Synthesis of chitosan nanoparticles and incorporation into hydrogels.
Ionotropic gelation method has been used before to obtain polymeric nanoparticles able to encapsulate bioactive molecules. This type of encapsulation gives protection and control the release of the active principle. In addition, this method is relatively easy to perform in the laboratory (Pedroso-Santana, 2020). Polymeric nanoparticles can be loaded into a hydrogel, which could give a second barrier of protection against environmental agents, increasing bioavailability of encapsulated molecules even more. Also, the stable physical structure of hydrogels could serve as an anchoring mechanism in the sites of action, establishing a static position for releasing of bioactive molecules. Considering the aforementioned, ionotropic gelation was used to obtain chitosan nanoparticles, with sizes around 200 nm (Figure 7 ), and to study their interaction with hydrogels. Due to the previous results of low swelling and poor biocompatibility, starch-PVA hydrogel was excluded from next experiments.
The nanoparticles were synthesized in a reproducible way, showing an average particle diameter of 205 nm and a polydispersion index (PdI) of 0,198 (Figure 7A ). DLS results were confirmed by STEM, demonstrating the nanoparticle spherical shape and the high homogeneity of the population (Figure 7B-E ). Considering a potential use of these nanoparticles for drug delivery, two positive characteristics of the population can be mentioned: this size is effective for the interaction with cellular structures, such as membrane and cytoplasmic organelles (Pedroso-Santana, 2018), and the size homogeneity correlates with a homogeneous drug-loading capacity (Li, 2015).
After characterization, the nanoparticles were centrifuged, and the pellet was dispersed in PBS. This aqueous dispersion containing agglomerates formed by the centrifugation step was used to submerge the hydrogels. Three hours later, the hydrogels were lyophilized and the physical interaction with the nanoparticles was visualized by SEM (Figure 8 ).
Starch hydrogel showed a higher quantity of nanoparticles adsorbed to the channel walls (Figure 8A-B ), in comparison to starch-CS (Figure 8C-D ). Considering that channel walls in both hydrogels are composed of starch, the lower number of nanoparticles in the second one could be due to the steric interference produced by the large chitosan beads. These beads introduce a barrier to the channel access (Figure 1K-L ), which would be interfering with the nanoparticle’s adsorption onto hydrogel walls. This limitation was overcome in starch-CS-GA hydrogels. Obtaining a clean and smooth structure allowed the homogeneous nanoparticle´s incorporation by the hydrogel (Figure 8E-F ). This hydrogel showed excellent interaction with the nanoparticles, based on their efficient incorporation and their regular distribution on the entire structure.
In order to use nanoparticles-loaded hydrogels in drug delivery applications, the system must retain a high quantity of nanoparticles and be able to release them in a controlled manner. In this way, the hydrogel structure guarantees the therapeutic effect of the drug at the site of action for a prolonged time. This will lead to a robust system for the administration of drugs with significant advantages such as: a controlled drug release and extended bioactivity (Wechsler, 2019). From the variants proposed in this work, Starch-CS-GA hydrogel showed the greatest response in terms of swelling rate, viability measured by MTT incorporation, and interaction with nanoparticles; therefore, this hydrogel was used for an in vivo experiment.
Starch-CS-GA hydrogel in vivo wound healing