Synthesized hydrogels.
Physical method was elected for the synthesis of hydrogels. This method does not use chemical cross-linking agents, which can affect entrapped drugs or biomolecules (Gulrez, 2011). Having a starch-based structure, hydrogel polymerization was induced with heat (1210C), followed by three freeze-thawing cycles. Three hydrogels were synthesized by this method: starch, starch-PVA and starch-CS. Different conditions (temperature, agitation and reagent quantities) were assayed until the semi-solid hydrogel structure was obtained. After the last freeze-thawing cycle, hydrogel samples were freeze-dried and analyzed by SEM.
A general first observation of microscopic images (Figure 1 ) confirms the obtaining of 3D networks in all the cases, but with several differences. Starch hydrogel has a cleaner structure, with well-defined channels (around 150 nm in diameter), which could capture therapeutic molecules or particles inside the matrix (Figure 1A-D ). Starch-PVA structure seems more rigid, with thinner channels and less network-like arrangement (Figure 1E-H ). While starch-CS has an interesting powder-like structure given by the existence of granules or beads that cover the channels and can be seen in the highest magnification images (Figure 1I-L ). Looking for a cleaner structure of starch-CS hydrogel, a second (chemical) synthesis reaction was performed, using GA as crosslinking agent. This procedure led to a clean 3D network (Figure 1M-P ), but less intricate and with wider channels than starch hydrogel.
Although variations in the procedure (reagents proportion, etc.) could determine different results in terms of network construction, it is interesting that microscopical structures of each of the three physical hydrogels suggests a way to apply it. Starch hydrogel, with clean channels and a complex structure seems optimum for drug or particle absorption and retention. Moreover, starch hydrophilic character will allow a fast load of water-dispersed particles or drugs, while the intricate hydrogel matrix could guarantee a relatively long-lasting release.
Additionally, starch-PVA hydrogel, with a hard and rigid structure has a bone-like appearance. An exploration of its capabilities in stem-cell or bone transplants applications could give positive results, since PVA have been reported before for this kind of uses (Dashtdar, 2015; Peng, 2019). A biodegradable matrix with a solid character, could be an efficient scaffold for cell grow and multiplication, while fulfilling a structural function as well.
The presence of big particles in the starch-CS network and the fact that they appeared spontaneously, is a very interesting feature. First, this suggests some level of separation between the polymers used for hydrogel synthesis, that is an absence of physical combination. This was the logical reason which led us to the posterior application of a chemical method. Their composition and internal structure must be known in order to explore potential applications.
In this way, energy dispersive X-ray spectroscopy analysis of starch-CS hydrogel was performed, showing channel walls with a typical carbohydrate composition, while important levels of nitrogen were detected on the beads (Figure 2 ). Because of chitosan chemical structure, an aminated polysaccharide (Li, 2018), nitrogen in the beads indicates their chitosan composition. Instead, the absence of nitrogen in the walls of the channels indicates their starch composition. The internal structure of the beads was known through its interaction with the microscope electron beam. After some minutes of the beam hitting the beads, they collapsed, and its empty interior was observed (Figure 3 ). This confirmed a capsule-like structure, suggesting potential uses in the encapsulation of molecules (proteins, bioactive compounds), which need to be explored in the near future.
A possible mechanism that led to beads formation could be related to the interaction of chitosan chains with ions present in the solution, during hydrogel synthesis. This is a mechanism similar to the reaction involved inionotropic gelation (Pedroso-Santana, 2020), but this must be confirmed and studied in detail, in next works.