3.1 Characterization of MFC, (MFC-Ag)NC, and (MFC-Ag)NC/PVA hydrogels
The absorbance spectra of MFC and (MFC-Ag)NC are shown in figure 2 measured by UV–vis spectroscopy. The spectra of (MFC-Ag)NC exhibited a strong absorbance band at 425 nm while there was no characteristic peak for MFC. The appearance of the peak at 425 nm is probably due to the surface plasmon excitation vibration effect of AgNPs which is following the previous studies [42]–[44]. It has also been observed that the UV absorption band is positioned at a long-wavelength range between 350-500 nm. The position and intensity of the absorption peak of silver NP may be depended on its size, shape, and surface capping agents [43]. Gu-Joong Kwon et al. have revealed that the spherical AgNPs can absorb light within the wavelength range of 400–430 nm [45]. They also mentioned that nanocellulose can act as both template and the capping agents of the (MFC-Ag)NPs. Ag-NPs are formed by the reduction of Ag+ ions by hydroxyl groups of cellulose followed by redox reaction by which metallic Ag is fixed within the cellulose porous structure.
Figure 3 shows the XRD pattern of MFC and (MFC-Ag)NC. The typical diffraction peaks assigned to cellulose and face-centered cubic (FCC) silver nanoparticles were observed clearly. The diffraction peaks at around 2θ = 12.08°, 19.96°, and 22.08° were observed due to the 101, 10\(\stackrel{-}{1}\), and 002 planes of cellulose respectively. The peaks at around 2θ = 34.74°, 40.62°, and 48.16° for (MFC-Ag)NC were noticed due to the well-crystallized silver nanoparticles with FCC structure [43]. It indicated that Ag+ has been successfully reduced with the cellulose matrix under microwave-assisted heating and formed a crystalline structure. However, the intensity of the peaks at 34.74°, 40.62°, and 48.16° is smaller compared to identified peaks of cellulose at position 19.96°, and 22.08°.
Figure 4 shows the FTIR spectra of (MFC-Ag)NC, PVA, and PVA/(MFC-Ag)NC hydrogels. The (MFC-Ag)NC displays the typical bands of cellulose [46], [47]. In general, the absorption bands of (MFC-Ag)NC at around 3383 cm−1 is attributed to the stretching vibration of the hydroxyl group; 2897, 1666, 1487, 1028, and 833 cm−1 peaks correspond to the C–H group, C=O stretching in cellulose, C–H bending mode, C–O–C stretching mode from the glucosidic units [46], and C–H rocking vibration of cellulose [48] respectively. The characteristic absorption bands of PVA hydrogel are found at 3358 cm−1 which is corresponded to the O-H stretching of the hydroxyl group of the PVA; sharp band at 1649 cm−1 is assigned to the C-O stretching of the acetate group, and 1492 cm−1 is attributed to combination frequencies of (CH-OH) of PVA. The peak at 1427 cm−1 represents the trigonal complex structure between PVA and borate [49], which means the formation of trigonal structure is dominant in this hydrogel. No remarkable changes were observed on the spectra of PVA/ (MFC-Ag)NC hydrogels compared to the PVA hydrogel. The characteristics bands of MFC and PVA are closer to each other. Probably, in the hydrogels, the individual bands of MFC and PVA were overlapped.
Scanning electron microscopy (SEM) provides major information about the surface morphology of hydrogels. Figure 5 exhibits the surface morphology of (MFC-Ag)NC (a and b), pure PVA (c), and PVA/(MFC-Ag)NC (d) hydrogel samples following water removal by freeze drying. The images of (MFC-Ag)NC showed that the prepared composite was in the micro range, exhibiting the diameter of the composite materials as 0.07, 0.12, 0.15, 0.17 µm. After the removal of water, the voids formed inside the pure PVA hydrogel was circular or oval in shape and exhibited a diameter of 5-7 µm. The addition of (MFC-Ag)NC has brought substantial differences to the hydrogels, particularly in terms of the diameter of the void which decreased to 1-2 µm. As the amount of (MFC-Ag)NC was increased the distance between PVA chain reduced which in turn lead to a dense and relatively compact structure. Furthermore, a random void structure was observed compared to the circular and oval structure of pure PVA hydrogel. That’s because of the random distribution of the (MFC-Ag)NC composite throughout the hydrogel structure.
3.2 Swelling Properties
Figure 6 shows the change of degree of swelling (DS) with time in distilled water, saline water, and various pH solutions. It has been seen that the swelling percentage of hydrogel is decreased with the increase of (MFC-Ag)NC loading (Figure 5a). From the SEM it is seen that the pore size decrease with the addition of (MFC-Ag)NC. As hydrogels retain water into their structure with the help of their beehive structure. As the pore size decreases, it affects the swelling property. So it can be said that with a higher percentage of (MFC-Ag)NC incorporation, the pore size decreases which led to the decrease of swelling of hydrogels [50]. The maximum swelling percentage was found to be 519.44% for 0 wt% (MFC-AG)NC. The addition of (MFC-Ag)NC till 1 wt% kept the swelling behavior similar to The maximum swelling was found at 48 hours but after this period the degree of swelling tends to reduce. This could be due to the disintegration of the hydrogel. Whenever polymer molecules are swelled in a particular solvent, the physical bonds between the molecules become weak [51]. Therefore, the PVA hydrogel started to disintegrate after a complete swell in distilled water. This also suggests that the bonding was weak and the cross-linking of the hydrogel was physical bonding.
The DS of the hydrogel intensely dropped in the saline (0.9% NaCl) water compared to distilled water. Moreover, the DS of hydrogel in saline water decreased after 1 wt% of (MFC-Ag)NC loading i.e. the hydrogel with 1 wt% (MFC-Ag)NC showed maximum DS capacity in the saline water. Outwardly, the degree of cross-linking in the hydrogel is influenced by the Na+ cation of saline water. Ag present in MFC may prohibit the entrance of Na+ ion to form crosslinking in hydrogel and hence DS increases. At a very higher percentage of (MFC-Ag)NC, (MFC-Ag)NC itself forms a crosslinked network with PVA and borax hydrogel causing the reduction of DS and also from the SEM it was evident that the pore size decreased after the addition of (MFC-Ag)NC compared to the pure PVA hydrogel which in-turn decreased the swelling degree to a continuous manner. The maximum DS of hydrogel is also found at 48 hours for saline water. More ionic density can reduce the gel stability which may act as the driving force for the breakdown of bonding between the PVA and (MFC-Ag)NC [52].
The DS of PVA hydrogel with 0 wt% (MFC-Ag)NC, 2 wt% (MFC-Ag)NC, and 5 wt% (MFC-Ag)NC was measured after 48 hours in different pH solutions ranging from 1 to 13 to determine the sensitivity of the hydrogel samples. As expected, the maximum DS value was obtained at pH 7 for all types of samples and all samples showed a downward trend in DS with the incorporation of (MFC-Ag)NC. Furthermore, the DS decreased in both acidic and basic solutions. At lower pH, the alcoholic groups of PVA and carboxylic acid groups of (MFC-Ag)NC are protonated which can minimize the swelling proportion because of the reduction of anion-anion repulsion. In distilled water, the groups converted into ions of alcohol and carboxylate that resulting in the maximum repulsive electrostatic forces which in turn increased the DS of the hydrogel. However, in the basic solution, the alcohol and carboxylic groups changed to negatively charged ions which caused greater repulsion and expected to have more swelling in the hydrogels, we observed a decreased order. The possible reason is the presence of Ag+ and Na+ ions which diminished the formation of negative carboxylate ions and hindered the effective electrostatic repulsion [53].
3.3 Tensile properties
The summary of mechanical properties of a series of hydrogels including tensile strength, Young’s modulus, and elongation at break with different (MFC-Ag)NC contents is listed in Table 1. The mechanical properties of (MFC-Ag)NC/PVA hydrogels were evaluated by uniaxial tensile measurements. The hydrogels with 0–5 wt% of (MFC-Ag)NC loading would lead to an increase in modulus and tensile strength. In (MFC-Ag)NC/PVA hydrogels, both covalent bonds and physical interactions between (MFC-Ag)NC and PVA chains might be stronger which led to the increase of tensile strength. This enhancement in mechanical behaviours would be related to the ability of (MFC-Ag)NC to change the energy dissipation process. Besides, the addition of a small fraction of (MFC-Ag)NC led to a significant increase in Young’s modulus (toughness). The moduli values are significantly higher than those reported by Han and co-workers [54], 0.9 KPa was reported for pure PVA-borax hydrogels and values ranging from 3.8 to 22.5 KPa was notified for gels reinforced with cellulose nanoparticles.
Table 1
Tensile properties of hydrogels with different PVA/(MFC-Ag)NC loading
PVA Hydrogel with (MFC-Ag)NC
|
Tensile Strength
(KPa)
|
Elongation
(%)
|
Young modulus
(KPa)
|
0wt%
|
10.8±1.4
|
280.9±4.2
|
5.4±1.1
|
0.5wt %
|
11.5±1.7
|
273.2±2.9
|
6.9±1.1
|
1wt %
|
12.1±1.2
|
261.5±3.3
|
8.4±1.1
|
2wt%
|
14.5±2.1
|
245.3±3.3
|
12.7±2.5
|
5wt%
|
16.3±4.3
|
209.1±1.3
|
23.9±1.7
|
This enhancement in mechanical properties can be attributed to the reinforcing influence of (MFC-Ag)NC. Liu et al. described the possible formation of a semi-interpenetrating network between nano-cellulose and PVA [55]. The strong interfacial interactions between the (MFC-Ag)NC and PVA impart restrictions on segments of the polymer chain during deformation leading to enhanced stiffness and strength. Entangled segments of nanocomposite act as a physical crosslinking agent, working in association with borax complexes towards maintaining the mechanical integrity of the hydrogels. This is supported by the increased gel content observed at (MFC-Ag)NC concentrations up to 5 wt%. At low degrees of crosslinking, polymer chains exhibit greater flexibility and freedom to move, increasing the possibility of bond reformation across the broken interface. Therefore elongation at break was found the highest in 0 wt% of (MFC-Ag)NC loading.
3.4 Thermogravimetric properties
Figure 8 shows the TGA and DTG curves of (MFC-Ag)NC and PVA hydrogels with various (MFC-Ag)NC contents. The TGA curve of (MFC-Ag)NC indicates higher stability toward thermal degradation. From 30-600 oC, weight loss percent of nanocomposite was observed and maximum degradation was observed from 242-380 oC. The TGA of (MFC-Ag)NC exhibited that the water content was approximately 7 wt%, and at 242-380 oC, 90% weight was lost which may be due to the degradation of the cellulose chain. PVA hydrogels with different percentages of (MFC-Ag)NC showed multiple steps of weight loss. The first steep weight loss started at 93°C and is attributed to the loss of water in the sample which led to 60-80% weight loss till 125 oC in this stage. 2 wt% and 5 wt% (MFC-Ag)NC loaded hydrogels showed lower weight loss compared to pure PVA hydrogel. This is evidence of the semi-interpenetrating network formation between PVA hydrogel and (MFC-Ag)NC. The strong electrostatic forces between (MFC-Ag)NC and PVA molecules offer obstruction during degradation of the polymer chains and lead to improved properties against thermal degradation. This data is also supported by a study performed by Sriupayo et al.[56]. The second step of degradation occurred at the temperature range from 135-225 oC. PVA started to degrade at around 130 oC. During degradation of PVA, the remaining water of gel is readily separated via vapor formation. The DTG graph shows the final degradation peaks at 366, 311, 307 and 303 oC for (MFC-Ag)NC, 0 wt% (MFC-Ag)NC/PVA, 2 wt% (MFC-Ag)NC/PVA, 5 wt% (MFC-Ag)NC/PVA hydrogels, respectively and the corresponding weight loss is 0.62 mg/min, 0.46 mg/min, 0.51 mg/min, 0.22 mg/min. The DTA curve shows the endothermic peaks are located at around 120, 124, and 190 oC (Figure 6) for 0 wt% (MFC-Ag)NC/PVA, 2 wt% (MFC-Ag)NC/PVA, 5 wt% (MFC-Ag)NC/PVA hydrogels, which fit well with the first step weight losses in the TG curve.
3.5 Anti-microbial activity
The antimicrobial activity of the hydrogel was observed against multi-drug resistant E. coli MZ20 (gram-negative), P. aeruginosa MZ2F (gram-negative), and S. aureus MZ18 (gram-positive) through the agar well diffusion method. As shown in Fig.9, the pure PVA hydrogel does not show any antibacterial activity. (MFC-Ag)NC/PVA hydrogels showed that the antibacterial ability increased with an increasing percentage of (MFC-Ag)NC. With the addition of (MFC-Ag)NC to the culture medium, it was expected to get attached to the cell wall of the bactericides and because of the nature of Ag nanoparticles, it destroyed the cell walls and affected the protein and other cellular components leakage and at last killed the cells. Ag nanoparticles can rapidly degenerate to form free radicals by redox reaction. During the reaction, Ag+ ion is formed which is responsible for the formation of reactive oxygen species (ROS) resulting the oxidative stress. Studies showed that nanoparticles can increase the level of ROS up to 50 fold. When the cells of gram-positive and gram-negative bacteria experience oxidative stress, a series of dysfunctions are observed in their lipid membrane, protein and DNA which stop cell growth and destroy them [57], [58].
Table 2 shows that the antimicrobial efficiency of (MFC-Ag)NC/PVA hydrogel depended on the concentration of the (MFC-Ag)NC regardless of the form of bacteria used. That’s why the best antibacterial effect was observed by (MFC-Ag)NC only. It is clear that, with a higher percentage of (MFC-Ag)NC content, the hydrogel samples exhibited an extended inhibition zone around the wells. 5% (MFC-Ag)NC/PVA hydrogels showed the maximum antimicrobial action against E. coli, P. aeruginosa, and S. aureus. The maximum width of the zone of inhibition of (MFC-Ag)NC/PVA hydrogels against E. coli, P. aeruginosa, and S. aureus were 4.1±0.2, 4.5±0.4, and 3.1±0.4mm respectively. By analyzing the outcomes we can also discover that (MFC-Ag)NC hydrogels exhibited better activity against gram-negative bacteria compared to gram-positive bacteria.
Table 2
Antimicrobial activity data of PVA hydrogels with different wt% of (MFC-Ag)NC against E. coli, P. aeruginosa, and S. aureus.
Tested Microorganisms
|
Zone of Inhibition of study compound (mm)
|
PVA hydrogel with (MFC-Ag)NC
|
(MFC-Ag)NC
|
0wt%
|
0.5wt%
|
1wt%
|
2wt%
|
5wt%
|
Escherichia coli
|
×
|
1.0±0.1
|
1.1±0.1
|
1.9±0.1
|
4.1±0.2
|
4.5±0.3
|
Pseudomonas aeruginosa
|
×
|
1.1±0.3
|
1.5±0.3
|
2.0±0.2
|
4.5±0.4
|
5.0±0.5
|
Staphylococcus aureus
|
0.7±0.1
|
1.8±0.1
|
2.0±0.1
|
2.3±0.5
|
3.1±0.4
|
3.9±0.5
|