Chitosan nanoparticles synthesis caught in action using microdroplet reactions

Chitosan nanoparticles are biocompatible, relatively non-toxic, biodegradable, and cationic in nature1,2. Thus, they are well accepted in biomedical applications such as drug delivery3,4,5. Ionic gelation is the most commonly used method for synthesising chitosan nanoparticles6. In this method, chitosan precursors are cross-linked using sodium tripolyphosphate (TPP). The method typically yields large sized (100–300 nm) particles with a high degree of polydispersity. Even though ionic gelation is a widely used method and factors governing the size and dispersivity of chitosan nanoparticles (such as the concentration of reactants, temperature, pH, and the level of deacetylation) are well known7 our basic understanding of the process at mechanistic level is poor. In the ionic gelation process, TPP crosslinks randomly oriented chitosan molecules, which, in turn, are connected to other similarly cross-linked moieties. Such intra- and inter- molecular cross-linking is rather uncontrolled and leads to polydispersity in the synthesised preparation. Here, we hypothesised that using confined reaction volumes of microdroplets and at preset temperatures; it should be possible to exert good control over the kinetics of nanoparticles synthesis if the reactions could be arrested rapidly at desired time points. Such an approach could enable time-lapsed capture of the synthesis process and provide us an opportunity to understand it at a more fundamental level. Thus, in our microdroplet experiments, synthesis reactions were arrested at varying time points to monitor the nucleation and growth of developing nanoparticles using DLS and SEM. For this purpose a simple yet novel technique, viz. fast dilution of the reaction mixture with DI water was employed. In a separate experiment, we had determined that with a 500-fold dilution, the reaction pH increased sharply from 2.84 ± 0.04 to 5.50 ± 0.05. Such a rise in pH could exert a strong endothermic effect8,9,10, resulting in extensive (90) de-protonation of chitosan11. Dilution could also result in increased intra-molecular distances, reducing the possibility of molecular interaction. The synthesis of chitosan nanoparticles tends to be arrested by DI water dilution owing to these reasons.