Starch-based hydrogels show relevant properties for tissue
engineering and loading of nanoparticulate systems.
Seidy Pedroso-Santanaa, Brian Ignacio Rivas
Tiznadoa, Noralvis Fleitas-Salazara,
Rafael Mauraa, Carolina
Gómez-Gaeteb, Alexis Debutc, Natalie
Parraa, Karla S. Vizuete
Armendarizc, Thelvia I. Ramosc,
Jorge R. Toledoa*.
aLaboratorio de Biotecnología y Biofármacos,
Departamento de Fisiopatología, Facultad de Ciencias Biológicas,
Universidad de Concepción, Barrio Universitario s/n, Concepción CP.
4030000, Chile.
bDepartamento de Farmacia, Facultad de Farmacia,
Universidad de Concepción, Barrio Universitario s/n, Concepción CP.
4030000, Chile.
cDepartamento Ciencias de la Vida y de la Agricultura,
Universidad de las Fuerzas Armadas ESPE, Av. General Rumiñahui s/n, PO
BOX 171-5-231B, Sangolqui, Ecuador.
*Corresponding author : Professor Jorge R. Toledo,
jotoledo@udec.cl
Abstract :
The synthesis of starch-based physical hydrogels in combination with
chitosan and polyvinyl alcohol, and their potential co-application with
chitosan nanoparticles was evaluated. The potential of starch-chitosan
hydrogel obtained by physical/chemical method for tissue engineering
uses was also studied in a mouse wound healing model. Although the
microscopical structure of each synthesized hydrogel suggests a possible
biological application, starch-polyvinyl alcohol hydrogel exhibited
rigid behavior with minor channel diameters, a lower swelling rate (less
than 300%), and negatively affected cell viability in a cytotoxicity
assay. Starch-chitosan hydrogel obtained by chemical crosslinking with
glutaraldehyde demonstrated the higher swelling rate (about 1100%),
cell viability values over 80%, and a homogeneous tri-dimensional
structure; along with an excellent interaction with chitosan
nanoparticles. This type of hydrogel was selected for an in vivoexperiment, showing significant differences in wound healing process
against a non-treated control, in terms of inflammation, exudate
production and tissue recovering.
Keywords : hydrogel, starch, chitosan, nanoparticles loading,
tissue engineering.
Introduction .
Hydrogels are polymeric three-dimensional structures, which absorb large
quantities of aqueous solutions (Hamedi, 2018). This property is useful
for drug delivery applications, based on retention and later release of
hydrophilic drugs. Several polymers, such as chitosan (Huang, 2018),
polyvinyl alcohol (Hou, 2015), starch (Biduski, 2018) and polyvinyl
pyrrolidone (Timaeva, 2020), are currently used for hydrogels synthesis
(Akhtar, 2016).
Starch is a natural polymer, obtained from plants, composed by
repetitive units of amylose and amylopectin (Ismail, 2013). Its
biocompatibility, biodegradability, high hydrophilicity, and low cost in
the market makes this material an appropriate option in the production
of hydrogels for biological applications (Perez, 2018). Starch-based
hydrogels can be synthesized by copolymerization of polysaccharide
monomers, using a chemical crosslinker, or under the action of physical
initiators like temperature (Ismail, 2013). Combination of starch with
other polymers is a way to obtain hydrogels with outstanding features in
terms of structure strength, pore diameter in the hydrogel matrix,
bio-adhesion, and response to environmental changes (Ou, 2017).
In general, hydrogel synthesis is carried out by physical or chemical
methods, or by a combination of both (Ou, 2017). Physical hydrogels,
also called self-assembling hydrogels (Fu, 2018), are relatively easy to
obtain, based on non-covalent interactions between polymeric chains
(ionic, electrical, etc.). One of the most used physical methods is
freeze-thawing; by which the formation of microcrystals is responsible
of the interaction among several polymeric planes, leading to a
network-like tridimensional structure (Gulrez, 2011).
On the other hand, chemical methods frequently use cross-linking agents
to link polymeric chains by covalent bonds (Gulrez, 2011). These
interactions occur between polymer functional groups (-COOH,
-NH2, etc.) and cross-linker groups. For example,
glutaraldehyde (GA) is a chemical cross-linker widely known by interact
with amines through its carbonyl (-CHO) groups (Crescenzi, 2003;
Fadouloglou, 2008). This reaction is easy to obtain, but if there are
proteins involved, its biological activity could be affected (Vrsanská,
2018).
The use of hydrogels in pharmaceutical applications provides drug
protection and controlled release (Lopez-Cordoba, 2019). Nevertheless,
considering the small sizes of bioactive molecules in comparison to the
size of hydrogel pores, the quantity of retained drug could be variable.
One approach to obtain higher values of retention and an increased
bioavailability is the formulation of active molecules inside nano and
microparticles (Lopez-Cordoba, 2019; Perez, 2018). Polymeric
nano/microparticles provide an additional protection to drugs along with
a larger interaction surface, which allows increased drug retention
inside hydrogel networks.
Here, we aimed to obtain different variants of starch-based hydrogels in
combination (or not) with other biocompatible polymers: chitosan and
polyvinyl alcohol. Physical and physical/chemical procedures were
applied to synthesize hydrogel structures, which were later loaded with
chitosan (CS) nanoparticles. Subsequently, in vitro and in
vivo experiments corroborated the relevance of the systems developed in
tissue engineering applications and for nanoparticulate systems loading.
Materials and methods .
Materials .
Low molecular weight deacetylated chitosan (50-190 KDa, 75-85%
deacetylated), sodium tri-polyphosphate (TPP), acetic acid, starch
(soluble, ACS reagent), polyvinyl alcohol (PVA) (31-50 KDa. 98-99%
hydrolized), glutaraldehyde (GA) and all the reagents used for molecular
biology and cell culture, were acquired from Sigma-Aldrich (USA).
Nitrocellulose membranes of 0.45 µm were from Merck (Germany).
Hydrogel synthesis .
Four variants of starch-based hydrogels were synthesized: 1) Starch, 2)
Starch-PVA, 3) Starch-CS, 4) Starch-CS-GA; similar to the procedures
previously described (Liu, 2010; Bursali, 2011). Starch solution 8%
(w/v) and Starch - PVA solution 5% - 5% (w/v), in distilled water,
were prepared by stirring at 500 rpm with subsequent autoclavation
(variants 1 and 2). The solutions were cooled at room temperature while
stirred at 500 rpm. Variant 3 was prepared by dissolution of 5% starch
and 5% CS (w/v) in acetic acid, 0.5% in water (v/v). The mixture was
stirred at 800 rpm until total resuspension. To prepare variant 4, 2.5
% starch and chitosan solutions (w/v) were mixed under agitation, and
400 μL of 0.4% GA solution (w/v) were added. The mix was stirred for
another 10 minutes at 800 rpm. All the solutions were poured into the
molds, frozen at -20 0C for 12 hours, and later
thawed. This was repeated 3 times to complete 3 freeze-thaw cycles.
Hydrogel characterization .
Structure and composition of lyophilized hydrogels were studied by
spectroscopy and microscopy techniques. Fourier transform infrared
spectroscopy (FTIR) in a Nicolet Nexus FTIR (USA) was used to obtain
hydrogel infrared spectra. The samples were analyzed at 250C, between 500-4000 cm-1. Using a
JEOL JSM-6380 LV (USA) microscope, hydrogel tridimensional structure was
observed by scanning electron microscopy (SEM), and its chemical
composition was analyzed by energy-dispersive X-ray spectroscopy (EDS).
Hydrogels swelling in the presence of water was also studied. Each dry
hydrogel sample was weighted before immersion in deionized water. At
certain times during 24 h, samples were weighted again and the swelling
rate (SR) was calculated using the formula: SR=(Ww-Wd)/Wd. Where Ww is
wet weight, and Wd is dry weight of hydrogel samples. SR values were
multiplied by 100 to obtain the percentual swelling rate of each
hydrogel.
Nanoparticle synthesis .
CS-TPP nanoparticles (NPs) were synthesized by ionotropic gelation
method as reported before (Canepa, 2017). Briefly: CS powder, 2 mg/mL,
was dissolved in acetic acid 2%, under magnetic stirring. The solution
was then filtered using 0.45 μm membrane filters. NPs were obtained by
dropwise addition of TPP at 1.5 mg/mL in water (2 mL), using a KDS 200
syringe infusion pump (USA), with 21 G1 1/2 needle and speed 15 mL/h,
under stirring. Resulting NPs suspension was stirred for 30 minutes and
later stored at 4 0C. Nanoparticle hydrodynamic
diameter was analyzed by Dynamic light scattering (DLS) using a Malvern
Zetasizer ZS-90 (UK). The size and shape were confirmed by scanning
transmission electron microscopy (STEM) using a TESCAN MIRA 3 (Czech
Republic).
Hydrogel interaction with nanoparticles .
In order to analyze hydrogel ability to absorb and retain nanoparticles,
lyophilized hydrogel fragments, 6 mm in diameter and 8 mm long, were
incubated with 50 mg of CS-TPP NPs in phosphate buffer saline (PBS) (3
mL), for 3 h at 25 0C. Then, the excess of PBS was
removed, and NPs-loaded hydrogels were lyophilized once again.
Interaction hydrogels/NPs was visualized by SEM.
Cell viability experiment .
Yellow tetrazolium (MTT) assay (van de Loosdrecht, 1994) was used to
study the effect of hydrogels on the viability of Human epidermal type 2
cells (HEp-2). In brief, lyophilized hydrogels fragments of 3 mm x 2 mm
and 2 mm thickness were incubated with HEp-2 cells in Dulbecco’s
Modified Eagle Medium (DMEM). Nontreated cells were used as viability
control and the treatment with Triton X-100 (1% v/v) was consider as
100% cytotoxicity control. The experiment was performed in a 96 well
plate, incubated at 37 0C and 5% CO2.
After 24 hours, an equal volume of medium containing MTT (2 mg/mL) was
added to each well. Incubation conditions were maintained for another 4
hours, before measuring absorbance at 570 nm in a Synergy HTX multi-mode
microplate reader (Biotek, USA).
In vivo studies .
The interaction of hydrogels with mice was studied in vivo in
order to analyze its effects in tissue regeneration. Healthy animals
(female CF-1, 30-40g, 4 weeks old) were separated in groups of 5 animals
each. Skin lesions were produced as previously described (Choi, 2008),
briefly, the animals were first anesthetized with ketamine/xylazine (100
mg/kg and 5 mg/kg, respectively) and the dorsal hairs were completely
shaved. Then, the area was cleaned with povidone iodide and a hot metal
ring (6 mm diameter) was used to induce a dorsal wound. The skin inside
the circular mark was excised with scissors. Lyophilized hydrogel
fragments (6 mm diameter, 2 mm thickness) were immediately placed on the
wound, without any other bandages or scaffolds. The wound areas were
measured and visually analyzed every 3 days, for 15 days. Animals
without hydrogel applied were used as control of the experiment.In vivo experiments were accomplished following biosafety and
bioethics principles. The Bioethics and Biosafety Committee, Biological
Sciences School, Universidad de Concepción, approved the procedure.
Results and Discussion .