Preparation and characterization of silver nanoparticles in methyl cellulose matrix and their antibacterial activity

Natural polysaccharides are excellent materials for nanometal composites preparation.^1–3) Polysaccharide starches serve as both reducing and capping agents. Prime use of polysaccharides during preparation of silver nanoparticles was as stabilizers. Contemporary research focuses on these macromolecules to act simultaneously as a reducing agent and a stabilizer; these properties are achieved by chemical modifications. Such modifications aim specifically to raise accessibility, solubility and reducibility of polysaccharide building units. Application of removable polysaccharide blocks (e.g., cellulosic fibres) in production of pure silver nanoparticles is another subject of present research.^4,5) This process is affordable, environmentally friendly and effective method how to prepare materials for various applications, as it does not need any seed, template or surfactant.^6,7) It is possible to influence the properties of the synthesized nanoparticles (NPs) depending on the conditions during preparation.^8–10) The colloid solutions of nanoparticles can be easily converted into solid films in order to prevent aggregation of the prepared NPs.¹¹⁾ Modifying the preparation conditions of the films or incorporating additives enables control of material properties of the prepared films.^12,13) One of the perspective polysaccharides is methyl cellulose (MC), which is very attractive because of its solubility in water, surface activity, and thermal gelation ability.^14,15) Moreover, cellulose and its derivates are nontoxic and non-allergenic to humans, therefore they are widely used in food, cosmetic, pharmaceutical, and biomedical applications.^16,17) Bhui et al. discovered that at 60 °C the as prepared silver NPs in MC are adsorbed and stabilized on the hydrophobic part of MC microfibril as small spherical particles.¹⁵⁾ The importance of silver NPs increases due to the increasing emergence of new strains of bacteria resistant to antibiotics. Small silver particles attack the cell walls disrupting vital functions of the cell. They are able to penetrate into the bacteria and interact with sulphur and phosphorus containing biomolecules such as DNA.¹⁸⁾ Furthermore, Ag⁺ ions get released from the NPs and disturb the ability of DNA molecules to replicate as they become condensed. Silver ions interact with thiol groups in proteins, which induces inactivation of the bacterial proteins.¹⁹⁾ AgNPs embedded in methyl cellulose matrix can broaden utility of this natural polysaccharide. The most valuable advantage these materials posess is their antibacterial activity against a wide spectrum of bacteria and moulds^18,20–22) — such properties are highly demanded in medicine and food packaging.^23–26)

2.1. Materials and methods

The NPs were prepared in the methyl cellulose matrix (Modernist Pantry E461) as shown in Fig. 1. The methyl cellulose powder was dissolved in distilled water in a ratio of 100 mL of water to 1 g of methyl cellulose. The solution was heated for 1 h at 80 °C to obtain a homogeneous suspension. Then the suspension was cooled to obtain a clear and transparent solution. Freshly prepared solution of 0.01 M AgNO₃⁴⁾ was added to the methyl cellulose solution and stirred for 24 h at 20 or 80 °C.

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2.2. Measurement techniques

The methyl cellulose solutions with Ag nanoparticles reduced from silver nitrate were studied by UV–vis spectroscopy. For comparison a spectrum of a solution containing only methyl cellulose without silver nitrate was prepared and measured (pristine solution). The results are shown on Fig. 2(a). The pristine methyl cellulose solution shows no significant absorption peaks in the measured range as could be expected as it is clear with no hints of coloration. The intense peak in the range from 425 nm, observed in case of the sample prepared at 80 °C, is a surface plasmon resonance (SPR) peak typical for spherical nanoparticles with diameter in order of tens of nanometres.²⁷⁾ Two distinct SPR peaks are present in the spectrum of the sample prepared at room temperature. This could be either caused by presence of spherical Ag structures of about 100 nm in diameter in which case a quadrupole SPR peak would show in addition to the original dipole one^28,29) or a preferential growth of the Ag particles in one axis, where cylindrical particles would form which then show SPR peaks associated with longitudinal and transversal plasmon resonance modes.³⁰⁾ To distinguish between these possibilities the NPs solutions were subjected to TEM observations. Figures 3(a) and 3(b) show micrographs of solutions prepared at room temperature. Small and large aggregates of — mostly spherical — nanoparticles are present in the micrographs. Figures 3(d) and 3(e) shows a micrograph of solution prepared at 80 °C. Silver is mostly present in form of discrete spherical particles with diameters of 10–40 nm; however, aggregates and clusters were observed as well. The size of the individual particles was identified by image analysis and histograms of size distribution were created. The size of the individual particles was found to be similar in both cases of the preparation conditions, but the image analysis method breaks up the NP clusters measuring sizes of individual particles. Since no preferential directional growth was observed in the micrographs, the breakup of the SPR peak in the case of samples prepared at room temperature can therefore be caused by tendency of the particles to form aggregates of diameters 100 nm and higher that are tight enough to enable propagation of the plasmon through the whole nanoparticle cluster, although the histograms of particle size distribution show no particles larger than 50 nm that would explain the occurrence of the quadrupole SPR absorption peak due to the image analysis method not measuring the size of whole clusters of particles.

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Both methyl cellulose solutions with silver nitrate addition were transferred to form of solid film by drying at 60 °C. Photographs of both red-brown transparent films can be seen in the schematic in Fig. 1. These solid films were measured by UV–vis spectroscopy; the results are shown in Fig. 2(b). The spectra of the films prepared from the solutions treated at room and elevated temperature do not show qualitative differences.

XPS spectra of both prepared solid films were measured on their surface. The elemental analysis did not find significant differences in composition between the films prepared at 20 and 80 °C. The elemental composition of the films corresponds with chemical composition of methyl cellulose with an addition of nearly 2% of silver (as shown in Table I). Peak separation of the silver peak at 367 eV shows the sample prepared at elevated temperature contains higher percentage of elemental silver [70%, Fig. 4(b)] compared to the film prepared at room temperature [42%, Fig. 4(a)].

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Antibacterial properties of both films were evaluated by inhibition zone method against strains E. coli and S. epidermidis. Both types of films show an increase in antibacterial activity compared to methyl cellulose without added silver — as can be seen in Fig. 5.

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The methyl cellulose films were stored for several weeks in darkness at room temperature and then re-dissolved in cold water. UV–vis spectra and TEM of the resulting solutions were measured in order to compare the newly released NPs to the NPs from the original methyl cellulose solutions. The spectra of the newly released NPs in Fig. 2(c) both show characteristics typical for small isolated silver NPs; the SPR peak at 425 nm is just stronger for the samples prepared at elevated temperature. TEM micrographs in Figs. 3(c) and 3(f) show spherical well separated nanoparticles with no indication to having tendency to form clusters. The change of the character of the UV–vis spectrum of the sample prepared at room temperature suggest a change in the structure of the material. The UV–vis spectra comparison suggests the larger structures are broken by the drying — redissolution process into smaller particles. As it is hard to imagine the NPs would recrystallize in the process into smaller particles the more plausible explanation is the clusters formed during the nitrate reduction get broken into separate NPs. During the solidification of the methyl cellulose its polymer chains get redistributed and polymer crystallites are formed. These structures could enter the cracks in the composition of the nanoparticle cluster and lead to separation of the particles from the clusters. In such way the size distribution of the NPs could be improved.

It was found out that the silver nanoparticles can be prepared in the MC matrix by in situ method. The higher temperature during the preparation was more effective to prepare isolated particles with good size distribution than the laboratory temperature. Converting the solution of MC with AgNPs into the solid film and subsequent redissolution does not seem to negatively affect the Ag NPs; in fact it led to improvements in the case of NPs prepared at room temperature. This method could therefore be used to store NPs without the risk of their aggregation.

This work was supported by the GA CR under the project P108/12/G108.