Poly(ethylene oxide)-b-Poly(propylene oxide) Amphiphilic Block

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Poly(ethylene oxide)-b-poly(propylene oxide) Amphiphilic Block Copolymer - Mediated Growth of Silver Nanoparticles and their Antibacterial Behavior Anna Perdikaki, Panagiota Tsitoura, Eleni C Vermisoglou, Nick Kanellopoulos, and Georgios Karanikolos Langmuir, Just Accepted Manuscript • DOI: 10.1021/la402083v • Publication Date (Web): 13 Aug 2013 Downloaded from http://pubs.acs.org on August 20, 2013

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Poly(ethylene oxide)-b-poly(propylene oxide) Amphiphilic Block Copolymer - Mediated Growth of Silver Nanoparticles and their Antibacterial Behavior Anna V. Perdikaki1, Panagiota Tsitoura2, Eleni C. Vermisoglou1, Nick K. Kanellopoulos1, and Georgios N. Karanikolos1* 1

Institute of Advanced Materials, Physicochemical Processes, Nanotechnology and

Microsystems (IAMPPNM), Demokritos National Research Center, Athens 153 10, Greece 2

Institute of Biosciences & Applications, Demokritos National Research Center, Athens 153 10, Greece *

Corresponding author email: [email protected]

Keywords: nanoparticles, silver, self-assembly, block copolymer, antibacterial, E. coli

Abstract Silver nanoparticles were grown in self-assembled amphiphilic poly(ethyleneoxide)/poly(propylene-oxide) triblock copolymers in selective solvents. Ternary systems of block copolymer, water, and p-xylene were used forming a dispersion of water droplets in oil (reverse micellar), as well as binary water/block copolymer solutions. Besides its stabilizing affect, the role of the copolymer as a reducing agent for the metal salt precursors was examined. It was found that block copolymer enabled reduction, carried out mainly by the PEO blocks, could take place only under particular conditions mostly related to the metal precursor, the block copolymer 1

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concentration, and the self-assembled micellar configuration. The effect of the triblock copolymers on growth and stabilization of gold nanoparticles was also examined. The antibacterial effect of the silver nanoparticles was investigated against Escherichia coli (E. coli) cells and their performance was evaluated through a series of parameterization experiments including effect of metal concentration, stability, activity over time, and dosage, while particular emphasis was given on the role of ions versus nanoparticles on the antibacterial performance.

1. Introduction Metal particles in the nanometer size range exhibit properties that are substantially different from those of the same material in bulk or in ionic form.1-3 Besides their fundamental interest, these particles are found more and more frequently in our common life due to their usage in a variety of commercially available products.4-5 Silver in particular, has been known for its antibacterial activity since ancient Greece yet, nowadays, the development of new bacteria strains that are resistant to current antibiotics demands for development of novel, high performance agents with improved antibacterial efficiency and minimal side effects.2,

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nanoparticles in medicine can be used to reduce infections e.g. in burn treatment and arthroplasty as well as to prevent bacteria colonization on prostheses, catheters, and dental materials.6-7 Furthermore, new applications that show promise include additives in textiles, food packaging, cosmetic and disinfectant products, and water treatment.8 Due to the significant antibacterial effect of silver nanoparticles, various methods have been developed for their synthesis, the most common being chemical reduction of metal salt precursors in solution. A variety of reducing agents have been 2


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used including strong, such as borohydrides, weak, such as citrates, or a combination of strong and weak ones through a sequential reduction process, according to which a strong reductant is first applied to produce small particles, which are subsequently enlarged by a weaker reducing agent.9-10 Synthesis using Tollens process has also been applied,6,

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according to which reduction of metal salts is being done using a

range of reducing saccharides, as a more environmentally friendly alternative. Interestingly, flame synthesis, as a non-solution based approach, has yielded good quality metal particles directly deposited on inert supports such as SiO2. Close control of particle size and morphology was achieved and nanosilver agglomeration could be hindered to a good extent by suitably tuning the reaction and deposition conditions. In this type of processing, the formation of an external oxide layer around the metal core is difficult to be eliminated resulting in metal/oxide hybrid particles.12 The antibacterial properties of silver ions have been known long ago, yet those of silver nanoparticles have been studied only recently following the rapid development of nanotechnology.13-16 In-vitro studies reveal that silver has the ability to attack normal cells, pathogens, and cancer cells and, despite the fact that in-depth investigation on the long-term effect in mammals is limited, there is evidence that the former are more resistant than microorganisms possibly due to the immune system of eukaryotic organisms that is capable of defending against external attack.17 Concerning the mechanism responsible for the antimicrobial properties of silver, it has been reported that the antibacterial action is based on inhibition of the respiration process, as silver binds onto the bacterial cell through interaction with the thiol groups found in the cell respiratory enzymes, and penetrates inside the cell converting DNA into a condensed form lacking replication ability.2, 18-20 Inactivation of proteins after attachment on the thiol groups is also a possible path, similar to what has been 3


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observed for heavy metals. Silver nanoparticles, due to their small size, have also the ability to penetrate inside the bacteria and interact with the sulfur-containing proteins, as well as with the phosphorus-containing molecules like DNA, while their extremely high surface area provides better contact with the microorganisms enhancing the antibacterial performance. Similarly to ions, it has been reported that nanoparticles attack the respiratory chain causing cell death, while inside the cell they can release ions from their surface, thus providing a dual antibacterial action.2,

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To this

extent, one should not neglect that ionic silver at high concentrations is toxic against eukaryotic cells, in contrast to silver in nanoparticle form that exhibits considerably lower toxicity even at increased concentrations.5, 12, 14, 19, 21 Therefore, it is important to comparably evaluate the antimicrobial efficiency of nanoparticles versus ions so as to maintain the proper balance between the two species, towards development of stable nanoparticle-based antibacterial systems exhibiting increased efficiency against pathogenic microorganisms, yet low toxicity/ecotoxicity for human cells and the environment. Herein, we have grown silver nanoparticles in stable self-assembled systems based on amphiphilic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-b-PPO-b-PEO) block copolymers (BCs, Pluronics). Amphiphilic BCs bear both polar and non-polar groups and spontaneously segregate upon contact with solvents of appropriate polarity forming stable self-assembled structures,22-23 thus providing an ideal stabilizing environment for growth of nanoparticles. To this extent, a variety of BCs have been utilized as templates for growth of silver nanoparticles, such as polystyrene-b-poly(acrylic acid),24 polyether-b-polyamide,25 polystyrene-bpoly(vinyl pyrrolidone),26 poly(styrene-b-isoprene-b-styrene),27-28 each one exhibiting a distinct set of properties and nanoparticle stabilization action. In our previous work, 4


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PEO-b-PPO-b-PEO templates were used to direct the growth of compound semiconductor nanocrystals and yielded exceptionally stable particle populations of different morphologies by enabling well-controlled nucleation and growth reactions inside the formed self-assembled nanodomains.29-30 These copolymers have also been used for metal nanoparticle formation in aqueous solutions and the mechanism of growth and stabilization was investigated.31-33 In the present work, under particular conditions, the copolymer molecules acted as a reducing agent through the PEO blocks, which are in contact with the water phase and the metal precursor, while they have the potential to also act as barriers and control the release of ions from the particle surfaces and the contact/interaction of the released or excess ions with cells. Using these BC systems, flexibility in altering nanoparticle characteristics was exemplified by comparatively testing different BC morphologies and growth parameters, namely, employing ternary systems consisting of two immiscible solvents, binary micellar mixtures, enabling BC-activated reduction, and particle formation using external reducing agent by microemulsion mixing, while growth of gold nanoparticles was also performed, thus providing multiple degrees of freedom in customized nanoparticle preparation. The antibacterial activity of the silver-loaded BC systems was investigated against Escherichia coli (E. coli) cells, where, among other parameters examined, experiments were performed by controllably altering the ion versus particle relative content and comparably evaluating the antibacterial activity of the resulting mixtures in order to provide insights concerning the effect of the balance between particles and ions on the bacterial growth. To this extent, the PEO-b-PPO-b-PEO block copolymers used exhibit unique function not only in passivating the particles and protecting them from agglomeration/degradation over time, but also in binding with silver in ionic 5


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form through complexation interactions,34 which is anticipated to affect activity. To this extent, and given that a well controlled irreversible reaction for nanoparticle growth was used, useful insights were extracted as to determine qualitatively and quantitatively which species (particles or ions) at the given nanoparticle formulation is mostly responsible for the antibacterial action, as well as data were obtained regarding the optimum content of ions and particles at a given block copolymer concentration. The PEO-b-PPO-b-PEO BCs are advantageous for this type of study as, under the condition tested, they do not exhibit any antibacterial action, in contrast to other analogous systems35, allowing for evaluation of the antibacterial effect originating exclusively from Ag. Their use as templates, even at low concentrations, proved very efficient in stabilizing the colloidal dispersion of the silver particles and ions and in maintaining the antibacterial effect significant for a long time after synthesis.

2. Experimental Section 2.1. Synthesis of nanoparticles in the PEO-b-PPO-b-PEO/water/p-xylene ternary system. Metal nanoparticles were prepared inside the reverse micelles of the L2 phase of the ternary system that consisted of P104 BC (molecular formula EO27PO61EO27 according to its nominal MW of 5900, 40% PEO content, BASF Corporation), pxylene (≥99.0%, Sigma Aldrich) as the non-polar continuous phase, and water as the dispersed polar phase in which the metal salts were dissolved. PEO is the water soluble block and PPO is the p-xylene soluble block. Information about the lyotropic properties of this ternary system can be found elsewhere.23 The metal precursors used were silver nitrate (AgNO3, Panreac), and hydrogen tetrachloroaurate (III) solution (99.9%, Sigma Aldrich). Silver nanoparticles were grown using also binary aqueous 6


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BC solutions consisting of P105 BC (with molecular formula EO37PO56EO37 according to its nominal MW of 6500, 50% PEO content, BASF Corporation), and potassium borohydride (KBH4, 99.9% Sigma Aldrich) as the reducing agent. Deionized water was used throughout the experiments. Gold nanoparticles were synthesized as follows: 0.617 g P104 BC was added to 1.83 mL p-xylene and the mixture was sonicated until it became homogenized and transparent indicating micelle formation. 0.008 mL of aqueous hydrogen tetrachloroaurate (III) solution of varying concentration was subsequently added to the above mixture and the final solution was sonicated so as to become homogenized and transparent as well. For the synthesis of silver particles, 1.96 mL p-xylene, 0.67 g P104, and 0.025 mL of aqueous silver nitrate solution were used and the composition of the reverse micellar system was 27.5 wt% P104, 71.5 wt% p-xylene and 1 wt% water. 2.2. Synthesis of silver nanoparticles in the PEO-b-PPO-b-PEO /water binary system. Silver nanoparticles in BC binary mixtures were prepared by adding 0.0715 g P105 to 1.6 mL water (3.5 wt% P105) followed by sonication until homogenization of the solution. 0.2 mL of aqueous AgNO3 solution was subsequently added followed by the addition of 0.2 mL of aqueous KBH4 under agitation. Aqueous silver ionic solutions and samples without AgNO3 precursor were also prepared as reference samples. 2.3. Characterization. UV–Visible (UV–Vis) absorption spectroscopy was performed using a HITACHI U-3010/U-3310 spectrophotometer in the range of 200–700 nm in order to observe changes in the absorption spectra for the different nanoparticle solutions, as well as to monitor bacterial growth. X-ray characterization was carried out in a Siemens D500 X-ray diffractometer. Samples for TEM analysis were

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prepared by depositing a drop of the metal nanoparticle solution on a carbon coated copper TEM grid. The instrument used was a FEI CM20 TEM operating at 200 kV. 2.4. Growth inhibition assay/antibacterial activity. The antibacterial activity of the nanoparticles was evaluated by a growth inhibition assay, using E. coli (strain DH5α) cells. All bacterial cultures were grown in LB medium [1% w/v tryptone, 0.5% w/v yeast extract, 0.5% w/v NaCl] at 37 °C under constant agitation (220 rpm). The growth inhibition assay was performed as follows: a small bacterial culture grown overnight was used to inoculate 10 mL of LB in glass Erlenmeyer flasks, at a dilution of 1:50. Subsequently, 1 mL of the various nanosilver solutions (except for the experiment concerning the different doses) was added, and the cultures were transferred to the incubator and left to grow aerobically at 37°C, as described above, for almost 6 hours. The bacterial growth was monitored by periodically taking 1 mL of sample from the cultures, and measuring the absorption optical density (O.D.) at 600 nm. A sample was also withdrawn immediately after addition of the nanoparticles to the cultures (“zero time”), and data were corrected for different background absorbance values of the silver nanoparticle solutions. In each experiment, a control E.coli culture (grown in parallel but without addition of nanoparticles) was also included. Two or three independent experiments were carried out for each set of data presented.

3. Results and Discussion 3.1. Growth of metal nanoparticles Silver nanoparticles were grown in PEO-b-PPO-b-PEO ternary mixtures consisting of water and p-xylene, as the two immiscible solvents, in order to achieve segregation of the polar PEO and less-polar PPO groups of the copolymer and enable 8


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self-assembly of stabilized water domains dispersed in oil. The composition of the reverse micellar solution was 27.5 wt% P104, 71.5 wt% p-xylene, and 1 wt% water. In this system, BC-enabled reduction of the Ag precursor (AgNO3) was found to occur in the water dispersed domains mainly through the PEO blocks, the concentration of which was 11 wt% in the final mixture. The mechanism of this type of reduction has been exemplified before for growth of Au particles using analogous BC systems without use of external reducing agents.34 UV-vis spectra of the obtained nanoparticles for different precursor concentrations in the final mixture are shown in Fig. 1a. The peaks are centered at ~440 nm, which is a characteristic absorbance wavelength for Ag nanoparticles, and the intensity increases for higher Ag concentrations. The TEM image in Fig. 1d shows spherical nanoparticles with a diameter of less than 10 nm corresponding to a Ag precursor concentration of 5x10-4 M. High-resolution TEM imaging (Fig. 1f) reveals the crystalline quality of the resulting Ag nanoparticles, while Fast Fourier Transform analysis indicated a dspacing of 0.235 nm, which corresponds to the (111) crystallographic plane of Ag. Notably, after homogenization of the solution, the particles continue to grow for several hours, indicating that the BC-enabled reduction process exhibits relatively slow kinetics. Indeed, as shown in Fig. 1b, the absorbance intensity after synthesis increases with time indicating that Ag+ ions continue to be reduced by the PEO blocks and be incorporated onto the mother particles inside the water droplets through particle-cluster coalescence.36-38 To this extent, assistance to the reduction was provided by an external reducing agent (KBH4), which was added in the Ag-containing microemulsion in the form of a KBH4-containing water-in-oil microemulsion. Both ternary systems had the same composition and were mixed together and homogenized by sonication for a 9


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(a) Ag absorbance intensity (a.u.)

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Figure 1. Nanoparticles grown in self-assembled P104 BC water-in-p-xylene ternary systems. (a) UV-vis spectra of Ag nanoparticles produced by BC-enabled reduction using different AgNO3 concentrations. (b) Evolution of BC-enabled reduction and KBH4-assisted reduction of Ag nanoparticles with time after preparation. (c) UV-vis spectra of Ag nanoparticles produced by KBH4-assisted reduction using different AgNO3 concentrations and the respective equimolar KBH4 amounts. (d) TEM image of Ag nanoparticles prepared by BC-enabled reduction using a 5x10-4 AgNO3 concentration in the final mixture. (e) Au nanoparticles produced by BC-enabled reduction using two Au precursor concentrations. (f) HR-TEM image of Ag nanoparticles. The inset corresponds to the Fast Fourier Transform of the lattice fringes revealing a d-spacing of 0.235 nm, which is attributed to the (111) crystallographic plane of Ag.

period of 15 min. Kinetic data of nanoparticle growth after synthesis (Fig. 1b) confirm that, after preparation, growth of particles formed by KBH4-aided reduction is considerably less significant compared to growth of the BC-reduced ones. This indicates that upon KBH4-aided reduction, the majority of Ag+ ions are already reduced and converted to nanoparticles during the sonication step. On the contrary, the BC-enabled reduction process exhibits slower kinetics and, as a result, the 15-min preparation period is not sufficient to fully reduce the ions, thus reduction and particle growth continues after preparation in higher conversion rate compared to the KBH4assisted reduction. UV-vis spectra of the particles resulted from KBH4-assisted reduction are shown in Fig. 1c. We observe that the absorbance intensity increases as the concentration increases, similarly to the BC-reduced particles, yet the peaks of the KBH4-reduced ones are sharper for all Ag concentrations tested indicating a finer 11


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nanoparticle dispersion., although the KBH4-based particles are more dilute due to the addition of the KBH4–containing microemulsion. Furthermore, a blue shift of the absorbance peak of the KBH4–based particles as the Ag concentration increases is evident, in contrast to the BC-reduced ones where a slight red shift is observed. This indicates that finer particle dispersion is formed with sizes averaging at lower values, in contrast to the BC-enabled reduction case where formation of aggregates takes place. Aggregation is the result of partial destabilization of the self-assembled domains caused by the oxidation of the PEO blocks, which act as Ag+ reductants, and the phenomenon is more pronounced for higher AgNO3 content as more PEO needs to be oxidized. In contrast, KBH4 helps preserving the quality of the copolymer by undertaking the reduction, thus promoting stabilization of the resulting particles. Enhancement of the stability is significant given the slow kinetics of the PEO-enabled reduction process, as described above, thus KBH4 reduces Ag before considerable destruction to the copolymer can take place. Using the same ternary systems we proceeded to growth of gold nanoparticles. The system used consisted of 27.3 wt% PEO-b-PPO-b-PEO, 72.3 wt% p-xylene and 0.4 wt% water containing hydrogen tetrachloroaurate (III) as the Au precursor. Similarly to Ag, upon BC-enabled reduction, gold ions (AuCl4-) are converted to Au0 through the PEO blocks (11 wt % PEO content) yielding Au nanoparticles. Fig. 1e shows absorbance spectra of the synthesized Au nanoparticles corresponding to 7x105

M and 1.4x10-4 M Au precursor concentration. The absorption bands, originated

from the surface plasmon of the gold nanoparticles,39 are centered at ~540 nm. Experiments using KBH4 as external reducing agent were also conducted and the resulting spectra for two different precursor compositions and equimolar KBH4 concentrations are shown in Fig. S1 of the Supporting Information. Similar to the Ag 12


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particles, the absorption bands exhibit single peaks, indicating the spherical particles are mainly produced,33 while a red shift is evidenced in the KBH4-based Au particles, from 532 to 538 nm, as concentration increases indicating that the average particle size increases.

3.2. Antibacterial activity of silver nanoparticles The PEO-b-PPO-b-PEO stabilized silver nanoparticles were tested for their antibacterial activity against E. coli cells. The nanoparticles were prepared using a P105 BC-in-water binary solution. Analogous systems have been shown before to stabilize Au nanoparticles.34 The BC content was 3.5 wt% in the final solution. The ternary p-xylene/PEO-b-PPO-b-PEO/water systems were also tried, in which silver nanoparticles were grown by BC-enabled reduction using a 3x10-4 M AgNO3 concentration in the final mixture. However, the addition of the particle solutions to the bacterial culture in the liquid medium affected the culture mainly due to the presence of the p-xylene, and destabilized the nanoparticle-containing “water-in-oil” microemulsion resulting in turbid mixtures that were difficult to be characterized by UV-Vis spectroscopy in order to quantify the bacterial growth inhibition. Notably, it was found that in the binary systems and under the BC concentrations used, BCenabled reduction did not take place. Therefore, growth of particles was assisted by the addition of KBH4 as to reduce the Ag+ ions to Ag0 according to the following reaction:40-41 AgNO3 + ΚΒΗ4 + 3H2O

Ag0 + B(OH)3 + 7/2H2 + KNO3

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The diffraction peaks (Fig. S2 in Supporting Information) confirm that the reaction yields crystalline Ag at room temperature.

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Figure 2. (a) Antibacterial activity of Ag nanoparticles using different Ag precursor concentrations and equimolar KBH4, monitored through the absorbance band at 600 nm. (b) Absorbance spectra of the nanoparticle samples. The inset shows the corresponding photographs of the nanoparticle solutions.

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The affect of Ag concentration was examined first. Bacterial growth in the liquid culture in the presence of different nanoparticle concentrations was monitored by the UV-Vis O.D. at 600 nm, which is widely used for indirect measurement of bacterial cell number. Fig. 2a compares the bacterial growth corresponding to three samples prepared using equal AgNO3 and KBH4 concentrations, namely, 10-3 M, 5x10-4 M, and 10-4 M, for a period of up to 6 h. It is evident that the bacterial growth was negatively affected by all three nanoparticle samples, with the effect being stronger as concentration increases. Indeed, for the sample corresponding to 10-3 M Ag, a minor increase in the O.D. at 600 nm is observed for all the duration of the experiment, while for a 10-times more dilute nanoparticle solution the effect was considerably weaker and the growth curve resembled that of the control sample. Notably, the curvature of the profiles changes from negative to positive from high to low concentrations, respectively, emphasizing the role of Ag in suppressing bacterial growth. To ensure that the only effect on the bacterial growth was due to Ag, we conducted control experiments using blank samples without addition of AgNO3, i.e. containing only PEO-b-PPO-b-PEO and KBH4 at same concentrations (5x10-4) as the ones used in the actual nanoparticle mixtures, and we observed that these components did not significantly affect the growth of the bacterial culture. Particularly concerning PEO-b-PPO-b-PEO, the fact that it does not have any antibacterial activity makes it suitable to study the effect of nanoparticles alone, in contrast to other surfactant stabilizers, such as PVP360, SDS, and Tween 80 that have been shown to affect bacterial growth.35 Fig. 2b shows the absorbance spectra and the corresponding visual appearance of the three nanoparticle samples used in the growth inhibition assay prior to addition to the bacterial culture. It is evident that all samples are optically transparent and the 15


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intensity of the 400 nm peak corresponding to Ag nanoparticles increases with concentration. In addition, the absorbance peak becomes wider yet it is shifted to lower wavelengths as concentration increases revealing that the particle dispersity increases but the average size of the majority of particles becomes smaller. Furthermore, the sample corresponding to 10-4 M Ag exhibits a considerably lower intensity compared to the other two, which is also supported by the visual appearance as shown in the inset photographs, justifying its weak antibacterial effect observed and shown in Fig 2a.

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The effect of Ag in nanoparticle versus ion form was subsequently investigated. Given the reported high toxicity of free Ag+ ions, such an analysis is critical in order to achieve the proper balance between activity and potential toxicity in nanoparticle-based antibacterial substances. Given that BC-enabled reduction of the Ag+ ions was not found to occur in the binary systems and at the copolymer content examined in this section, control of the ion and nanoparticle relative concentrations was achieved by tuning the amount of the KBH4 reducing agent added in the Ag precursor BC solution. Therefore, keeping the AgNO3 concentration constant, the effect of the KBH4 concentration and thus, taking into account the reaction stoichiometry by Eq. 1, the content of free Ag+ ions in the antibacterial activity was explored. For this purpose, a sample corresponding to 5x10-4 Ag concentration was tested that was prepared by using three different KBH4 concentrations, namely, a smaller one (10-4 M) ensuring the existences of excess free Ag+ ions, a larger one (103

M) indicating that no ionic Ag exists, and an equimolar amount (5x10-4 M).

Photographs of the three nanoparticle samples indicating the nanoparticle existence are shown in Fig. S3 in the Supporting Information. Fig. 3 shows the corresponding bacterial growth, as monitored by the O.D. at 600 nm, versus time. It is evident that the existence of Ag+ ions enhances the activity, as the sample corresponding to lower KBH4 concentration causes almost complete growth inhibition, while the one with excess KBH4, though still exhibiting some antibacterial effect, its action is suppressed considerably. This indicates that ions play the primary role in the antibacterial performance. Depending on the relative content of the reducing agent and BC, these ions can be dispersed in the aqueous phase, can form complexes with the BC chains through ion-PEO complexation, as has been indicated before for Au,34 and can be

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bound onto the nanoparticle surface. To this extent, the BC chains can also act as barrier to control the generalized release of Ag+ ions from the particle surfaces.

2 Samples 1 month after synthesis:

1,5 O.D. at 600 nm (a.u.)

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1

5x10-4 M Ag NPs

5x10-4 M Ag ions no P105

5x10-4 M Ag ions with P105

Ag ions 1 day no P105

Ag ions Ag ions 1 day 1 month with P105 with P105

0,5

0

Ag NPs 1 month

Ag NPs 1 day

Ag ions 1 month no P105

Figure 4. Antibacterial effect of Ag+ ions versus nanoparticles one day and one month after preparation. The nanoparticle samples correspond to a Ag precursor and KBH4 concentration of 5x10-4 M. Bars from left to right correspond to bacterial growth at 0 min, 40 min, 1h, 2h, 3h, 4h, and 5h after nanoparticle addition to the bacterial culture.

To further emphasize on the effect of Ag+ ions, we conducted experiments using AgNO3 without reducing agent as to ensure that all Ag amount provided is in ionic form. Fig. 4 shows the antibacterial activity of these ionic samples in the presence and absence of BC, in contrast to nanoparticle samples prepared using the same Ag concentration (5x10-4) by addition of KBH4. The experiments were repeated 18


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with parallel comparison between freshly prepared and one month old Ag samples, so as to investigate possible changes with time. Notably, almost complete growth inhibition was induced by the ionic samples, and the same behavior was observed by testing the samples one month after preparation. Furthermore, the samples containing exclusively Ag+ ions did not show any coloration immediately after preparation and one month later (inset of Fig 4) irrespective of whether BC was added or not. This indicates that the binary BC mixture used does not induce BC-enabled reduction of ionic Ag+ into Ag nanoparticles, in contrast to what has been observed for Au nanoparticles using analogous systems.31,

34

This is further confirmed by UV-vis

measurements of the blank samples one day and one month after preparation, which are appended in the Supporting Information (Fig. S4). In addition, the ternary systems described above (Fig. 1) were able to induce BC-enabled reduction of Ag ions using a BC with lower PEO content (P104 with 40 wt% versus P105 with 50 wt% used in the binary systems), which is the block with the dominant role in the reduction,34 yet at higher overall PEO concentration in the final solution (11 wt% versus 1.7 wt% used in the binary systems). Consequently, BC-enabled reduction is a function of the metal precursor, the BC concentration, and the mixture configuration. Concerning the latter, ternary reverse micellar systems consisting of BC-stabilized water droplets dispersed in a non-polar continuous medium direct the water-soluble PEO blocks to be highly concentrated around the water nanodomains as to avoid interaction with the non-polar continuous phase. Because of this highly localized configuration, the effect of the PEO blocks in reducing the metal precursor which is dissolved in the dispersed water domains is anticipated to be much stronger than in the binary mixtures, where both BC and metal precursor are widely distributed in the whole volume of the solution as water is the continuous medium. 19


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(a) -4

Absorbance (a.u.)

10 M Ag 1month -4 10 M Ag 1day -3 10 M Ag 1day -4 5x10 M Ag 1day -3 10 M Ag 1month -4 5x10 M Ag 1month

350

400

450

500

550

600

650

700

Wavelength (nm)

(b) 1,8

CONTROL 2 years

O.D. at 600 nm (a.u.)

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1,3

1 day

0,8

0,3

-0,2

0

50

100

150

200

250

300

350

400

Time (min)

Figure 5. (a) Absorbance spectra of Ag nanoparticles corresponding to 10-4 M, 5x10-4 M and 10-3 M AgNO3 and KBH4 concentration one day and one month after preparation. (b) Growth inhibition effect of Ag nanoparticles solution two years after preparation using a 5x10-4 M AgNO3 and KBH4 concentration.

20


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The stability of the BC-bound nanoparticles and their antibacterial activity over time up to two years after synthesis was examined next. Fig. 5a shows absorbance spectra of Ag nanoparticle solutions with concentrations of 10-4 M, 5x10-4 M, and 10-3 M one month in comparison to one day after preparation. The absorption bands after one month are wider, the intensity increases, and the peaks are shifted to higher wavelengths. For instance, a red-shift is observed in the sample corresponding to 5x10-4 concentration from 392 nm to 412 nm. This is indicative that particle dispersity and average size increase, which is attributed to particle agglomeration taking place with time, and to conversion of unreduced ions into Ag0 and subsequent incorporation into the mother particles or formation of additional clusters that subsequently grow into particles. Concerning the latter, it is evident that after synthesis and despite the fact that equimolar amounts of Ag precursor and reducing agent were used, there still may exist unreduced Ag+ ions in the solution that react gradually with KBH4. It is possible that the presence of the BC provides an encapsulating protective shell to the ions through complexation, thus preventing instantaneous reduction when the reducing agent is added. However, the antibacterial activity of the particles one month after preparation remains almost unchanged compared to the particles one day after preparation, as shown in Fig. 4, indicating that the changes in the nanoparticle solutions with time revealed by the UV-vis spectra are minor when it comes to antibacterial performance and do not affect the antibacterial activity. This stability and prolonged activity of the particles is attributed to the role of the PEO-b-PPO-b-PEO as stabilizer despite the fact that a relatively small copolymer concentration was used (3.5 wt%). To evaluate the nanoparticle solutions over a bigger timeframe, we conducted bacteria experiments two years after preparation of the nanoparticles (Fig. 5b, 5x10-4 M). Notably, despite the long period after synthesis, 21


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the nanoparticles are still active emphasizing on the fact that the BC enhances stability and maintains activity. The decrease in the activity is attributed to the reduction of unreacted ionic species and to the increase of the average nanoparticle size by agglomeration effects, as discussed above, which lowers the content of surface-bound ions due to the decrease of the overall nanoparticle surface area. To this extent, one could further enhance the antibacterial performance over time by using higher copolymer concentration and by appropriately tuning the nanoparticle concentration, and dosage of a given nanoparticle solution. Concerning the latter, the effect of nanoparticle dosage in the suppression of bacterial growth was examined. To do this, the concentration of the nanoparticle solution to be added in the bacterial culture was remained constant and the amount of the solution injected to a fixed bacterial culture volume was altered. This study was important for the purpose of enhancing activity, by tuning dosage, without changing nanoparticle characteristics, while when activity is tuned by changing metal precursor concentration before particle growth (Fig. 2), this inevitably changes particle characteristics as well. Fig. S5 in the Supporting Information shows the timedependent change in O.D. at 600 nm over a period up to 6 h for different dosages of a nanoparticle solution with a concentration of 5x10-4 M Ag added in 10 mL of bacterial culture. It is evident that, despite the fact that a relatively dilute nanoparticle solution was used, the antibacterial activity is enhanced as nanoparticle dosage increases and, using a volume of 2 mL of the solution under consideration, almost complete inhibition of bacterial growth was achieved. Consequently, optimum efficiency can be achieved by executing combined experiments by manipulating nanoparticle solution concentration upon synthesis, and thus particle characteristics, and dosage during the antibacterial application. 22


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4. Conclusions Noble metal nanoparticles were prepared using amphiphilic PEO-b-PPO-bPEO triblock copolymers through BC-enabled reduction, which was primarily attributed to the PEO blocks, and directed reduction by an external reducing agent (KBH4). Ternary systems consisting of two immiscible solvents able to accommodate the dual affinity of the copolymer blocks, where the growth was directed in the waterin-oil dispersed reverse micelles, and binary BC/water mixtures were used. It was found that BC-enabled reduction could be activated in the first case, whereas in the latter case particle formation could only take place after addition of KBH4, indicating that activation of BC-enabled reduction is a function of the metal precursor, the BC concentration, and the micellar mixture configuration. The BC-stabilized Ag nanoparticles exhibited significant stability and antibacterial activity against E. coli. It was found that Ag+ ions present in the solution, complexed with the copolymer chains, or bound onto the nanoparticle surface play the primary role in the bacterial growth inhibition activity. To this extent, results concerning the balance between relative ion and nanoparticle concentrations were extracted and the respective antibacterial activity was evaluated, which is important in the efforts to compensate for toxicity issues raised by the exclusive use of ionic silver. Besides, the developed nanoparticle mixtures are environmentally benign since they involve water and polymers that are not only nontoxic but have been approved for pharmaceutical use. Data on nanoparticle concentration and dosage were also obtained as to provide additional degrees of freedom to optimize performance. Consequently, the stable and active nanoparticle systems developed and the parameterization results reported are promising towards development of novel safe and ecofriendly nanoparticle substances for high performance antibacterial applications. 23


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Acknowledgment We thank Dr. Konstantinos Giannakopoulos and Dr. Nikos Boukos for the TEM imaging, and Dr. Athanasios Kontos for assistance with the UV-Vis spectroscopy measurements. We also thank Dr. Kostas Iatrou for useful suggestions and discussions. AP acknowledges the NCSR Demokritos for a PhD scholarship and GNK acknowledges support by the European Union through a Marie Curie international reintegration grant (FP7, grant agreement n° 210947).

Supporting

Information

Available:

Additional

nanoparticle

characteristics

including UV-vis spectra of Au particles obtained by KBH4-assisted reduction, X-ray diffraction pattern of Ag, comparison between nanoparticle and ionic solutions over time, and effect of dosage on the antibacterial activity. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table of Contents Image:

Metal particle

Polar dispersed phase Non-polar continuous phase

PEO PPO

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Poly(ethylene oxide)-b-Poly(propylene oxide) Amphiphilic Block

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