Proteomic Insights into the Interaction of Chitosan Nanoparticles with Canine MDCK Epithelial Cells

Chitosan is considered an excellent biomaterial for epithelial healing treatment. However, information on its molecular interaction with cells at a molecular level is still lacking. Thus, in the present study, homemade synthesized chitosan nanoparticles (CS NPs) and their physicochemical characterization were examined; we found that NPs had average sizes of 15, 30, and 125 nanometers by modifying some variables in the synthesis protocols. It is worth noting that a crystalline structure was found on the smallest NPs, with an average size of 15 nm, as observed in high-resolution transmission electron micrographs. To study the in vitro interaction of CS NPs with Madin-Darby canine kidney (MDCK) cells, co-culturing was performed, and cell viability was assessed. We found that NPs were not toxic at concentrations of up to 400 µg/mL during the first 24 h. Additionally, quantitative mass spectrometry revealed the overexpression of several proteins induced by the co-culture of CS NPs with MDCK cells, and a proteomic analysis suggested two important things: possible clathrin-mediated endocytosis could be the interaction mechanism of CS NPS with MDCK, and proteins related to cytoskeleton formation and organization were overexpressed. Moreover, wound healing assays revealed that <125 nm> CS NPs yielded the best closure rates, where mitomycin was added to make sure that only cell migration occurred.