2.1. Materials
The GG, PMVE-MAn (Mw =216 kDa), CS (from shrimp shells, > 75% deacetylated), CA (≥95%), Pentasodium tripolyphosphate (TPP), thiazolyl blue tetrazolium bromide (MTT), fetal bovine serum (FBS), trypsin–EDTA, RPMI 1640 medium, Dimethyl sulfoxide (DMSO), and Phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St. Lo., USA). DPPH (1,1-Diphenyl-2-picrylhydrazyl), 1% glacial acetic acid, Ethanol 96%, Tween 80%, and Muller Hinton Agar were purchased from Merck Chemical Co. (Darmstadt, Germany). The mouse embryonic fibroblast cell line (NIH-3T3), and Stock cultures, Escherichia coli (PTCC-1270), Staphylococcus aureus (PTCC-1112) were purchased from Pasture Institute (Iran).
2.2. Synthesis of CSNPs and CA-loaded CSNPs
CA-loaded CSNPs were prepared using ionic gelation assay with minor change [21]. In briefly, CA (0.3 mL, 1 v/v %) was added slowly into preheated CS solution (0.4% w/v) under magnetic stirring. After that, TPP solution (0.3% w/v) was added gradually into CA-CS solution under magnetic stirring (at 800 rpm) for 20 min at room temperature to obtain CA-loaded CSNPs. Tween 80% was used as a surfactant. The nanodispersion was then centrifuged at 10,000 × g for 1 h and washed by distilled water (4 times) to remove the unloaded CA.
2.3. Particle size, zeta potential and encapsulation efficiency (EE) studies
The particle size and zeta potential of CSNPs were calculated with a dynamic light scattering (DLS) (Zetasizer Nano-ZS90, Malvern Instruments Ltd., Worcestershire, UK) under vacuum condition. All evaluations were performed at room temperature. The EE % of CA-loaded CSNPs was calculated by UV–Vis spectrophotometer (Spectrum SP-UV500DB) at 285 nm.
2.4. Preparation of composite hydrogels
GG (0.2% w/v) /PMVE-MA (2% w/v) hydrogel samples were prepared as follows; aqueous solutions comprising various weight ratios of GG, and PMVE-MAn were put in 24-well plates. Solutions were freeze-dried for 72 h. The final hydrogel samples were taken from the 24-well plates. The freeze-dried hydrogel samples were put into an oven at 75 °C for 3 days and 8 h for each day. The final obtained hydrogels were cited in a high content of water for 24 h. Finally, these hydrogel samples were frozen in liquid nitrogen and after that freeze-dried for 48 h.
Table 1. Preparation chart of GG/P (Guar Gum/PMVE-MA) hydrogels.
Hydrogel Sample Code
|
GG
(0.2% w/v)
|
PMVE-MAn (2% w/v)
|
GG/P20
|
80
|
20
|
GG/P40
|
60
|
40
|
GG/P70
|
30
|
70
|
2.5. Characterization
FTIR spectra were recorded for GG, PMVE-MA, and final hydrogels with Thermo Avatar 370 spectrometer (Tensor27, Bruker Co., Ettlingen, Germany) in transmittance mode amongst 400-4000 cm−1. The morphology of hydrogels was analyzed by a scanning electron microscopy (SEM, Hitachi High-Tech HITACHI, Tokyo, Japan). To investigate the thermal stability of produced hydrogels, thermogravimetric analysis (TGA) was performed with a LINSEIS SPA PT 1600 device (Germany) at a range of 20-600 °C, at a heating rate of 10 °C/min and under N2.
2.6. Swelling degree
To measure the swelling degree of hydrogel samples, gravimetric analysis was performed [22]. In short, at first the dry weight of the hydrogels was measured (Wd), after that the hydrogels were immersed in 30 ml of PBS solution at room temperature. After that, the swollen hydrogels were weighed after 2, 4, 7, 10, 12, 24, 36,7 2, and 96 h (Ws). The swelling degree (SD) was measured according with the Eq:
2.7. Mechanical Characteristics
Using a universal testing system (INSTRON series 3366), the mechanical properties of the samples were investigated. The initial elastic modulus was calculated from the slope of the initial linear segment of stresss-train curves, Tensile strength was taken as the maximum force divided by the minimum cross-sectional area of the specimen to find a precise tensile strength. The strain value corresponding to the maximum stress was calculated as a strain at break.
2.8. Cell viability study
The MTT technique was accomplished to explore the cell viability of hydrogel samples [23–25]. In summary, the hydrogel samples were cut into 12 mm2 and immersed in 75% ethanol for 60 min to be sterilized. After that, 1 mL of cell suspension including 5 × 104 cells was seeded onto sterilized hydrogel samples and incubated in RPMI-1640 including 10% FBS at 37 °C with 5% CO2. After 1, 3, 5 and 7 days, 200 μL DMSO was added to dissolve formazan crystals formed inside cells. Then, absorbance was determined using spectrophotometer (UV-2550, Shimadzu, Japan) at 570 nm.
2.9. Antioxidant capacity
The antioxidant capacity of the optimal hydrogel and optimal hydrogel containing CA-loaded CSNPs was calculated with the DPPH radical scavenging assay during 9 h of incubation [26]. In summary, 0.1 mM of DPPH solution in ethanol was prepared. Next, 1 mL of DPPH solution was added to 3 mL of solution containing hydrogel and hydrogel/CA-loaded CSNPs. Finally, after 30 min, the absorbance was measured at 517 nm assessed with a UV-Vis spectrophotometer (Spectrum SP-UV500DB) after 3, 6, and 9h. The DPPH radical scavenging was calculated with the Eq:
Where AbsDPPH was the absorbance of DPPH ethanolic solution and Absextract was the absorbance of the hydrogels.
2.10. Antibacterial activity
Antibacterial activities of the optimal hydrogel and optimal hydrogel containing CA-loaded CSNPs against S. areus and E. coli bacteria were measured using disk diffusion methods [8]. In short, 3-5 mm disk of hydrogel sample was exposed on Mueller-Hinton agar plate that cultured by 1.5 × 108 CFU/mL concentration of each bacterial suspension. After 24 h, inhibition of bacterial growth area about the hydrogel sample disk was determined and expressed.
2.11. Statistical analysis
Statistical differences (P < 0.05) were analyzed using one-way ANOVA followed by Tukey’s test for multiple comparisons by GraphPad Prism 5. Obtained results were expressed as mean ± standard deviation (SD). The level of significance was considered P < 0.05.