Hyaluronan/chitosan nanofilms assembled layer-by-layer and their antibacterial effect: A study using Staphylococcus aureus and Pseudomonas aeruginosa
Graphical abstract
Introduction
Nosocomial infections are those developed in patients during their stay in a hospital. Such infections occur in 5–10% of all hospitalizations in Europe and North America; this percentage, however, reaches more than 40% in parts of Asia, Latin America, and sub-Saharan Africa [1]. Nosocomial infections are generally caused by opportunistic microorganisms, among which the most prevalent Gram-positive bacteria are different strains of Staphylococcus aureus and Enterococcus genus [2]. The most predominant Gram-negative bacteria are Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacteriaceae family and Acinetobacter genus [2]. Both S. aureus and P. aeruginosa have become the most important cause of nosocomial infections in recent years. Their pathogenicity is mainly due to the ability to form biofilms on biomedical devices and prosthesis [3], [4]. In the latter case, thin surface coatings for both attachment reduction and larger bacterial killing rates are mandatory [5].
Due to its antibacterial properties, biocompatibility and biodegradability, chitosan (CHI) is an excellent alternative to coat biomedical devices to prevent nosocomial infections [6]. It has been successfully used against both gram-negative and gram-positive bacteria [7]. Two mechanisms have been proposed as the main cause of antibacterial action by CHI. One is that the polycationic nature of chitosan interferes with bacterial metabolism by electrostatic interactions at the negatively charged cell surface of bacteria, and the other one is blocking of transcription of RNA from DNA by adsorption of penetrated CHI to DNA molecules [7].
In the last years, different chitosan-based nanofilms assembled layer-by-layer (LbL) have been used as antibacterial coatings, either as a single, pristine film [5], [8], [9], [10] or enhanced with antimicrobial species, such as peptides [11], [12] or silver nanoparticles [13]. The simplicity, versatility, and nanoscale control that LbL assembly provides make it one of the most widely used technologies for coatings applied in biomedicine and other fields [14]. LbL is a simple bottom-up technique, which consists of alternating physisorption of oppositely charged polyelectrolytes. For biomedical applications, the hyaluronan (HA, a polyanion) can be used in combination with CHI (a polycation) for the synthesis of antibacterial coatings by the LbL technique: HA forms a soft, highly hydrated, and nontoxic film, whereas CHI has the antimicrobial characteristics [10].
Previous works have assembled HA and CHI by LbL for biomedical applications. Richert et al. [15] studied the influence of salt concentration, Chua et al. [11] coupled HA/CHI films with surface-immobilized cell-adhesive arginine-glycine-aspartic acid (RGD) peptide, and Cui et al. [16] quaternized CHI with glycidyltrimethylammonium chloride (GTMAC) to build up microcapsules. Richert et al. [15] obtained an 80% reduction of E. coli adhesion after 30 min of cell culture. In the case of Chua et al. [11] and Cui et al. [16], for a cell culture of 4 h, they reported an 80% decreasing of S. aureus and ∼100% reduction of Escherichia coli, respectively. In our work, we focus on the synthesis and optimization of the antibacterial effect up to 8 h of culture of HA/CHI nanofilms assembled by LbL. Optimization was performed by varying specific pH values of polyelectrolyte solutions, which control the ionization degree of each biopolymer. Moreover, due to the exponential growth trend exhibited by the thickness of our nanofilms, the surface density of amino groups, which are associated with the antibacterial effect, was also enhanced by increasing the number of HA/CHI bilayers. Surface properties of HA/CHI assemblies were monitored in each alternating deposition; the final physicochemical properties of our samples were correlated with their antibacterial effect against the human pathogenic microorganisms S. aureus and P. aeruginosa.
Section snippets
Materials
Polyethylenimine (PEI, 50 wt.% solution in water, MW ≈ 7.5 × 105 g/mol), hyaluronic acid sodium salt (HA, from Streptococcus equi sp, MW ≈ 1.58 × 106 g/mol), chitosan (CHI, 75–85% deacetylated, low MW ≈ 5 × 104 g/mol), methylene blue (MB, MW ≈ 373.90 g/mol), and rose Bengal (RB, MW ≈ 1017.64 g/mol) were purchased from Sigma-Aldrich, USA. Sodium hydroxide (NaOH), sodium chloride (NaCl), and hydrochloric acid (HCl) were purchased from Synth Brazil. All chemicals were used without further purification, and solutions
Results and discussion
Si substrate preparation was evaluated by measuring the water contact angle after each step (Fig. S1). After plasma treatment, cleaned samples changed their wettability into a strong hydrophilic behavior, from 54° to 16°, due to the silanol groups formed by plasma oxidation [17], [18]. In the same sense, after PEI deposition, samples changed their wettability into a more hydrophobic behavior, from 16° to 80°. This is because PEI is a highly positive charged polycation, which readily attaches to
Conclusions
HA/CHI nanofilms assembled by LbL technique with different pH values of the biopolymer stem-solutions, which control their ionization degree, were studied in detail as antibacterial coatings. Due to the important impact in medicine, as mains precursors of nosocomial infections, S. aureus and P. aeruginosa were used to test the nanofilms as antibacterial surfaces. We observe a good correlation between the nanofilm surface physicochemical properties and the obtained antibacterial effect; we have
Acknowledgements
We would like to thank Analytical Resources and Calibration Laboratory (LRAC) from Faculty of Chemical Engineering and Multi-user Lab (LAMULT) from Institute of Physics “Gleb Wataghin”, both from UNICAMP, for providing the analytical facilities. 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 2013/02300-1), CNPq and CAPES. J.
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