2007, 3, 168–171. (b) Sharma, V. K.; Yngard, R. A.; Lin, Y. Adv. Colloid Interface Sci. 2009, 145, 83–96. (c) Marambio-Jones, C.; Hoek, E. M. V. J. Nanopart. Res. 2010, 12, 1531–1551. (d) Chaloupka, K.; Malam, Y.; Seifalian, A. M. Trends Biotechnol. 2010, 28, 580–588. (6) (a) Lee, K.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 19220–19225. (b) Schoefield, C. L.; Haines, A. H.; Field, R. A.; Russel, D. A. Langmuir 2006, 22, 6707. (c) Yoosaf, K.; Ipe, B. I.; Suresh,
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