3.1. Swelling ratio and gel fraction
The sodium alginate/acrylic acid composite hydrogels conjugated to silver nanoparticles were successfully synthesized through a simultaneous chemically ion cross-linking and physically microwave polymerization of sodium alginate acrylic acid monomers, respectively. However, based on used acrylic acid and silver ion contents and applied microwave time, their characteristics were differed considerably. For instance, the swelling ratio of the obtained hydrogels varied from 10 to 506.7%, and their gel fraction laid between 0.07 and 0.91. All gained hydrogels exhibited noticeable bactericidal activities due to the presence of silver in their matrice, and were able to encapsulate and load various compounds such as cephalexin, from 36.89 to 92.58%. The characteristics of all samples prepared in different conditions were shown in Table 1 and the ANOVA analysis results were summarized in Table 2.
Table 2
ANOVA analysis and p-values and F-ratio of linear, quadratic and binary interaction terms in suggested models for prepared sodium alginate/acrylic acid composite hydrogels conjugated to silver nanoparticles
Characteristics
|
Linear
effects
|
X1
|
X2
|
X3
|
Quadratic
effects
|
X11
|
X22
|
X33
|
Interaction
effects
|
X12
|
X13
|
X23
|
swelling
|
P-value
|
0.000
|
0.000
|
0.000
|
0.000
|
0.000
|
0.000
|
0.000
|
0.008
|
0.000
|
0.234
|
0.458
|
0.000
|
F-ratio
|
302.91
|
320.91
|
166.80
|
534.15
|
163.31
|
46.40
|
321.43
|
11.11
|
199.71
|
1.61
|
0.60
|
565.49
|
gel fraction
|
P-value
|
0.000
|
0.000
|
0.000
|
0.000
|
0.025
|
0.007
|
0.708
|
0.756
|
0.000
|
0.021
|
0.135
|
0.000
|
F-ratio
|
4106
|
10846
|
76.6
|
31.63
|
4.8
|
11.65
|
0.15
|
0.10
|
34.68
|
7.5
|
2.64
|
90.07
|
cephalexin load
|
P-value
|
0.000
|
0.000
|
0.000
|
0.355
|
0.000
|
0.000
|
0.013
|
0.017
|
0.001
|
0.000
|
0.373
|
0.507
|
F-ratio
|
39.23
|
107.20
|
33.69
|
0.94
|
26.59
|
60.42
|
9.03
|
8.27
|
12.11
|
35.81
|
0.87
|
0.47
|
clear zone
|
P-value
|
0.000
|
0.000
|
0.001
|
0.671
|
0.24
|
0.005
|
0.078
|
0.681
|
0.167
|
0.800
|
0.291
|
0.071
|
F-ratio
|
15.31
|
35.85
|
20.23
|
0.19
|
4.93
|
12.60
|
3.84
|
0.18
|
2.07
|
0.07
|
1.24
|
4.08
|
The ability to be swollen in thermodynamically compatible media is the most favorable characteristic of hydrogels. The water molecules penetrate into the polymeric network of hydrogels and the rubbery phase of hydrogels, which is first be expanded and filled by penetrated molecules of solvent and detach from glassy segments. In contradiction of the favorable osmotic force, the opposite elasticity force occurs that stopovers the extending and deformation of the hydrogel networks. The equilibrium in swelling will be occurred as the elasticity and osmotic forces balance (Ganji et al. 2010).
The ANOVA analysis for swelling ratio of prepared hydrogels indicated that all selected process parameters affected the swelling ratio of hydrogels significantly, especially in linear form. The interactions of acrylic acid with silver nitrate and microwave time were the insignificant terms on this characteristic. Thus, these two terms were removed from initial model, and the final reduced model were offered as Eq. 4.
Swelling ratio (%) = -742.5 + 63.40 X1 + 2255.1 X2 + 108.4 X3 − 4.879 X!2 − 876.8 X22 + 9.89 X32 − 314.9 X2. X3 (Eq. 4)
The coefficient of determination (R2) for this model was 97.26%. Thus, it can be concluded that this model can successfully predict about 97% of swelling ratio changes of hydrogels. Moreover, the insignificant p-value of lack of fit (p-value = 0.824), can confirm the suitability of this model in predicting the swelling ratio of sodium alginate/acrylic acid composite hydrogels. By comparing the F-ratio of significant terms, it could be also concluded that the interaction effect of silver nitrate and microwave time had the greatest effects on this characteristic (Table 2).
The main effects plot of independent variables on swelling ratio of hydrogels were shown in Fig. 1a. Since the interaction of silver nitrate with microwave time was only the significant term on swelling ratio, just the contour plot of the variation of this response by silver nitrate and microwave time at the certain level of acrylic acid (3.985g) was visualized as Fig. 1b. As can be seen in Fig. 1a, using greater acrylic acid contents in formulation of hydrogels, could yield the product with larger swelling ratio. An optimum content was obtained for silver nitrate leading to the hydrogels with the highest swelling ratio. Thus, the swelling ratio of hydrogels increased by rising the silver ions up to certain levels, and greater silver ions decreased this characteristic, considerably. The swelling ratio of hydrogels also decreased by increasing microwave time. However, the effect of microwave time on swelling ratio was different at various levels of silver ion, as simultaneous increases or decreases of microwave time and silver ion content resulted to the production of hydrogels with less swelling ability.
Generally, the microwave exposure of hydrogels for long time lead to a considerable increase in polymerization degree of the acrylic acid monomers. Therefore, the hydrogel network becomes more cohesive and stronger and water penetration into the network will be limited (Makhado et al. 2018; Kretschmann et al. 2007). Furthermore, the previous researches also confirmed that the presence of silver ions has positive impact on construction of stable acrylic acid hydrogel matrices, by accelerating the polymerization of acrylic acid. Thus, the gained stable hydrogel matrices would have greater water absorption and swelling ability. However, at high silver ion contents, various cross-linking can be occurred between silver ions and carboxylic groups of (poly) acrylic acid, leading to form O-Ag-O bonds, in which, with the formation of these bonds, the free hydroxyl groups on (poly) acrylic acid would be reduced. Consequently, the chance for hydrogen bonding between hydrogels carboxylic groups and water decreased, resulted in an extensive decline in water absorption of hydrogels (Serrano-Aroca and Deb 2020; Kowalski et al. 2019). The increase of swelling ratio by acrylic acid content would also be related to the formation of more impregnated poly acrylic acid matrices into sodium alginate networks. Therefore, the amount of empty media of hydrogel networks, which can be filled by water, increased (Serrano-Aroca and Deb 2020; Quintanilla de Stéfano et al. 2020). Moreover, the swelling of hydrogels lasts until equilibrium state, where the Gibbs free energy of hydrogel is minimized. According to the theory of Flory–Rehner, the Gibbs free energy will be minimized if the osmotic and elasticity forces become equal. An increase in hydrogels’ cross-linking density causes the formation of smaller chains. The shorter chains have less elasticity force as compared to longer ones. Thus, the equilibrium between elasticity and osmotic forces occurs at less swelling ratios (Quintanilla de Stéfano et al. 2020).
The ANOVA analysis of gel fraction of prepared hydrogels showed that all considered process parameters affected the gel fraction of hydrogels pointedly, particularly in linear form. The quadratic effects of both silver nitrate and microwave time, and the interaction effect of acrylic acid and microwave time, were insignificant terms on gel fraction changes of samples. Thus, these three terms were removed from initial model and the final reduced model for estimating the hydrogels’ gel fractions was shown as Eq. 5, with the R2 equal to 96.90%.
Gel fraction = -0.0627 + 0.09253 X1 + 0.3005 X2 + 0.07332 X3 − 0.001391 X12 − 0.00483 X1X2- 0.06396 X2X3 (Eq. 5)
Moreover, the insignificant p-value of lack of fit (p-value = 0.28), can confirm the correctness of model in predicting the gel fraction of produced composite hydrogels. By comparing the F-ratio of significant terms, it can also be concluded that the linear effect of microwave time had the highest influence on this characteristic (Table 2).
The main effects plot of independent variables on swelling ratio of hydrogels were shown in Fig. 2a. The contour plots of gel fraction changes by both silver nitrate-microwave time (fixed middle level of acrylic acid, Fig. 2b), and acrylic acid-silver nitrate (fixed middle level of microwave time, Fig. 2c) were shown due to the significant effects of acrylic acid-silver nitrate and silver nitrate-microwave time interactions. According to Fig. 2a, increasing of either acrylic acid or silver nitrate and microwave time raised the gel fraction of obtained hydrogels. Moreover, simultaneous increase or decrease of acrylic acid and silver nitrate, at constant microwave exposure time, or simultaneous increase or decrease of silver nitrate and microwave time, at fixed level of acrylic acid, also caused a decrease in gel fraction of samples (Figs. 2b,c).
Increasing the acrylic acid content led to construction the denser and more robust hydrogel matrices, which are insoluble in water. Furthermore, increasing the silver nitrate augmented the O-Ag-O cross-linking between the monomers and thus, made them insoluble in water. The exposure of hydrogels to microwave for extended time also increased the in polymerization degree of the acrylic acid monomers and produced stronger water insoluble matrices (Pourjavadi et al. 2006).
3.2. Cephalexin Load
All synthesized hydrogels could efficiently adsorb cephalexin, ranged from 43.75 to 98.75%. The ANOVA analysis for cephalexin load of samples indicated that the linear effects of acrylic acid and silver nitrate concentrations, the quadratic effects of all studied independent variables and the interaction effect of acrylic acid with silver nitrate contents were significant (p-value < 0.05) on this response. However, the acrylic acid content was the most effective parameter on drug loading of samples. The final reduced model predicting the cephalexin loading of hydrogels was found as Eq. 6. The linear effect of microwave time was not removed from the model due to its significant quadratic effect. The R2 for this model was 96.66%.
Cephalexin load (%) = 24.82–11.53 X1 + 65.8 X2 + 9.63 X3 + 1.278 X12 − 35.37 X22 − 1.836 X32 + 5.146 X1X2 (Eq. 6)
The obtained high R2, and insignificant p-value of lack of fit (p-value = 0.179), could confirm the precision of model in calculating the cephalexin load of produced composite hydrogels. By comparing the F-ratio of significant terms, it can also be concluded that the linear effect of acrylic acid had the utmost impact on this characteristic (Table 3).
Table 3. The kinetics of cephalexin adsorption by optimum composite hydrogels (the R2 and main coefficients for correlated general absorption models)
The main effects plots (Fig. 3a) indicated that while increasing the acrylic acid content up to certain level decreased the drug load of samples, further uses of acrylic acid in formulation of hydrogels increased their drug absorption, considerably. The reverse trend was also observed for silver nitrate and microwave exposure time, in which increasing of these two parameters up to certain level improved their drug loading efficiencies, however, additional contents of silver ions or extra duration of microwave irradiation decreased their drug absorption efficiencies. The contour plot of cephalexin absorption changes by acrylic acid and silver nitrate (at fixed middle level of microwave time was also shown in Fig. 3b. According to Fig. 3b, simultaneous increase or decrease of these two parameters could enhance the drug absorption efficiency of hydrogels.
Using high concentrations of acrylic acid (especially at certain microwave exposure time) caused the production of composite hydrogels with weak cross-links. These weak cross-linked acrylic acid-based hydrogels could also be obtained at less silver ions concentrations as well as less microwave irradiation time, since the anionic groups on hydrogels become less protonated, and the hydrogen bonding between the functional groups would be weakened. Thus, the pore size in the hydrogel matrix would be increased, causing a considerable enhance in the efficiency of hydrogels’ absorption. Increasing the silver ions also ionized the background polymers, upturned their electrostatic repulsions, weakened their matrices and made them more penetrable against drugs (Quintanilla de Stéfano et al. 2020). Previous researches also have shown that drug diffusion into hydrogels with high cross-linking densities matrices was difficult, because the hydrogels with more cross-links were more compacted and possessed very small less penetrable pores (Kowalski et al. 2019).
3.3. Antibacterial activity
All synthesized hydrogels showed antibacterial activity against s. aureus, with growth inhibitory zone ranged from 10 to 21 mm. The ANOVA analysis of antibacterial activity designated that the linear effects of acrylic acid and silver nitrate and the quadratic effect of acrylic acid were just the significant terms, on changes of this characteristic in 95% confidence interval (p-value < 0.05). The quadratic effect silver nitrate content and also its interaction with microwave time were also significant in 90% confidence interval (0.1 > p-value > 0.05). The final reduced model after removing the insignificant terms (p-value > 0.1) was shown as Eq. 7, in order to predict the variation of antibacterial activity of hydrogels.
Growth Inhibition zone (mm) = 21.22 + 2.479 X1 − 13.92 X2 − 2.381 X3 − 0.2239 X12 + 7.90 X22 + 2.35 X2X3 (Eq. 7)
According to obtained R2 for Eq. 7 (R2 = 92.39%), it can be concluded that this model can predict more than 92% of antibacterial activities of hydrogels against s. aureus in term of their growth inhibited zone diameter.
The main effects plots (Fig. 4a) for changes of this response by selected independent parameters point out that while increasing the acrylic acid content up to certain level increased the antibacterial activity of samples, further uses of acrylic acid in formulation of hydrogels affected reversely, and decreased this ability of samples. Increasing the silver nitrate ions as well as decreasing the microwave time also strengthened the bactericidal activity of hydrogels. The contour plot of growth inhibitory zoon of samples changed by silver nitrate content and microwave time (at fixed middle level of acrylic acid) was also shown in Fig. 4b. As can be seen in Fig. 4b, simultaneous increase of both silver nitrate and microwave irradiation time could enhance the bactericidal activity of hydrogels.
According to F-ratio of terms, the silver nitrate content was the most effective agent on antibacterial activity of obtained hydrogels. This result was predictable due to high antibacterial activity of silver. Some previous researches also reported an antibacterial activity for acrylic acid and its di-block copolymers (Gratzl et al. 2014). Thus, it seems that intensifying of hydrogels’ antibacterial activities by increasing the acrylic acid could be related to this antibacterial activity of acrylic acid residues in hydrogel matrices. Increasing the irradiation time also decreased the antibacterial activity of hydrogels, due to increasing the cross-linking bonds between acrylic acids and decreasing the monomer residues. Considerable antibacterial activities of prepared samples at high silver nitrate contents and extended irradiation time could be related to the reduction of silver ions to silver nano-particles by microwave emissions and their stabilization between the dense cross-linked matrices of hydrogels (Xia et al. 2012; Singh and Dhaliwal 2020).
3.4. Optimization and model confirmation
The acrylic acid and silver nitrate contents, as well as the microwave irradiation time, were numerically optimized in order to produce the hydrogels with the highest swelling ratio, gel fraction, cephalexin load and, s. aureus growth inhibited zone, using multiple goal optimization process. Thus, according to optimization analysis, using the highest contents of either acrylic acid (7.8 g) or silver nitrate (1.5 g) at less microwave exposure time (1 min) can give the hydrogels with the maximum highest swelling ratio (655%), gel fraction (> 0.99), cephalexin load (> 0.99) and, s. aureus growth inhibited zone (24.97 mm).
For confirming the presented models, three samples were prepared in obtained optimum conditions (acrylic acid = 7.8 g, silver nitrate = 1.5 g and microwave exposure time = 1 min), and were quantified. The swelling degree, gel fraction, cephalexin load and antibacterial clear zone diameter of these samples were 650.5 ± 14.5%, 0.98 ± 0.07, 0.99 ± 0.03% and, 26 ± 2 mm, respectively. The insignificant differences between the experimental data and predicted ones by model (655%, 0.99, 0.99% and 24.97 mm, in turn), confirmed the suitability and correctness of models.
3.5. Complementary characterizations of optimum sodium alginate/acrylic acid composite hydrogels conjugated to silver nanoparticles
3.5.1. The absorption and release kinetics of cephalexin by optimum sodium alginate/acrylic acid composite hydrogels conjugated to silver nanoparticles
The absorption or release of bioactive compounds by/from most hydrogels are occurred mostly due to diffusion phenomena. However, the absorption/release profile for a swelled composite hydrogels are complex, depending on the relative diffusion rates of either water or bioactive compound from/toward hydrogel networks. If the diffusion into the hydrogel takes place in gentle rates as compared to the hydrogel chains’ relaxation, the process will be controlled by diffusion. Otherwise, the process will be relaxation-controlled. The irregular pattern between the mentioned states is known as Non-Fickian Diffusion.
In order to determine the cephalexin absorption kinetic model of optimum composite hydrogel, they were immersed into an aqueous solution containing certain amount of cephalexin (0.25 g/L), for 5 h, and cephalexin content of aqueous media was measured at fixed time intervals. The results were correlated to common kinetics models namely, zero-order, first-order, second-order, Michaelis-Menten and logarithmic models. The linearized model coefficients and coefficient of determination (R2) for each model were calculated. These results were shown in Table 3. Due to the highest obtained R2 for zero order kinetics, it can be concluded that the cephalexin absorption by synthesized optimum composite hydrogels obeyed zero-order kinetic model. Thus, this model was chosen as the best model to describe absorption behavior of optimum hydrogel for cephalexin at its studied concentrations. In zero-order kinetics, the absorption only depends on time and is constant at various concentrations of active compound. The absorption occurs rapidly until the saturation concentration at equilibrium state reaches (Bhasarkar and Bal 2019).
The release kinetics of cephalexin from optimum hydrogels (into deionized water, pH = 7.0 ± 0.1) were also evaluated after completely saturation and removal of accumulated surface molecules of cephalexin. The cephalexin contents of deionized water were measured at certain time intervals. Similar to cephalexin absorption survey, the results were correlated to common kinetics models and the obtained linearized model coefficients and coefficient of determination (R2) for each model were summarized in Table 4. From Table 4 and obtained R2 values for each model, due to the greatest R2 for Higuchi model, it can be concluded that the release of cephalexin from the synthesized hydrogels, follows this model. Based on Higuchi model, the release is considered as diffusion process based on Fick’s law. The diffusion occurs at microscopic or molecular scale through the hydrogel networks and the diffusion rate decreases as the release process continues, since the drug should pass elongated path in order to reach the media and be released. The Higuchi model is valid for most of the water-soluble or water-insoluble drugs which are released from semi-solid matrices like hydrogels (Peppas and Narasimhan 2014).
Table 4
The kinetics of cephalexin release from optimum composite hydrogels (the R2 and main coefficients for correlated general release models)
|
Common release Models
|
|
Cephalexin release
|
|
C-C0=-Kt
Linear
|
Equation
|
C = 0.1009t + 0.0741
|
Zero order
|
R2
|
0.9514
|
|
Ln C-LnC0 = Kt
1st order
|
Equation
|
LnC = 0.112t-0.8406
|
First order
|
R2
|
0.7979
|
|
C-C0 = Kt 0.5
|
Equation
|
C = 0.3522t (0.5)-0.173
|
Higuchi Model
|
R2
|
0.9977
|
|
C 1/3-C0 1/3=Kt
|
Equation
|
C (1/3) = 0.0533t + 0.5849
|
hixson crowell
|
R2
|
0.735
|
|
Ln C-LnC0 = K Ln t
Logarithmic
|
Equation
|
LnC = 0.8183ln t + 1.8078
|
korsmeyer peppas model
(power law model)
|
R2
|
0.9872
|
3.5.2. FT-IR and UV-Vis absorbance Spectrum of optimum hydrogels
The FT-IR spectra of the optimum sodium alginate/acrylic acid composite hydrogels conjugated to silver nanoparticles was shown in Fig. 5A. The absorption spectrum from 3950 to 3500 cm− 1 was pointed out to stretching sodium alginate NH2. The peak at 3431 cm− 1 was related to the hydroxyl group. The broad peak from 3350 to 2500 cm− 1 indicated the presence of OH scratching of acrylic acid. The peaks at 1634 and 1433 cm− 1 were appeared respectively due to symmetric carboxylate group and asymmetric sodium alginate polymer group. The low peak at 1500 cm− 1 was assigned to stretching C-O. The peaks from 1650 to 1750 cm− 1 were corresponded to stretching carbonyl group of acrylic acid. The broadband from 1300 to 1000 cm− 1 was referred to the C-O group of alginate. The peak at 950 cm− 1 was related to O-H bonding of hydrogel monomers.
Figure 5B shows the UV–Vis absorbance spectra of optimum composite hydrogels conjugated to silver nanoparticles. The observed peak at about 400 nm was related to the surface Plasmon resonance of silver nanoparticles. Thus, the formation of silver nanoparticles from silver ions inside the hydrogels matrices was confirmed (Singh and Dhaliwal 2020).
3.5.3. SEM images of optimum hydrogels
The surface morphology of optimum hydrogel was visualized by scanning electron microscopy images, which were taken in different scales, from 100 µm to 500 nm (Fig. 6). The hydrogels’ SEM images obviously showed the relatively wide sized distribution of produced silver nanoparticle on the surface of the hydrogel. The presence of fine pores on hydrogels’ surface, which can be seen in TEM images, made them efficient candidate for uptakind the various bioactive compounds. The energy dispersive x-ray spectrum of samples also confirmed the presence of Ag, Ca, Cl, Na, N, O and C on the surface of prepared hydrogels.