Covalently and ionically, dually crosslinked chitosan nanoparticles block quorum sensing and affect bacterial cell growth on a cell-density dependent manner.

In our efforts to improve the quality and stability of chitosan nanoparticles (NPs), we describe here a new type of chitosan NPs dually crosslinked with genipin and sodium tripolyphosphate (TPP) that display quorum quenching activity. These NPs were created using a simplified and robust procedure that resulted in improved physicochemical properties and enhanced stability. This procedure involves the covalent crosslinking of chitosan with genipin, followed by the formation of chitosan NPs by ionic gelation with TPP. We have optimized the conditions to obtain genipin pre-crosslinked nanoparticles (PC-NPs) with positive ς-potential (~ +30 mV), small diameter (~130 nm), and low size distributions (PdI = 0.1-0.2). PC-NPs present physicochemical properties that are comparable to those of other dually crosslinked chitosan NPs fabricated with different protocols. In contrast to previously characterized NPs, however, we found that PC-NPs strongly reduce the acyl homoserine lactone (AHL)-mediated quorum sensing response of an Escherichia coli fluorescent biosensor. Thus, PC-NPs combine, in a single design, the stability of dually crosslinked chitosan NPs and the quorum quenching activity of ionically crosslinked NPs. Similar to other chitosan NPs, the mode of action of PC-NPs is consistent with the existence of a "stoichiometric ratio" of NP/bacterium, at which the positive charge of the NPs counteracts the negative ς-potential of the bacterial envelope. Notably, we found that the time of the establishment of the "stoichiometric ratio" is a function of the NP concentration, implying that these NPs could be ideal for applications aiming to target of bacterial populations at specific cell densities. We are confident that our PC-NPs are up-and-coming candidates for the design of efficient anti-quorum sensing and a new generation antimicrobial strategies.