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.