Building and quantification of a bioactive coating containing LL37-Alginate complexes

The healing of infected or chronic wounds is a current challenge in the healthcare field, requiring the development of new therapeutic approaches. In this context, a bioactive coating can be adsorbed on the surface of a dressing to improve its performances. Such coating may contain the LL37 peptide which possesses interesting wound healing and antibacterial properties. To immobilize LL37 in this coating, a layer-by-layer assembly method was developed by successive adsorption of oppositely-charged polyelectrolytes on the surface. LL37 was first complexed with negatively-charged alginate (ALG) into protein-polyelectrolytes complexes (PPCs) before being integrated into the assembly with positively-charged chitosan (CHI). The development of this coating construction method triggers the need to develop a robust quantification method to be able to determine the amount of LL37 immobilized in the coating. The aims of this work were to validate the good construction of the coating using the LbL assembly and to develop a quantification method to assess the amount of LL37 present in the coating. A combination of two quantification tests was used: the bicinchoninic acid (BCA) assay and the NanoOrange (NO) assay. Surface characterization methods such as quartz crystal microbalance and X-ray photoelectron spectroscopy were also used to support this development. Fluorescent LL37 was finally used to enhance the performances of the quantification approach. As BCA and NO tests are sensitive to both LL37 and CHI, a system of two equations can be computed to evaluate the LL37 and CHI masses. However, the NO test was shown not to quantify the multilayer entirely. This observation triggered the need to identify other options or accurate quantification. Nonetheless, by using the BCA test alone, it is possible to approximate the LL37 mass with an error range of about 10%. This LL37 mass reaches 3 μg/cm2 after the deposition of 25 bilayers of [CHI-PPCs(LL37-ALG)]. The use of fluorescent LL37 was finally explored to increase the accuracy of the quantification method. The present work led to promising developments towards a robust quantification of the components of multilayer films featuring anti-bacterial and wound healing properties. It constitutes a useful step towards the development and application of innovative dressings.