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.

Effect of the ultrastructure of chitosan nanoparticles in colloidal stability, quorum quenching and antibacterial activities.

We have fabricated two types of crosslinked chitosan-based nanoparticles (NPs), namely (1) ionically crosslinked with tripolyphosphate (TPP), designated as IC-NPs and (2) dually co-crosslinked (ionically and covalently with TPP and genipin, respectively) termed CC-NPs. The two types of NPs were physichochemically characterized by means of DLS-NIBS, synchrotron SAXS and M3-PALS (zeta potential). First, we found that covalent co-crosslinking of ionically pre-crosslinked nanoparticles yielded monodisperse CC-NPs in the size range of ∼200 nm, whereas the parental IC-NPs remained highly polydisperse. While both types of chitosan nanoparticles displayed a core-shell structure, as determined by synchrotron SAXS, only the structure of CC-NPs remained stable at long incubation times. This enhanced structural robustness of CC-NPs was likely responsible of their superior colloidal stability even in biological medium. Second, we explored the antimicrobial and quorum sensing inhibition activity of both types of nanoparticles. We found that CC-NPs had lower long-term toxicity than IC-NPs. In contrast, sub-lethal doses of IC-NPs consistently displayed higher levels of quorum quenching activity than CC-NPs. Thus, this work underscores the influence of the NP's ultrastructure on their colloidal and biological properties. While the cellular and molecular mechanisms at play are yet to be fully elucidated, our results broaden the spectrum of use of chitosan-based nanobiomaterialsin the development of antibiotic-free approaches against Gram-negative pathogenic bacteria.

Affinity protein-based FRET tools for cellular tracking of chitosan nanoparticles and determination of the polymer degree of acetylation.

Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agriculture, and industry. Its bioactive properties are determined by parameters such as the degree of acetylation (DA), but current techniques to measure the DA are laborious and require large amounts of substrate and sophisticated equipment. It is also challenging to monitor the fate of chitosan-based nanoparticles (CS-NPs) in vitro because current tools cannot measure their enzymatic or chemical degradation. We have developed a method based on the Förster resonance energy transfer (FRET) that occurs between two independent fluorescent proteins fused to a CS-binding domain, who interact with CS polymers or CS-NPs. We used this approach to calibrate a simple and rapid analytical method that can determine the DA of CS substrates. We showed unequivocally that FRET occurs on the surface of CS-NPs and that the FRET signal is quenched by enzymatic degradation of the CS substrate. Finally, we provide in vitro proof-of-concept that these approaches can be used to label CS-NPs and colocalize them following their interactions with mammalian cells.