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