*2.6. Rheological Analysis*

The hydrogels were evaluated to determine the viscoelastic parameters G0 and G00. The elastic modulus (G0 ) shows the elasticity of the crosslinked bonds and the total elasticity of the material, while the loss or viscous modulus (G") provides a perspective on the viscosity of the material. The ratio between G0 and G00 indicates the total resistance to deformation of the material [46].

The mechanical properties of hydrogels are important when selecting material for biomedical and other applications. They depend on the composition of the hydrogel and its water content. Hydrogels with a higher water content generally have a better permeability and biocompatibility [47]. However, there are some disadvantages, as a high degree of swelling is accompanied by a decrease in mechanical strength. For many applications, the combination of a high swelling degree and good mechanical properties is very important. Many approaches have been used to improve the mechanical properties of hydrogels, including the copolymerization of hydrophilic and hydrophobic monomers, increasing the crosslinking density, and varying the polymerization conditions [48].

Figure 8 shows the variation of the elastic and viscous (G0 and G00) moduli as a function of angular frequency (ω) and the absorbed dose for the XGCMCGO hydrogels. We observed that G0 was greater than G00 for all the hydrogel compositions. This is a critical requirement for hydrogels, as a G0 greater than the G00 suggests that the hydrogel has a higher elastic behavior [49].

Rheological analysis confirms the crosslinking or degradation processes that occur depending on the composition of the polymer mixture. Moreover, the effect of irradiation at various irradiation doses is highlighted, and the G0 and G" is dependent on the composition of the hydrogel and the absorbed dose. A rheological analysis was performed on swollen hydrogel samples. The determined values of G0 and G" are shown in Table 4.

**Figure 8.** Elastic modulus (G0 ), as a function of the angular frequency (ω), of the (**a**) XGCMCGO (50:50), (**b**) XGCMCGO (80:20), and (**c**) XGCMCGO (70:30) hydrogels and their evolutions as a function of the absorbed dose. Viscous modulus (G"), as a function of the angular frequency (ω), of the (**d**) XGCMCGO (50:50), (**e**) XGCMCGO (80:20), and (**f**) XGCMCGO (70:30) hydrogels, and their evolutions as a function of the absorbed dose.

For the XGCMCGO (80:20) hydrogel, the G0 increased with an increase in the absorbed dose, with a maximum value for G0 = 1052 Pa at 15 kGy. The XGCMCGO (50:50) hydrogel had a G0 = 869 Pa at 2.5 kGy. When this polymeric blend was irradiated with 15 kGy, the G0 decreased very drastically down to 12 Pa. In the case of the XGCMCGO (70:30) hydrogel, G0 = 913 Pa was obtained at 2.5 kGy, and at 15 kGy, G0 = 766 Pa.

The G0 decreased with the absorbed dose in the cases of the XGCMCGO (50:50) and (70:30) hydrogels. We concluded that the decrease in G0 reflected the reduction of the crosslinking density (νe), while the decrease in G" was due to the inhibition of the viscous behavior, showing a complete degradation of the polymer blend.

The XGCMCGO (80:20) hydrogel had a very well defined elastic behavior depending on the absorbed dose. In a recent study, a superabsorbent hydrogel based on CMC/starch/GO presented a value of G0 = 8050 Pa [50]. In another study, hydrogels obtained from CMC by redox polymerization for soft-tissue healing applications had a value of G0 = 814 Pa [45]. As shown by the rheological analysis and the sol-gel analysis, the XGCMCGO (80:20) hydrogel had the best properties.

### **3. Conclusions**

We prepared novel and complex XGCMCGO hydrogels by e-beam crosslinking to be used as a potential substrate in biomedical engineering. Irradiation of XGCMC polymeric blends with e-beams proved to be a suitable technique, especially when GO was incorporated into their composition. Irradiation of polymeric mixtures with a wide range of doses allowed us to obtain hydrogels with different properties.

XGCMCGO hybrid hydrogels had better mechanical strength compared to the pure CMC hydrogel, and a better flexibility and improved swelling behavior than the simple XG hydrogel.

The gel fraction and the swelling properties of the prepared hydrogels depended on the composition of the polymers and the absorbed dose.

The hydrogels showed superabsorbent capacity and reached equilibrium in less than 6 h, especially the XGCMCGO (70:30) hydrogel, which reached the highest swelling degree of about 6000%, at 15 kGy. The crosslinking process predominated compared to the degradation process.

By characterizing the network structure, we observed that the hybrid hydrogels with high XG concentration showed the best structural properties. The crosslinking density increased with the absorbed dose.

The mesh size obtained from the experimental data of rheological analysis was in the range of 123–210 nm; these values were comparable to those of several hydrogels.

The interaction between the hydrogel components was highlighted by FT-IR analysis, and the increase in absorption band intensities for the characteristic functional group, as well as their shifting toward lower wavenumbers, were correlated with the crosslinking degree, which decreased at a dose of 15 kGy.

For all hydrogel compositions, G0 > G00, thus suggesting that the hydrogels had a higher elastic behavior. The G0 had values in a wide range of 12–1052 Pa, with the maximum value obtained at a dose of 15 kGy for the XGCMCGO (80:20) hydrogel composition.

Therefore, the prepared XGCMCGO superabsorbent hydrogels belong to a class of environmentally friendly materials, and might have potential practical applications in many areas, such as in biomedical engineering and hygienic products.

### **4. Materials and Methods**

### *4.1. Materials*

Sodium carboxymethylcellulose (CMC, Mw = 2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> g/mol), *<sup>N</sup>*0*N*-methylenebis-acrylamide (NMBA 99%, Mw = 154.17 g/mol), acrylic acid anhydrous 99% containing MEHQ as inhibitor (AA, Mw = 72.06 g/mol), and NaOH were purchased from Merck KGaA, Darmstadt, Germany. A commercial xanthan gum (XG—food grade, produced by Jungbunzlauer, Wien, Austria) in powder form with a molecular weight of Mw = 1.6 <sup>×</sup> <sup>10</sup><sup>6</sup> g/mol was used. Ultra-highly concentrated single-layer graphene oxide (6.2 g/L) was purchased from Graphene Laboratory Inc., New York, NY, USA.

### *4.2. Synthesis of Hydrogels and E-Beam Irradiation*

In this experiment, three different hydrogels based on XGCMCGO with different content of XG and CMC in the presence of AA, NaOH, and NMBA were prepared. XG (5 wt %) and CMC (2 wt %) were dissolved in DI water at room temperature. After complete solubilization of the XG and CMC, they were mixed with each other in different compositions. For each XG:CMC ratio, 75 mL of the mixture was prepared. The ratios of XG:CMC in the mixtures were 50:50; 80:20, and 70:30. For a better understanding of the experiments, sample compositions are presented in Table 5.


**Table 5.** Sample composition details.

Then, 15 mL of each homogeneous solution of XGCMCGO was packed in a hermetically sealed polyethylene zip bag and subjected to e-beam irradiation at predetermined doses (2.5–15 kGy). The e-beam sample irradiation was performed in air at room temperature (25 ◦C) using a linear electron accelerator (National Institute for Laser, Plasma and Radiation Physics, Măgurele, Romania) at a fixed beam energy of 6 MeV (average beam current of 10 µA, pulse length of 3.75 µs, pulse repetition rate of 50 Hz, and average dose rate of 1 kGy/min) [51]. The dosimetry was performed using graphite calorimeters.

### *4.3. Sol-Gel Analysis*

The hydrogel samples were dried in a vacuum oven to a constant weight and then immersed in DI water for 48 h at room temperature (25 ◦C). After 48 h, the swollen hydrogels were removed from the water and dried at 30 ◦C to a constant weight. The gel fraction (GF) and soluble fraction (s) were calculated as follows:

$$\mathbf{G}(\%) = \begin{pmatrix} \mathbf{W\_d} \\ \overline{\mathbf{W\_i}} \end{pmatrix} \tag{1}$$

$$\mathbf{s} = \mathbf{1} - \mathbf{G} \tag{2}$$

where W<sup>i</sup> is the initial weight of dried sample after irradiation, W<sup>d</sup> is the weight of the dried insoluble part of sample after immersion for 48 h, and s is soluble fraction of the polymer. All measurements were carried out in triplicate for each sample, and all values were expressed as mean value and standard deviation of three independent samples.

The gelation doses (the doses necessary to produce the first insoluble gel fraction) and the degradation vs. crosslinking ratios for the XGCMCGO hydrogels were calculated using a customized computer program for sol-gel analysis (Gelsol95), which was based on the Charlesby–Rosiak formula [26]:

$$\mathbf{s} + \sqrt{\mathbf{s}} = \frac{p\_0}{q\_0} + \left(\mathbf{2} - \frac{p\_0}{q\_0}\right) \left(\frac{\mathbf{D\_V} + \mathbf{D\_g}}{\mathbf{D\_V} + \mathbf{D}}\right) \tag{3}$$

where *p*<sup>0</sup> is the degradation density (i.e., average number of main chain scissions per monomer unit and per unit dose), *q*<sup>0</sup> is the crosslinking density (i.e., fraction of monomer units crosslinked per unit dose), D is the absorbed dose (kGy), D<sup>g</sup> is the gelation dose (kGy), and D<sup>V</sup> is the virtual dose (kGy) (the dose necessary to transform the real sample into a sample with the molecular weight distribution of Mw/M<sup>n</sup> = 2).

The radiation yields of crosslinking and degradation (scission) were calculated using the following equations:

$$\mathbf{G(X)} = \frac{4.9 \cdot 10^2 \cdot \mathbf{c}}{\mathbf{M}\_{\mathbb{C}} \cdot \mathbf{D} \cdot \boldsymbol{\rho}} \tag{4}$$

$$\mathbf{G(S)} = \mathbf{G(X)} \cdot \mathbf{2} \frac{p\_0}{q\_0} \tag{5}$$

where G(X) is the radiation yield of crosslinking (expressed as number of moles of crosslinking bonds per Joule), G(S) is the radiation yield of chain scission (mol/J), M<sup>C</sup> (kg/mol) is the average molecular weight between two successive crosslinks, c (g/L) is the polymer concentration in irradiated solution, D is absorbed dose (J/kg), and ρ (kg/m<sup>3</sup> ) is the polymer density [52].

### *4.4. Swelling Degree*

The swelling properties of hydrogels were explored by placing the dried hydrogels in DI water at room temperature for 48 h to reach swelling equilibrium. At specified times, the swelled hydrogels were taken out of the distilled water, blotted with paper, weighed, and immersed again.

The swelling degree (SD(%)) was calculated as a function of the dry (Wd) and swollen (Ws) hydrogel weights using Equation (6) [53]:

$$\text{SD}(\%) = \frac{(\text{W}\_{\text{s}} - \text{W}\_{\text{d}})}{\text{W}\_{\text{d}}} \cdot 100 \tag{6}$$

### *4.5. Diffussion of Water*

The most basic law of Fick's was used for the explanation of swelling kinetics and diffusion of the polymeric structures. The following equation was used to determine the nature of diffusion of water into hydrogels [54]:

$$\mathbf{F} = \frac{\mathbf{M\_{\!\!\! }}}{\mathbf{M\_{\!\!\! }}} = \mathbf{k} \mathbf{t}^{\prime} \tag{7}$$

where F is the fraction of swelling due to the water uptake, M<sup>t</sup> is the adsorbed water at time t, M<sup>∞</sup> is the adsorbed water at equilibrium, k is a proportionality constant, and *n* is the diffusional exponent. The first 60% of the water uptake data were fitted to Equation (7), and the corresponding values of k and n were obtained.

### *4.6. ATR-FTIR Spectroscopy*

The changes in chemical structure of the crosslinked hydrogels were investigated. ATR-FTIR spectra of unirradiated and irradiated samples were taken with a PerkinElmer Spectrum 100 FTIR Spectrometer. The samples for FTIR analysis were first dried in a vacuum oven at 30 ◦C for 72 h. The samples were subjected to wavenumbers ranging from 4000 to 600 cm−<sup>1</sup> at ambient temperature and a resolution of 4 cm−<sup>1</sup> , averaged from 50 scans/sample.

### *4.7. Characterization of Network Structure*

Network parameters of the XGCMCGO hydrogels, such as the average molecular weight between two crosslinks (Mc), crosslinking density (νe), and mesh size (ξ), were determined by using the swelling and rheological measurements. Using elastic modulus (G0 ) values determined from rheological measurements and based on the rubber elasticity theory, Mc could be determined using the following equation [55]:

$$\mathbf{M}\_{\mathbf{C}} = \frac{\mathbf{A}\rho \mathbf{R} \mathbf{T} (\mathbf{v\_{2r}})^{2/3} (\mathbf{v\_{2s}})^{1/3}}{\mathbf{G}'} \tag{8}$$

where R is the universal gas constant (8.314 m<sup>3</sup> Pa/molK), T is the absolute experimental temperature (298.15 ◦K), ν2r is the polymer volume fraction after e-beam crosslinking, ν2s is the polymer volume fraction of the crosslinked hydrogel in swollen state, ρ (kg/m<sup>3</sup> ) is the polymer density, and the factor A equals 1 for an affine network and 1–2/φ for a phantom network.

The effective crosslink density (νe) of the hydrogels was calculated using Equation (9):

$$\mathbf{v\_e} = \frac{\rho}{\mathbf{M\_{C}}} \tag{9}$$

The polymer volume fractions (ν2r and ν2s) were determined using Equation (10):

$$\mathbf{v\_{2r(s)}} = \frac{\left[1 + \left(w\_{2r(s)} - 1\right) \cdot \rho\_{\text{hydoted}}\right]^{-1}}{\rho\_{\text{solvent}}} \tag{10}$$

where ρhydrogel and ρsolvent are the densities of the hydrogel and solvent (kg/m<sup>3</sup> ), respectively; and *w*2r(s) is the weight of the hydrogel after e-beam crosslinking after swelling (g). The weight swelling ratio of hydrogels after crosslinking (*w*2r) was calculated as: *w*2r = hydrogel mass after irradiation/hydrogel dry mass. The weight swelling ratio of hydrogels after swelling (*w*2s) was calculated as: *w*2s = hydrogel mass after swelling/hydrogel dry mass.

The mesh size of the polymer network (ξ) was determined using Equation (11) [56]:

$$\boldsymbol{\xi} = \mathbf{v}\_{2s}^{-1/3} \cdot \left[ \mathbf{C}\_{\mathbf{n}} \left( \frac{2 \mathbf{M}\_{\mathbf{C}}}{\mathbf{M}\_{\mathbf{r}}} \right) \right]^{-1/2} \cdot \mathbf{l} \tag{11}$$

where C<sup>n</sup> is the Flory characteristic ratio, M<sup>r</sup> is the average molecular weight of the repeating unit, and l is the carbon–carbon bond length (0.154 nm).

### *4.8. Dynamic Rheological Measurements*

Dynamic rheological measurements of the hydrogels were performed by employing an MFR 2100 Micro Fourier Rheometer (GBC, Australia) equipped with a home-made temperature control jacket connected to a Lauda E100 circulating water bath.

The operating parameters of the instrument during rheological investigation were as follows: gap between plates—400 µm; displacement amplitude—0.03 µm (to fall into the linear viscoelasticity domain); frequency domain—0.005–2.000 Hz (with a step of 0.005 Hz, which led to angular frequencies, in rad/s, of 2π times higher than the corresponding frequencies taken in Hz); equilibration time for each of the isothermal measurements—20 min; and 30 scans per rheogram.

The dynamic rheological parameters of storage modulus (G0 ) and loss modulus (G00) were determined to evaluate the stability of the hydrogel network. All rheological measurements were performed in triplicate at the same constant temperature of 23 ◦C, and all values were expressed as mean value and standard deviation.

**Author Contributions:** Conceptualization, I.C., M.D. and A.S.; methodology, I.C. and M.D.; investigation, I.C., M.D., A.S. and M.M.; data curation, I.C., M.D. and M.M.; writing—original draft preparation, I.C.; writing—review and editing, M.D. and A.S.; supervision, M.D. and A.S.; visualization, M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Romanian Ministry of National Education and Research, by Installations and Special Objectives of National Interest, and by the Nucleu LAPLAS VI Program (Contract No. 16N/08.02.2019). The APC was funded by Installations and Special Objectives of National Interest.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

