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 .