Layer-by-layer assembling of hyaluronan/chitosan nanofilms and their antibacterial effect: a study using Staphylococcus aureus and Pseudomonas aeruginosa

摘要

The layer-by-layer (LbL) is a simple, versatile and cost-effective technique to form thin films on different materials. This method consists of alternating physisorption of oppositely charged polyelectrolytes. As hyaluronan (HA) and chitosan (CHI) are oppositely charged biopolymers (negative and positive, respectively), they are excellent candidates to be used for surface coatings obtained by this technique (Fig. 1A). Due to the antimicrobial characteristics of CHI by NH_3~+ groups, bilayers of HA/CHI can be used as antibacterial coatings. In that sense, this work is about the synthesis of antibacterial nanofilms of HA/CHI biopolymers assembled by LbL, varying specific pH values of polyelectrolyte solutions (Fig. 1B). HA/CHI assemblies can be monitored by measuring their respective surface properties, in each alternating deposition. The final surface physicochemical properties can be correlated with their obtained antibacterial properties. In this work, we considered two human pathogenic microorganisms, Staphylococcus aureus and Pseudomonas aeruginosa. The antibacterial effect of HA/CHI nanofilms was evaluated using the spread plate counting method. These results were correlated with the morphology of nanofilms (characterized using SEM and AFM), as well as with their chemical properties studied by UV-Vis and XPS spectroscopy. Fig. 1C shows the antibacterial effect of the HA/CHI nanofilms. In the case of S. aureus assay, the strain was very sensible to the pH of synthesis of samples, which is strongly related with the free ammonium groups on the top CHI layer of samples. Compared to the control, the Si substrate, sample pH = 4.5 reduced 1 and 2 orders of magnitude of CFU/cm~2 at 4 and 8 h of cell culture, respectively. The antibacterial effect of the pH = 3 was very notorious in both times of culture, reducing 3 orders of magnitude for 4 h and 5 orders for 8 h. In the case of pH = 2, there was not important reduction of the bacterial density, for both times culture. In the case of P. aeruginosa assay, three samples did not show important antibacterial effect. The resistance of P. aeruginosa can be explained by its outer membrane, which would protect its cell wall from the killer contact to of free protonated ammonium groups of films. Figure 1. A) Schematic representation of the buildup of HA/CHI multilayer thin films by LbL: immersion of the charged substrate in the correspondent polysaccharide electrolyte, alternating with rinsing steps. B) lonization curves of HA and CHI obtained by the Henderson-Hasselbach equation; pKa of HA is 3.0 while that of CHI is 6.5. The pH values of nanofilms synthesis condition are also indicated: 4.5,3 and 2. C) Antibacterial effect of HA/CHI nanofilms. The number of viable bacteria on the samples at 4 and 8 hours as determined using the spread plate method.** denotes a significant difference compared to the Si substrate at 4h (p ＜ 0.01); ## denotes a significant difference compared to the Si substrate at 8h(p＜ 0.01).