STD-NMR ANALYSIS OF INTERMOLECULAR INTERACTIONS BETWEEN CHITOSAN-BASED HYDROGELS AND BIOINSPIRED IONIC LIQUIDS

Edible films derived from natural polymers are emerging due to their safety, biodegradability, tailored properties, and multiple functionalities compared to petroleum-based synthetic films. The film's 3D network structure is formed by covalent or noncovalent bonding in an adequate liquid dispersion, to ensure the mechanical characteristics of the films after solvent evaporation. These characteristics can change according to constituents of the polymer, such as: type of polymer matrix, nature of the cross-links, size of the polymeric chains, etc. Polymer dispersions and films can have a very diverse set of properties, such as swelling, rheological behavior, and interactions with charged surfactants. One of the most promising natural polysaccharides to produce edible films is chitosan (CS), the deacetylated form of chitin derived from crustaceans. This polymer is useful due to its biodegradation, antimicrobial activity, and good film-forming capacity. Ionic liquids can be added to improve the physical, mechanical, and antimicrobial properties of composite materials as dispersions and final films. ILs are versatile and present advantageous properties, such as thermal stability, homogeneity and microbiological properties, that can be improved according to the cation-anion combination. When 3D polymer networks are formed in the presence of ILs, a complex set of intermolecular interactions is possible. These interactions will modify the physicochemical properties of the final soft material and even allow the occurrence of emergent properties. An effective method to analyze the interactions between ligands and hydrogels is through Saturation Transfer Difference - Nuclear Magnetic Resonance (STD-NMR) analysis. The STD-NMR experiment utilizes the Nuclear Overhauser Effect (NOE), in which the signal intensity of a proton is affected by cross-relaxation with a nearby perturbed proton by irradiation. When a proton is saturated through a selective radiofrequency pulse, the signals of nearby protons are either enhanced in small molecules or reduced in large molecules during the relaxation process. In this context, the research aims to investigate the intermolecular interactions between chitosan and imidazolinium ionic liquids, with anions derived from natural sources, such as eugenol and cinnamaldehyde, using STD-NMR. The synthesis of ILs from decimethylimidazol ([DEC(MIM)]) requires a step corresponding to the preparation of the precursor. The precursor [DEC(MIM)][Br] was synthesized from 1-methylimidazole and bromodecane in a reflux system at 65 ºC for 24 hours. After this synthesis, the ion exchange reaction with the IRN78 resin was carried out.  The solution of the precursor in ethanol was passed through the ion exchange column in order to exchange the bromide (Br^-) for hydroxide (HO^-). The exchange reaction is accompanied by the spot test with AgNO3, which allows observing the gradual disappearance of bromide. Then, the ion exchanged product [DEC(MIM)][OH] reacts with eugenol or cinnamic acid in a reaction at room temperature under stirring for 24 hours to furnish [DEC(MIM)][EUG] and [DEC(MIM)[CIN], respectively.

STD-NMR experiments were performed at 25°C in a BRUKER spectrometer operating at 600 MHz. The [DEC(MIM)][EUG] and [DEC(MIM)][CIN] were solubilized at 50 µM in a solution containing 5 µM chitosan and D₂O. The on-resonance irradiation was performed at 3.07, 7.47 and 8.61 ppm, while off-resonance irradiation was applied at 40.0 ppm. The saturation was set at D20 = 0.5; 0.75; 1.0; 2.0; 4.0; 6.0 and 8.0s. Next, an STD build-up curve was generated, demonstrating the STD intensity as a function of the saturation time. The exponential curves resulting were fitted according with STD equation to obtain STD% for signals monitored. From this,  epitope maps were generated for [DEC(MIM)][EUG]-chitosan and [DEC(MIM)][CIN]-chitosan systems in aqueous solution. These maps demonstrate the regions of closest proximity and allow observing ligand-polymer interactions on a magnitude scale based on different colors. Based on the STD-NMR analysis, stronger cross-linking was observed between the ILs and chitosan. For both ionic liquids, a strong interaction was observed between the anion and the polymer surface, providing important insights into the spatial proximity between the ionic liquids and the chitosan. It was possible to see the difference in the IL-chitosan intermolecular interaction pattern according to the anion of the IL.

From [DEC(MIM)][CIN]-chitosan system, it was observed interaction between chitosan and the cation hydrophobic side alkyl chain, which is formed by 10 carbons.  When compared to the [DEC(MIM)][EUG]-chitosan, chitosan interacted more with the anion. These results indicate that IL anion influences the IL-polymer intermolecular interactions in the polymeric dispersion and, consequently, in the film-formation mechanism. When the chitosan-based films were produced in the presence of ILs, the influence of IL was evidenced in the mechanism of dispersion formation and consequently in the shape of the final biopolymer films. The STD-NMR results were corroborated with the contact angle analysis. It was observed that films with ILs derived from eugenol presented a greater affinity for apolar substances. On the other hand, the film with IL derived from cinnamaldehyde has a greater affinity for polar substances, which can be related to chitosan interacting more with the cation chain than with the anion itself.