Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa
Introduction
Layer-by-layer (LbL) assembly is a simple, versatile and cost-effective technique to form thin films on different materials (Decher, 1997). This method consists of alternating physisorption of oppositely charged polyelectrolytes (Borges & Mano, 2014). In particular, hyaluronan (HA) and chitosan (CHI) are oppositely charged biocompatible polysaccharide electrolytes (negative and positive, respectively) used for surface coatings employing the LbL technique (Salomäki & Kankare, 2009).
Bilayers of HA/CHI have been successfully used as antibacterial coatings (Chua, Neoh, Kang, & Wang, 2008) because HA forms a soft, highly hydrated, and nontoxic film (Necas, Bartosikova, Brauner, & Kolar, 2008), whereas CHI has the antimicrobial characteristics (Chirkov, 2002) (Fig. 1a). One of the most important interactions during the formation of polyelectrolyte multilayers (PEMs) assemblies using the LbL technique is provided by electrostatics; pH and ionic strength (IS) of the polyelectrolyte solutions are thus key synthesis variables in this technique (Boddohi, Killingsworth, & Kipper, 2008). Changes in these two parameters induce modifications in the growth mechanism, morphology, thickness, roughness, and surface wettability of the thin films obtained by LbL (Borges & Mano, 2014). Changes in pH modify the degree of ionization of CHI amino groups and HA carboxylic groups (Fig. 1b) (Sakiyama, Takata, Kikuchi, & Nakanishi, 1999) while increasing IS leads to the swelling of multilayer assemblies (Borges & Mano, 2014). In this context, we addressed the influence of pH and IS on the final characteristics of our HA/CHI nanofilms assembled by LbL technique.
Furthermore, the electrostatics characteristics of the nanofilm surfaces are expected to affect the initial adhesion of microorganisms. For that reason, the as-synthesized HA/CHI nanofilms were tested as antibacterial biomaterial using Xylella fastidiosa – one of the top 10 plant pathogenic bacteria in molecular plant pathology (Mansfield et al., 2012) – as a model. X. fastidiosa is the causal agent of several diseases in economically important plants, such as plum, almond, peach, coffee, grapevine and citrus (Alves, Marucci, Lopes, & Leite, 2004). It is thus important to find a suitable antibacterial surface that protects these economically important crops. X. fastidiosa was chosen as a model for our antibacterial activity test since its adhesion mechanisms on different surfaces have been studied in detail (Janissen et al., 2015, Lorite et al., 2013). In particular, the initial adhesion of the rod-shaped bacteria takes place through the poles with few cells completely in contact with the surface, which is gradually covered by extracellular polymeric substances (EPS) – mainly polysaccharides – produced by the bacteria. Moreover, X. fastidiosa biofilm, although composed of hundreds of bacteria living together in a matrix, is held to the substrate surface by only a few cells. Thus, any antibacterial effect of the substrate surface would be minimized by the floating architecture of the biofilm.
For gram-negative bacteria such as X. fastidiosa, one of the most accepted antibacterial mechanisms for CHI is the leakage of intracellular constituents of the microorganisms caused by the interaction between positively charged CHI molecules and negatively charged microbial cell membranes (Shahidi, Arachchi, & Jeon, 1999). X. fastidiosa represents thus a tough test for antimicrobial surfaces such as our nanofilms; the small surface contact area at the pole and the extensive secretion of polysaccharides by the cells and consequent surface coverage might easily hinder the nanofilm antimicrobial effect. In our study, the observed antibacterial effects of the obtained HA/CHI nanofilms were correlated with their physicochemical properties; these were controlled by means of the specific values of pH and IS of the polyelectrolytes solutions. This work can also generate alternatives and environment-friendly strategies to inhibit other type of pathogenic gram-negative bacteria, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum, by studying possible antibacterial effects of HA/CHI nanofilms on the adhesion of X. fastidiosa.
Section snippets
Polyelectrolyte solutions
Three polymers were used for the preparation of electrolyte solutions: polyethylenimine (PEI, 50 wt.% solution in water, molecular weight ≈7.5 × 105 g/mol), hyaluronic acid sodium salt (HA, from Streptococcus equi sp., molecular weight ≈1.58 × 106 g/mol), and chitosan (CHI, low molecular weight ≈5 × 104 g/mol, 75–85% deacetylated) (Sigma–Aldrich, USA). Polyelectrolyte solutions were prepared by dissolving the respective polymer in ultrapure water (Milli-Q® system) with a resistivity of 18.2 MΩ cm at
Results and discussion
In order to determine the chemical composition of PEMs, ATR-FTIR analyses were carried out as shown in Fig. 3a. The spectra of 4.5pH/0.075IS and 4.5pH/0.10IS presented just two transmittance bands at 2853.5 and 2925 cm−1, which are attributed to CH stretching mode (Alkrad, Mrestani, Stroehl, Wartewig, & Neubert, 2003). In the case of 3.0pH/0.10IS, two additional modes were observed, one at 3105 cm−1 and other in the range of 3200–3500 cm−1, which correspond to NH with CO combination (Alkrad et
Conclusions
Layer-by-layer nanofilms of HA/CHI were assembled varying the pH and IS of polyelectrolyte solutions and their physicochemical and antibacterial properties were studied in detail. Due to the important impact in agriculture and the detailed knowledge of its life cycle, the X. fastidiosa microorganism was used as a model of gram-negative bacteria. At pH 4.5, both PEMs (4.5pH/0.075IS and 4.5pH/0.10IS) presented antibacterial effect. When IS was increased at this pH, the antibacterial behavior of
Conflict of interests
The authors declare that they have no competing interests.
Acknowledgements
The authors acknowledge the support from Analytical Resources and Calibration Laboratory (LRAC) from Chemical Engineering Department and the Multi-User Lab (LAMULT) from Institute of Physics “Gleb Wataghin”, both at UNICAMP, which provided invaluable access to their equipments. We would also like to thank the National Nanotechnology Laboratory (LNNano) for granting access to the electron microscopy facilities. This work was financially supported by FAPESP (grant numbers 2010/51748-7 and
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