Paper Title
Immobilization onto Chitosan Nanoparticle of Peptides Promoting Wound Healing

Abstract
Chitosan, a biopolymer derived from chitin, containing randomly distributed units of N-acetyl-D-glucosamine and D-glucosamine being antimicrobial and biocompatible, have extensively been used, among in other applications, as starting material for wound-healing products. The biological properties of the polymer are influenced by the molecular mass (MW), i.e. the number of units in the chain, and by the degree of acetylation (DA), i.e. the ratio between D-glucosamine and N-acetyl-D-glucosamine. The glucosamine units, that provide positive charges to chitosan, are believed to be responsible for high mucoadhesive properties of the polymer, because of ionic interactions with negatively charged sialic acid and sulphonic acid substructures of the mucus. The positive charges of chitosan are also essential for the permeation property. Chitosan nanoparticles (CNP), having around 300 nm in diameter and a zeta potential around 43 mV, were prepared based on the ionic gelation of chitosan (MW 200 kDa, DA 20%) with tripolyphosphate anions. In one-pot reaction, the CNPs were cross-linked and arginine–glycine–aspartic acid–serine (RGDS) was immobilized, to improve cell attachment required by a faster wound-healing process. Testing the antibacterial activities of pristine CNP and RGDS-CNP against Escherichia coli and Pseudomonas aeruginosa have revealed that by cross-linking of chitosan and immobilization of RGDS, the antibacterial properties are preserved (minimum inhibitory concentration less than 0.25 μg/mL and minimum bactericidal concentration around 1 μg/mL). In vitro assays, using human dermal fibroblasts, showed the absence of toxicity of both CNP and RGDS-CNP, and an increase of 7 times of promotion of migration of human dermal fibroblasts treated with RGDS-CNP in comparison with the pristine CNP. Funding: This work was supported by the grant Biotechnological tools implementation for new wound healing applications of by-products from the crustacean seafood processing industry (ChitoWound) [PN3-P3-284]. Keywords - Antimicrobial, Chitosan Nanoparticles, Cross-Linking, Immobilization, Rgds, Wound-Healing