Layer-By-Layer Deposition of Bioactive Polyelectrolytes with Incorporation of Antimicrobial Agents as a New Strategy to Develop Bioactive Textile

Polyelectrolyte multilayer coatings have become a new and general way to functionalize a variety of materials. Particularly, the Layer-by-Layer (LbL) method is a technique developed for the coating of solid surfaces. The LbL technique presents a unique mean to construct surface coatings that can conform to a variety of biomaterial surfaces and serve as matrices enabling controlled delivery of bioactive molecules from surface. As the deposition process is achieved in aqueous medium, incorporation of active agents is possible since the coatings obtained by LbL are less densely packed and this is advantageous for diffusion through the coating. The coating is constructed by the alternate adsorption of oppositely charged polyelectrolytes at the surface of the material, easily obtained when it is dipped in polyelectrolyte solutions. A deposition cycle creates a layer, and these cycles can be repeated as often as needed. This study aims to obtain novel bioactive textiles with potential application as wound-dressings. The biopolymers chosen for the functionalization of cotton (substrate), were chitosan (CH) and alginate (ALG). The multilayer coating of cotton with CH and ALG is constructed by the adsorption of CH and ALG with opposite charge on the surface of cotton substrates. The successive deposition of multilayers of CH and ALG was analyzed by three different techniques. Contact angle between a water droplet and the surface of the sample, cationic dye staining method and analysis by ATR-FTIR (Fourier Transform Infrared spectroscopy with Attenuated Total Reflection). These techniques showed that there was alternating deposition between CH and ALG and the presence of electrostatic bonds between the layers. In order to evaluate the antibacterial activity of the functionalized cotton, the Japanese standard JIS L 1902:2002 for the halo method (qualitative assay), and the absorption method (quantitative test) were assessed. These tests revealed an antibacterial effect on the functionalized cotton for both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Klebsiella pneumoniae). In addition a method was optimized for incorporating L-cysteine (L-cys) between the layers of CH and ALG deposited on cotton samples by the LbL, in order to obtain a better antimicrobial effect. Several strategies were used and the best results were obtained by the method where the ALG turns into a gel in the presence of calcium, since L-cys can be incorporated directly between the layers of CH and ALG without any covalent bond. Thus, the bioactive L-cys agent was immobilized without losing its bioactive characteristics. These new samples were analyzed for the antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae according with the previously used standard, and the results showed an increase in the antibacterial effect due to the presence of L-cys. This new coating method has the great advantage to able to select other types of bioactive agents without needing further optimization. In this way, L-Cys was replaced by antimicrobial peptides (AMPs). The reason for the use of AMPs is related with the continuous use of antibiotics which resulted in multiresistant bacterial strains all over the world. Consequently, there is an urgent need to search for alternatives for antibiotics. The AMPs are the new generation of antimicrobials. Four AMPs of different features were used. The depth in which each AMPs is incorporated between the layers was determined by energy dispersive analysis of X-rays (Energy Dispersive X-ray EDS). Results showed, that all AMPs used have a higher antimicrobial effect when compared with previous samples (with and without L-Cys) for both microorganisms and are non-cytotoxic to normal human dermal fibroblasts at the tested Concentrations. This confirms that this new functionalization approach of cotton coated with layers of CH and ALG by the LbL technique with incorporated AMPs leads to good antibacterial and cytotoxicity results, which make them suitable to be used as wound dressings.