*2.4. Characterization of Fe3+-Crosslinked Layered SG/CMC IPN Hydrogels*

The FTIR spectra of each sample were taken using an FTIR spectrometer (Spectrum Two FTIR, Perkin Elmer), and obtained with a resolution of 0.5 cm−1 using 8 scans and a wavenumber range of 4000–600 cm−1 . All samples were mixed with KBr powder in a certain ratio (1:100) and then compressed to form pellets.

### *2.5. Thermogravimetric Analysis (TGA)*

The hydrogels were washed by soaking in an excess of 70% ethanol for 2 days and were then dried at 60 ◦C and lyophilized. Lyophilized hydrogels were grinded in a blender. Thermogravimetric Analysis (TGA) was performed using a Perkin Elmer Pyris1 thermogravimetric analyzer. The thermal analyzer was operated under nitrogen atmospheric pressure and regulated by compatible PC commands. The dried sample (10 mg) was placed in a crucible and heated in an enclosed system with a linear temperature increase at a rate of 10 ◦C/min over a temperature range of 25 to 600 ◦C.

### *2.6. Field Emission Scanning Electron Microscopy (FE-SEM) Analysis*

The cross-sectional morphology of SG/CMC IPN hydrogels were observed using FE-SEM (JSM-7800F Prime, JEOL Ltd., Akishima, Japan). The samples were quickly frozen and then lyophilized for 24 h. For observation, the surface of cross-sectioned hydrogel was coated with a thin layer of platinum at 10 mA for 60 s in a vacuum. SEM/EDS analysis of hydrogels was performed with FE-SEM (AURIGA, Carl Zeiss, Oberkochen, Germany) with an Energy selective Backscattered (EsB) detector. All hydrogels were platinum-coated at 10 mA, 120 s before examination using field emission scanning electron microscope.

### *2.7. Compression Test*

Compression test was performed by using Instron E3000LT (Instron Inc., Norwood, MA, USA) by preparing a hydrogel disk with a height of 15 mm and a diameter of 20 mm. The sample was placed on a plate and compressed at a rate of 5 mm/min. Compressive stress was recorded when the sample was compressed with a strain of 70%. The measurement was performed in triplicate. All the hydrogels used in the measurements were prepared in the same manner to the rheological analysis.

### *2.8. Rheological Experiments*

The rheological properties of the hydrogel were analyzed by oscillating angular frequency sweep and temperature ramp tests using a DHR-2 rheometer (TA Instruments, New Castle, DE, USA) equipped with a 20-millimeter parallel plate. The angular frequency was swept from 0.1 to 100 rad/s at a strain of 0.5% at 25 ◦C. The hydrogel samples were prepared as disks and were analyzed with a parallel plate measuring 20 mm in diameter, and the gap between plates was adjusted to 1.3 mm. A stress strain amplitude sweep test was conducted on samples from 0.1% to a maximum strain of 100% at 1.0 Hz to determine the limit of the linear viscoelastic region. All hydrogel samples used for the measurement were prepared with the same mold and the same amount of water. Each measurement was performed in triplicate. The temperature ramp test was conducted at a constant angular frequency of 10 rad/s and a constant strain of 1.0%. It was also carried out at 10 to 70 ◦C with an insulating cover to maintain the temperature.

### *2.9. Equilibrium Swelling Ratio and pH Sensitivity Measurements*

The swelling properties of SG/CMC IPN hydrogels were investigated in various pH buffer solutions (pH 2, 4, 6, 8, 10). Each of the dried SG/CMC gels were immersed in pH buffer solutions at 37 ◦C to induce an equilibrium state. Then, the mass of the swollen hydrogel was weighed at various times after removing excess surface water. The swelling ratio was determined using following equation:

$$\text{Swelling ratio } \text{(\%)} = \frac{\text{w}\_{\text{s}} - \text{w}\_{\text{d}}}{\text{w}\_{\text{s}}} \tag{1}$$

where ws is the weight of the swollen hydrogel and wd is the weight of the dried hydrogel in a vacuum oven prior to PBS immersion. After separating the hydrogel from the solution for accurate weight measurement, the solution remaining on the surface was gently wiped off using a laboratory tissue. Each measurement was performed in triplicate.

### *2.10. Drug Loading and Drug Release*

5-Fluorouracil (5-FU) was used as the model drug to analyze drug release properties of SG/CMC IPN gels in different pH conditions. To put it simply, 1 mg of 5-FU was dissolved in 1 mL of SG/CMC polymer solution. The concentration of 5-FU in the hydrogels was fixed at 1 mg/mL. The obtained hydrogels were incubated in 40 mL of various pH buffer solutions (pH = 1.2, 7.4) at 37 ◦C with 50 rpm of constant stirring. At each interval time points, 500 µL of released medium was taken out for UV–Vis spectroscopic analysis and then same volume of fresh solution was added. The concentrations of 5-FU were analyzed using a spectrophotometer (UV2450, Shimadzu Corporation, Kyoto, Japan) at a wavelength of 266 nm. The cumulative amount of the drug was calculated by the following equation:

$$\text{Cumulative amount of the drug} = \text{C}\_{\text{n}}V + \sum\_{i=1}^{i=n-1} \text{C}\_{i}V\_{i} \tag{2}$$

where *V* is the release of medium volume, *Vi* is the sampling volume, and *Cn* and *Ci* are the 5-FU concentrations in the release medium and extraction sample. All measurements were conducted in triplicate.

### *2.11. In Vitro Cytotoxicity*

The cytotoxicity of the hydrogels was evaluated using WST-8 assays using human embryonic kidney 239 cells (HEK-293, Korean Cell Line Bank, Korea). For direct cytotoxicity tests, HEK-293 cells were seeded into 24-well culture plates at a concentration of 3 × 104 cells per well with minimum essential medium (MEM, WELGENE, Gyeongsan-si, Korea) containing 10% fetal bovine serum and 1% penicillin/streptomycin; then, 5 mg of hydrogel sample was added to the wells and plates were incubated at 37 ◦C in an atmosphere containing 5% CO2. MEM medium was used as a negative control, and 10% (*v*/*v*) dimethylsulfoxide (DMSO) dissolved in the MEM medium was used as a positive control. After a 48-hour incubation, WST-8 assay reagent (QuantiMax, BIOMAX, Seoul, Korea) was added to each well and absorbance was measured at 450 nm. Cell viability was determined using the following formula:

$$\text{Cell viability } (\%) = \frac{\text{Absorbance of cells with hydrogen}}{\text{Absorbance of negative control cells}} \tag{3}$$

All assays were repeated in triplicate for each sample.

### **3. Result and Discussion**

### *3.1. Characterization of SG/CMC IPN Hydrogels*

The coordination of the Fe3+-crosslinked layered SG/CMC IPN hydrogels were confirmed through FTIR analysis. Figure 1 shows the FTIR spectra of SG, CMC, and S1C1 gels, and Table 2 shows the shift of the characteristic absorption peaks of the FTIR spectra. Here, the coordination of SxCx with Fe3+ ions was explained by S1C1 gel, and similar peak shifts occurred in the other SxCx gels. The FTIR spectrum of succinoglycan exhibited absorption peaks at 3326 cm−1 , corresponding to the –OH stretching bands, and the C=O stretching carbonyl esters of the acetate group showed absorption peaks at 1728 cm−1 [36–38]. Additionally, the absorption peaks at 1629, 1382, and 1074 cm−1 were attributed to the asymmetric C=O stretching vibration of the succinate and pyruvate functional groups, the symmetric stretching vibration of the carboxylate –COO– group in the acid residue, and the asymmetric C-O-C stretching vibration, respectively [36,37]. The FTIR spectrum of CMC showed a broad absorption band at 3408 cm−1 , which was related to the stretching frequency of the –OH group, and the peaks at 1625, 1422, and 1076 cm−1 were related to the asymmetric, symmetric stretching vibrations of the carboxylate groups, and C–C bending of CMC, respectively [39,40].

**Figure 1.** FTIR spectra of SG gel, S1C1 gel, and CMC gel. **Figure 1.** FTIR spectra of SG gel, S1C1 gel, and CMC gel.

2.

C=O stretch

However, when the Fe3+ ions are crosslinked with the polymer, the S1C1 gel absorption peaks shifted to 3433, 1600, and 1049 cm−1 , respectively. When the polymer coordinated with the Fe3+ ions, the –OH stretching band of the polymer mixture significantly red shifted to 3433 cm−1. This proves that the –OH group and Fe3+ ion of the polymer mixture are coordinated [41]. In addition, the specific peak associated with the carboxyl group was shifted to 1600 cm−1, and the asymmetric C-O-C stretching peak by backbone sugar shifted to 1049 cm−1. The shift of these peaks is also due to the coordination of the carboxyl group of the polymer mixture with the Fe3+ ions [42,43]. In conclusion, the Fe3+ ion was strongly coordinated by hydrogen bonds between the hydroxyl groups and carboxyl groups of SG and CMC. A schematic representation of the presumable coordination mechanism of the However, when the Fe3+ ions are crosslinked with the polymer, the S1C1 gel absorption peaks shifted to 3433, 1600, and 1049 cm−1 , respectively. When the polymer coordinated with the Fe3+ ions, the –OH stretching band of the polymer mixture significantly red shifted to 3433 cm−1 . This proves that the –OH group and Fe3+ ion of the polymer mixture are coordinated [41]. In addition, the specific peak associated with the carboxyl group was shifted to 1600 cm−1 , and the asymmetric C-O-C stretching peak by backbone sugar shifted to 1049 cm−1 . The shift of these peaks is also due to the coordination of the carboxyl group of the polymer mixture with the Fe3+ ions [42,43]. In conclusion, the Fe3+ ion was strongly coordinated by hydrogen bonds between the hydroxyl groups and carboxyl groups of SG and CMC. A schematic representation of the presumable coordination mechanism of the SG and CMC polymer solutions and FeCl3 based on the above findings is shown in Figure 2.

SG and CMC polymer solutions and FeCl3 based on the above findings is shown in Figure

**)**

1600

**SG CMC S1C1**

OH stretch 3326 3408 3433 [36,37,39,41]

Carboxymethyl ether - 1625 [40]

asymmetric C-O-C stretch 1074 1076 1049 [36,37,44]

**Reference**

**Table 2.** Shift of the main peaks in the FTIR spectra of SG gel, S1C1 gel, and CMC gel.

**Peak Assignment Peak Wavenumber (cm−1**

Acetate 1728 -

**Table 2.** Shift of the main peaks in the FTIR spectra of SG gel, S1C1 gel, and CMC gel.

**Figure 2.** Schematic illustration of model of SG/CMC IPN hydrogels formation mechanism. **Figure 2.** Schematic illustration of model of SG/CMC IPN hydrogels formation mechanism.

#### *3.2. Thermogravimetric Analysis (TGA) and Derivative Thermogravimetry (DTG) 3.2. Thermogravimetric Analysis (TGA) and Derivative Thermogravimetry (DTG)*

Figure 3 illustrates thermogravimetric analysis (TGA) and the derivative thermogravimetry (DTG) curves of SG gel, S1C1 gel, and CMC gel. As observed, the mass loss seen near the initial 120 °C in all the hydrogels was due to the water evaporation in the hydrogel [26,45]. The SG gel exhibited a 43.4% mass loss in the temperature range of 211–397 °C, and the major DTG picks appeared twice at 264 and 509 °C. The second phase was related with the degradation of the thermally stable structure formed by cross linkage and strong bonds in succinoglycan [17]. Additionally, the weight of the CMC gel decreased significantly between 220 and 495 °C. Thereafter, the maximum DTG peak was observed at 278 °C in the DTG curve, and the degradation steps in this range of CMC gel occurred due to the cellulose backbone cleavage and fragmentation [46]. In comparison, the weight loss in the S1C1 gel was observed at 210 and 392 °C, with maximum DTG peaks at 247 Figure 3 illustrates thermogravimetric analysis (TGA) and the derivative thermogravimetry (DTG) curves of SG gel, S1C1 gel, and CMC gel. As observed, the mass loss seen near the initial 120 ◦C in all the hydrogels was due to the water evaporation in the hydrogel [26,45]. The SG gel exhibited a 43.4% mass loss in the temperature range of 211–397 ◦C, and the major DTG picks appeared twice at 264 and 509 ◦C. The second phase was related with the degradation of the thermally stable structure formed by cross linkage and strong bonds in succinoglycan [17]. Additionally, the weight of the CMC gel decreased significantly between 220 and 495 ◦C. Thereafter, the maximum DTG peak was observed at 278 ◦C in the DTG curve, and the degradation steps in this range of CMC gel occurred due to the cellulose backbone cleavage and fragmentation [46]. In comparison, the weight loss in the S1C1 gel was observed at 210 and 392 ◦C, with maximum DTG peaks at 247

and 523 °C. The weight loss in this range was 38.9%, showing a more moderate weight

observed for SG gel and CMC gel at 386 and 323 °C, respectively, whereas a 50% weight loss was observed for S1C1 gel at 398 °C. Therefore, it was found that the thermal stability of the hydrogel was increased through IPN formation in the process of coordinating SG and CMC with Fe3+. This is the first report to increase the thermal stability of a non-beaded

CMC-based hydrogel using succinoglycan, an unmodified natural polysaccharide.

and 523 ◦C. The weight loss in this range was 38.9%, showing a more moderate weight loss at high temperatures, and it was shown that the formation of a hydrogel with metals reduced the skeletal decomposition of SG and CMC. In addition, a 50% weight loss was observed for SG gel and CMC gel at 386 and 323 ◦C, respectively, whereas a 50% weight loss was observed for S1C1 gel at 398 ◦C. Therefore, it was found that the thermal stability of the hydrogel was increased through IPN formation in the process of coordinating SG and CMC with Fe3+. This is the first report to increase the thermal stability of a non-beaded CMC-based hydrogel using succinoglycan, an unmodified natural polysaccharide. *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 15 *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 15

**Figure 3.** (**a**) TGA and (**b**) DTG curves of SG/CMC IPN gel. **Figure 3.** (**a**) TGA and (**b**) DTG curves of SG/CMC IPN gel. **Figure 3.** (**a**) TGA and (**b**) DTG curves of SG/CMC IPN gel.

### *3.3. Compression Mechanical Properties of SG/CMC IPN Hydrogels 3.3. Compression Mechanical Properties of SG/CMC IPN Hydrogels 3.3. Compression Mechanical Properties of SG/CMC IPN Hydrogels*

Compression tests were performed to investigate the effect of SG on the mechanical properties of Fe3+ crosslinked layered SG/CMC IPN gels. Figure 4 shows the compressive strain curves. Compared to CMC only gels, SG/CMC IPN gels exhibit a higher compressive stress. As the ratio of SG content from S1C3 to S3C1 increased, the compressive stress of SG/CMC IPN gels improved (S3C1 gel: 0.0483 MPa > S1C1 gel: 0.0412 MPa > S1C3 gel: 0.0128 MPa > CMC gel: 0.0057 MPa). This result is exhibited because SG is able to maintain its physical consistency, even under extreme conditions [19,20]. It has been reported that the addition of SG or modified-SG increases the mechanical strength of the hydrogel [17,47]. Therefore, these results indicate that SG effectively enhanced the mechanical strength of the Fe3+ crosslinked layered SG/CMC IPN gels. It has been reported to increase the mechanical strength of CMC-based hydrogels using synthetic polymers or modified polymers [26,27,29]. However, it is significant that the mechanical strength of CMC-based hydrogel as a non-bead form was increased by using unmodified natural polysaccharide. Compression tests were performed to investigate the effect of SG on the mechanical properties of Fe3+ crosslinked layered SG/CMC IPN gels. Figure 4 shows the compressive strain curves. Compared to CMC only gels, SG/CMC IPN gels exhibit a higher compressive stress. As the ratio of SG content from S1C3 to S3C1 increased, the compressive stress of SG/CMC IPN gels improved (S3C1 gel: 0.0483 MPa > S1C1 gel: 0.0412 MPa > S1C3 gel: 0.0128 MPa > CMC gel: 0.0057 MPa). This result is exhibited because SG is able to maintain its physical consistency, even under extreme conditions [19,20]. It has been reported that the addition of SG or modified-SG increases the mechanical strength of the hydrogel [17,47]. Therefore, these results indicate that SG effectively enhanced the mechanical strength of the Fe3+ crosslinked layered SG/CMC IPN gels. It has been reported to increase the mechanical strength of CMC-based hydrogels using synthetic polymers or modified polymers [26,27,29]. However, it is significant that the mechanical strength of CMC-based hydrogel as a non-bead form was increased by using unmodified natural polysaccharide. Compression tests were performed to investigate the effect of SG on the mechanical properties of Fe3+ crosslinked layered SG/CMC IPN gels. Figure 4 shows the compressive strain curves. Compared to CMC only gels, SG/CMC IPN gels exhibit a higher compressive stress. As the ratio of SG content from S1C3 to S3C1 increased, the compressive stress of SG/CMC IPN gels improved (S3C1 gel: 0.0483 MPa > S1C1 gel: 0.0412 MPa > S1C3 gel: 0.0128 MPa > CMC gel: 0.0057 MPa). This result is exhibited because SG is able to maintain its physical consistency, even under extreme conditions [19,20]. It has been reported that the addition of SG or modified-SG increases the mechanical strength of the hydrogel [17,47]. Therefore, these results indicate that SG effectively enhanced the mechanical strength of the Fe3+ crosslinked layered SG/CMC IPN gels. It has been reported to increase the mechanical strength of CMC-based hydrogels using synthetic polymers or modified polymers [26,27,29]. However, it is significant that the mechanical strength of CMC-based hydrogel as a non-bead form was increased by using unmodified natural polysaccharide.

**Figure 4.** Compressive stress–strain curves of hydrogels with different SG concentrations. **Figure 4.** Compressive stress–strain curves of hydrogels with different SG concentrations. **Figure 4.** Compressive stress–strain curves of hydrogels with different SG concentrations.

hydrogel, the oscillation angular frequency sweep test was performed. The oscillatory angle frequency sweep experiment was conducted from 0.1 to 100 rad/s at a fixed strain of 0.5. As can be seen in Figure 5, the storage modulus value G′ of all the hydrogels is larger

As shown in Figure 5a, to obtain information on the mechanical properties of the hydrogel, the oscillation angular frequency sweep test was performed. The oscillatory angle frequency sweep experiment was conducted from 0.1 to 100 rad/s at a fixed strain of 0.5. As can be seen in Figure 5, the storage modulus value G′ of all the hydrogels is larger

*3.4. Rheological Behavior of SG/CMC IPN Hydrogels*

#### *3.4. Rheological Behavior of SG/CMC IPN Hydrogels Polymers* **2021**, *13*, x FOR PEER REVIEW 9 of 15

As shown in Figure 5a, to obtain information on the mechanical properties of the hydrogel, the oscillation angular frequency sweep test was performed. The oscillatory angle frequency sweep experiment was conducted from 0.1 to 100 rad/s at a fixed strain of 0.5. As can be seen in Figure 5, the storage modulus value G0 of all the hydrogels is larger than the loss modulus value G00. This indicates that the hydrogel is not in a fluid sol state, but a stable solid gel state. Additionally, compared with CMC gel, hydrogel containing SG has a larger storage modulus G0 at all frequencies. Comparing the actual G0 value, the CMC gel is about 1010 Pa. However, the G0 value of the S1C1 gel is 6060 Pa, and the G0 value of the S3C1 gel with the highest SG ratio is 6560 Pa. This is an increase of about 6.0 times and 6.5 times, respectively, compared to the CMC gel. This means that the addition of SG to the CMC-based hydrogel improves the mechanical strength, and the mechanical properties can be adjusted according to the amount of SG. than the loss modulus value G″. This indicates that the hydrogel is not in a fluid sol state, but a stable solid gel state. Additionally, compared with CMC gel, hydrogel containing SG has a larger storage modulus G′ at all frequencies. Comparing the actual G′ value, the CMC gel is about 1010 Pa. However, the G′ value of the S1C1 gel is 6060 Pa, and the G′ value of the S3C1 gel with the highest SG ratio is 6560 Pa. This is an increase of about 6.0 times and 6.5 times, respectively, compared to the CMC gel. This means that the addition of SG to the CMC-based hydrogel improves the mechanical strength, and the mechanical properties can be adjusted according to the amount of SG.

**Figure 5.** Rheological observations from the oscillation angular frequency sweep test: (**a**) Storage modulus (G′, filled symbols) and loss modulus (G″, empty symbols) of hydrogels; (**b**) The loss tangent (tan δ) of hydrogels. **Figure 5.** Rheological observations from the oscillation angular frequency sweep test: (**a**) Storage modulus (G0 , filled symbols) and loss modulus (G00, empty symbols) of hydrogels; (**b**) The loss tangent (tan δ) of hydrogels.

The loss tangent (tan δ) was shown to measure the viscoelastic properties of CMC and SxCx gels (Figure 5b). Figure 5b shows that the SG/CMC IPN gels have low tan δ values at all frequencies. This shows that the gel was stably formed. In particular, the S1C1 gel prepared with a 1:1 ratio of SG and CMC had the lowest tan δ value, indicating the formation of the most mechanically strong hydrogel. Therefore, in subsequent experi-The loss tangent (tan δ) was shown to measure the viscoelastic properties of CMC and SxCx gels (Figure 5b). Figure 5b shows that the SG/CMC IPN gels have low tan δ values at all frequencies. This shows that the gel was stably formed. In particular, the S1C1 gel prepared with a 1:1 ratio of SG and CMC had the lowest tan δ value, indicating the formation of the most mechanically strong hydrogel. Therefore, in subsequent experiments, S1C1 was set as the general SxCx.

ments, S1C1 was set as the general SxCx. Temperature ramp rheological studies of hydrogels were conducted at a constant frequency and strain. In Figure 6, the Tm value where the G″ value is higher than the G′ value did not occur in all the hydrogels. However, it can be seen that the G′ value of the CMC gel became rapidly insignificant. Therefore, the application in various fields is limited. However, in the case of the S1C1 gel with 2% of SG added to the CMC hydrogel, it Temperature ramp rheological studies of hydrogels were conducted at a constant frequency and strain. In Figure 6, the Tm value where the G00 value is higher than the G0 value did not occur in all the hydrogels. However, it can be seen that the G0 value of the CMC gel became rapidly insignificant. Therefore, the application in various fields is limited. However, in the case of the S1C1 gel with 2% of SG added to the CMC hydrogel, it can be seen that the G0 value is clearly maintained. This means that due to the strong mechanical strength of SG, it did not deteriorate during heating to 70 ◦C.

can be seen that the G′ value is clearly maintained. This means that due to the strong me-

chanical strength of SG, it did not deteriorate during heating to 70 °C.

chanical strength of SG, it did not deteriorate during heating to 70 °C.

**Figure 6.** Temperature dependence of hydrogel through rheological observation of the oscillation temperature ramp test. (**a**) Storage modulus (G0 , filled symbols) and loss modulus (G00, empty symbols) of hydrogels. (**b**) The loss tangent (tan δ) of hydrogels. **Figure 6.** Temperature dependence of hydrogel through rheological observation of the oscillation temperature ramp test. (**a**) Storage modulus (G′, filled symbols) and loss modulus (G″, empty symbols) of hydrogels. (**b**) The loss tangent (tan δ) of hydrogels.

### *3.5. Field Emission Scanning Electron Microscopy (FE-SEM) 3.5. Field Emission Scanning Electron Microscopy (FE-SEM)*

The FE-SEM image of the SG/CMC gel cross-section is shown in Figure 7. Figure 7a–c were each hydrogel observed at the same magnification and Figure 7d was the S1C1 gel observed at high magnification. A three-dimensional interconnected porous structure was observed on the cross-section surface of the SG gel. These pores can be related to the degree of cross-linking and mechanical strength [47]. The SEM image of the CMC gel shows an increase in the surface area of the hydrogel due to the coarse, small, and frequent porous structure. This large surface area supports the superabsorbency of CMC-based hydrogels by facilitating water molecules to diffuse into the polymer network [48]. On the other hand, the S1C1 gel shows both the interconnected porous structure of the SG gel and the frequent porous structure of the CMC gel. Figure 7d shows the structure of the same S1Cl gel when viewed at a relatively high magnification. As shown in Figure 7d, the layered structure was shown, which may be due to the diffused Fe3+ ions cross-linked with the polymer in each layer during SG/CMC IPN gel formation. Fe3+ ions form a first cross-link at the interface between FeCl3 and SG/CMC, and then permeate from the top to the bottom of the SG/CMC polymer mixture solution to form a layered hydrogel. The FE-SEM image of the SG/CMC gel cross-section is shown in Figure 7. Figure 7a– c were each hydrogel observed at the same magnification and Figure 7d was the S1C1 gel observed at high magnification. A three-dimensional interconnected porous structure was observed on the cross-section surface of the SG gel. These pores can be related to the degree of cross-linking and mechanical strength [47]. The SEM image of the CMC gel shows an increase in the surface area of the hydrogel due to the coarse, small, and frequent porous structure. This large surface area supports the superabsorbency of CMC-based hydrogels by facilitating water molecules to diffuse into the polymer network [48]. On the other hand, the S1C1 gel shows both the interconnected porous structure of the SG gel and the frequent porous structure of the CMC gel. Figure 7d shows the structure of the same S1Cl gel when viewed at a relatively high magnification. As shown in Figure 7d, the layered structure was shown, which may be due to the diffused Fe3+ ions cross-linked with the polymer in each layer during SG/CMC IPN gel formation. Fe3+ ions form a first crosslink at the interface between FeCl3 and SG/CMC, and then permeate from the top to the bottom of the SG/CMC polymer mixture solution to form a layered hydrogel.

than the loss modulus value G″. This indicates that the hydrogel is not in a fluid sol state, but a stable solid gel state. Additionally, compared with CMC gel, hydrogel containing SG has a larger storage modulus G′ at all frequencies. Comparing the actual G′ value, the CMC gel is about 1010 Pa. However, the G′ value of the S1C1 gel is 6060 Pa, and the G′ value of the S3C1 gel with the highest SG ratio is 6560 Pa. This is an increase of about 6.0 times and 6.5 times, respectively, compared to the CMC gel. This means that the addition of SG to the CMC-based hydrogel improves the mechanical strength, and the mechanical

The loss tangent (tan δ) was shown to measure the viscoelastic properties of CMC and SxCx gels (Figure 5b). Figure 5b shows that the SG/CMC IPN gels have low tan δ values at all frequencies. This shows that the gel was stably formed. In particular, the S1C1 gel prepared with a 1:1 ratio of SG and CMC had the lowest tan δ value, indicating the formation of the most mechanically strong hydrogel. Therefore, in subsequent experi-

Temperature ramp rheological studies of hydrogels were conducted at a constant frequency and strain. In Figure 6, the Tm value where the G″ value is higher than the G′ value did not occur in all the hydrogels. However, it can be seen that the G′ value of the CMC gel became rapidly insignificant. Therefore, the application in various fields is limited. However, in the case of the S1C1 gel with 2% of SG added to the CMC hydrogel, it can be seen that the G′ value is clearly maintained. This means that due to the strong me-

properties can be adjusted according to the amount of SG.

**Figure 5.** Rheological observations from the oscillation angular frequency sweep test: (**a**) Storage modulus (G′, filled sym-

bols) and loss modulus (G″, empty symbols) of hydrogels; (**b**) The loss tangent (tan δ) of hydrogels.

ments, S1C1 was set as the general SxCx.

**Figure 7.** FE-SEM images illustrating the microstructures of cross-sectioned hydrogels: (**a**) SG gel; (**b**) S1C1 gel (Low magnification); (**c**) CMC gel; (**d**) S1C1 gel (High magnification). **Figure 7.** FE-SEM images illustrating the microstructures of cross-sectioned hydrogels: (**a**) SG gel; (**b**) S1C1 gel (Low magnification); (**c**) CMC gel; (**d**) S1C1 gel (High magnification).

EDS confirmed that Fe3+ ions were uniformly distributed in the polymer mixture (Figure S1). As shown in Figure S1d, when the S1C1 gel is cut into cross-sections, Fe3+ ions are

Figure 8 shows the pH-sensitive swelling behavior of SG/CMC IPN gel at an acidic and alkaline pH. The swelling equilibrium appeared on an average of 6 h, and the hydrogel was immersed in a pH buffer of five points for 2 days for sufficient swelling. SG and CMC-based hydrogels are known to have a large influence on swelling depending on the pH. This pH-dependent swelling behavior is due to the carboxyl groups of SG and CMC.

SG/CMC IPN hydrogel was formed through the uniform penetration of Fe3+ ions.

*3.6. Swelling Behavior of SG/CMC IPN Hydrogels*

EDS confirmed that Fe3+ ions were uniformly distributed in the polymer mixture (Figure S1). As shown in Figure S1d, when the S1C1 gel is cut into cross-sections, Fe3+ ions are evenly distributed from the top to the bottom of the hydrogel. This means that the SG/CMC IPN hydrogel was formed through the uniform penetration of Fe3+ ions.

### *3.6. Swelling Behavior of SG/CMC IPN Hydrogels*

Figure 8 shows the pH-sensitive swelling behavior of SG/CMC IPN gel at an acidic and alkaline pH. The swelling equilibrium appeared on an average of 6 h, and the hydrogel was immersed in a pH buffer of five points for 2 days for sufficient swelling. SG and CMC-based hydrogels are known to have a large influence on swelling depending on the pH. This pH-dependent swelling behavior is due to the carboxyl groups of SG and CMC. At an acidic pH (pH 2, 4), most carboxylic acid groups are protonated. In addition, the hydrogen bonding interaction between the carboxylate and hydroxyl groups is enhanced. Therefore, the SG/CMC IPN tends to become denser and, consequently, the swelling value decreases [49]. At a higher pH (pH 6, 8), some carboxylic acid groups are ionized; therefore, the hydrogen bonds are broken [49,50]. Therefore, the electrostatic repulsion between the carboxylic acid groups is increased compared to an acidic pH. Therefore, the SG/CMC IPN tends to swell more. The reason that the swelling ratio is reduced in a basic solution (pH 10) is that Fe3+ ions prevent the anion–anion repulsion of the carboxylic acid group [50]. Similar results have been reported in many previous reports [49–52]. *Polymers* **2021**, *13*, x FOR PEER REVIEW 11 of 15 At an acidic pH (pH 2, 4), most carboxylic acid groups are protonated. In addition, the hydrogen bonding interaction between the carboxylate and hydroxyl groups is enhanced. Therefore, the SG/CMC IPN tends to become denser and, consequently, the swelling value decreases [49]. At a higher pH (pH 6, 8), some carboxylic acid groups are ionized; therefore, the hydrogen bonds are broken [49,50]. Therefore, the electrostatic repulsion between the carboxylic acid groups is increased compared to an acidic pH. Therefore, the SG/CMC IPN tends to swell more. The reason that the swelling ratio is reduced in a basic solution (pH 10) is that Fe3+ ions prevent the anion–anion repulsion of the carboxylic acid group [50]. Similar results have been reported in many previous reports [49–52].

**Figure 8.** The influence of different pH conditions (pH 2, 4, 6, 8, and 10) on the equilibrium swelling ratio of SG, CMC gel, and SG/CMC IPN hydrogel at 37 °C. **Figure 8.** The influence of different pH conditions (pH 2, 4, 6, 8, and 10) on the equilibrium swelling ratio of SG, CMC gel, and SG/CMC IPN hydrogel at 37 ◦C.

Additionally, then, the hydrogels showed a difference in the swelling ratio according to the SG/CMC ratio, but all showed a similar swelling behavior. The three-dimensional structure of the hydrogel prepared using the IPN crosslinking method is expected to provide a wide space for expansion according to the pH because SG and CMC are formed based on hydrogen bonding. These swelling test results suggest that SG/CMC IPN hydrogels using a double-crosslinking strategy can effectively maintain the superabsorbency of CMC while maintaining the swelling changes with pH changes. Additionally, then, the hydrogels showed a difference in the swelling ratio according to the SG/CMC ratio, but all showed a similar swelling behavior. The three-dimensional structure of the hydrogel prepared using the IPN crosslinking method is expected to provide a wide space for expansion according to the pH because SG and CMC are formed based on hydrogen bonding. These swelling test results suggest that SG/CMC IPN hydrogels using a double-crosslinking strategy can effectively maintain the superabsorbency of CMC while maintaining the swelling changes with pH changes.

### *3.7. Drug Release Profiles of 5-FU 3.7. Drug Release Profiles of 5-FU*

pattern of 5-FU according to the pH.

Figure 9 shows the drug release behavior for 16 h in pH 1.2 and pH 7.4 buffer solutions. 5-FU was released within 7 h at pH 1.2, of which 54% was released from the S1C1 gel. In contrast, at pH 7.4, 5-FU was released more rapidly than at pH 1.2. The S1C1 gel completely released the loaded drug 7 h after the start of the release. In Figure 9c, 5-FU was released from the S1C1 gel to clearly show the difference in release according to pH conditions. The buffer was changed from pH 1.2 to pH 7.4 3 h after the start of the release experiment. After changing to the pH 7.4 condition, 5-FU was rapidly released and then Figure 9 shows the drug release behavior for 16 h in pH 1.2 and pH 7.4 buffer solutions. 5-FU was released within 7 h at pH 1.2, of which 54% was released from the S1C1 gel. In contrast, at pH 7.4, 5-FU was released more rapidly than at pH 1.2. The S1C1 gel completely released the loaded drug 7 h after the start of the release. In Figure 9c, 5-FU was released from the S1C1 gel to clearly show the difference in release according to pH conditions. The buffer was changed from pH 1.2 to pH 7.4 3 h after the start of the release experiment. After changing to the pH 7.4 condition, 5-FU was rapidly released and then completely

completely released within 7 h. These results clearly show the difference in the release

released within 7 h. These results clearly show the difference in the release pattern of 5-FU according to the pH.

**Figure 9.** Cumulative release of 5-FU from of SG, CMC gel, and SG/CMC IPN hydrogels at 37 °C: (**a**) pH 1.2 condition; (**b**) pH 7.4 condition; (**c**) Change the pH from 1.2 to 7.4 after 3 h of experiment. **Figure 9.** Cumulative release of 5-FU from of SG, CMC gel, and SG/CMC IPN hydrogels at 37 ◦C: (**a**) pH 1.2 condition; (**b**) pH 7.4 condition; (**c**) Change the pH from 1.2 to 7.4 after 3 h of experiment.

This pattern occurs because of the carboxylic acid groups present in SG and CMC. At pH 1.2, the hydrogel shrinks because the carboxyl group is protonated, and hydrogen bonding is strengthened. Therefore, it can slow the release of the drug. On the other hand, at pH 7.4, the release rate is increased by increasing the hydrophilicity of the polymer because the carboxylic acid group is ionized. Therefore, these results clearly suggest that SG/CMC IPN gels with higher drug release under neutral conditions can be used to deliver drugs to carriers [53]. This is the first report of pH-dependent drug release in gel form instead of bead form using unmodified natural polysaccharide. Table S1 summarizes This pattern occurs because of the carboxylic acid groups present in SG and CMC. At pH 1.2, the hydrogel shrinks because the carboxyl group is protonated, and hydrogen bonding is strengthened. Therefore, it can slow the release of the drug. On the other hand, at pH 7.4, the release rate is increased by increasing the hydrophilicity of the polymer because the carboxylic acid group is ionized. Therefore, these results clearly suggest that SG/CMC IPN gels with higher drug release under neutral conditions can be used to deliver drugs to carriers [53]. This is the first report of pH-dependent drug release in gel form instead of bead form using unmodified natural polysaccharide. Table S1 summarizes the results of other previously published papers.

### the results of other previously published papers. *3.8. Cytotoxicity of Hydrogels*

*3.8. Cytotoxicity of Hydrogels* The manufactured hydrogel must be non-toxic for use in drug delivery and biomedical applications. The in vitro cytotoxicity of SG/CMC IPN gels was assessed using a WST-8 assay. Cell viability was evaluated and investigated by adding 5 mg of hydrogel to the cultured HEK-293 cells, followed by further culturing [54]. As shown in Figure 10, the cell viability of HEK-293 cells after 2 days of additional culture was more than 99.5% for all the hydrogels, showing no significant difference from the negative control group. In particular, in the case of the three SG/CMC IPN gels, cell viability was similar to that of CMC gels, which is known to be non-toxic. It has been reported that hydrogels that form IPN The manufactured hydrogel must be non-toxic for use in drug delivery and biomedical applications. The in vitro cytotoxicity of SG/CMC IPN gels was assessed using a WST-8 assay. Cell viability was evaluated and investigated by adding 5 mg of hydrogel to the cultured HEK-293 cells, followed by further culturing [54]. As shown in Figure 10, the cell viability of HEK-293 cells after 2 days of additional culture was more than 99.5% for all the hydrogels, showing no significant difference from the negative control group. In particular, in the case of the three SG/CMC IPN gels, cell viability was similar to that of CMC gels, which is known to be non-toxic. It has been reported that hydrogels that form IPN through Fe3+ coordination do not have cytotoxcity [55–59]. On the other hand, the positive control treated with DMSO showed a cell viability of 25%. The results indicate that the SG/CMC IPN gel does not cause negative effects on HEK-293 cells and can be used for biomedical applications such as drug delivery.

through Fe3+ coordination do not have cytotoxcity [55–59]. On the other hand, the positive control treated with DMSO showed a cell viability of 25%. The results indicate that the SG/CMC IPN gel does not cause negative effects on HEK-293 cells and can be used for

biomedical applications such as drug delivery.

**Figure 10.** Cytotoxicity of SG/CMC IPN hydrogels against HEK 293 cells. **Figure 10.** Cytotoxicity of SG/CMC IPN hydrogels against HEK 293 cells.

### **4. Conclusions**

**4. Conclusions** In this study, SG/CMC hydrogels were prepared by applying the IPN polymerization of SG to CMC gels via a double cross-linking strategy of two polymers using ionic crosslinking between carboxyl groups and Fe3+. This hydrogel enables an effective pH-responsive drug delivery with significantly improved mechanical strength while maintaining the super absorbency, which is the unique advantage of CMC gel. The resulting SG/CMC IPN gel could control the rheological properties, the cross section, and pore size of the gels, depending on the relative ratio of SG. In addition, the prepared hydrogel exhibited a pH-responsive swelling property, and the drug release of 5-FU where the release was significantly increased at a physiological pH (pH 7.4) compared to an acidic condition (pH 1.2). In addition, the SG/CMC IPN hydrogel was not cytotoxic. Overall, our results mentioned above suggest that the prepared SG/CMC IPN hydrogels have potential for use in a variety of biomedical applications such as cosmetics, food engineering, and controlled In this study, SG/CMC hydrogels were prepared by applying the IPN polymerization of SG to CMC gels via a double cross-linking strategy of two polymers using ionic cross-linking between carboxyl groups and Fe3+. This hydrogel enables an effective pH-responsive drug delivery with significantly improved mechanical strength while maintaining the super absorbency, which is the unique advantage of CMC gel. The resulting SG/CMC IPN gel could control the rheological properties, the cross section, and pore size of the gels, depending on the relative ratio of SG. In addition, the prepared hydrogel exhibited a pH-responsive swelling property, and the drug release of 5-FU where the release was significantly increased at a physiological pH (pH 7.4) compared to an acidic condition (pH 1.2). In addition, the SG/CMC IPN hydrogel was not cytotoxic. Overall, our results mentioned above suggest that the prepared SG/CMC IPN hydrogels have potential for use in a variety of biomedical applications such as cosmetics, food engineering, and controlled drug delivery systems.

drug delivery systems. **Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1, Figure S1: (a) SEM image of S1C1 gel, (b–d) EDS mapping image for C, O, and Fe, Table S1: Summary of gel characteristics, compressive test, TGA/DTG, and pH-dependent drug release results obtained in this **Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/polym13183197/s1, Figure S1: (a) SEM image of S1C1 gel, (b–d) EDS mapping image for C, O, and Fe, Table S1: Summary of gel characteristics, compressive test, TGA/DTG, and pH-dependent drug release results obtained in this study with other previously published papers.

study with other previously published papers. **Author Contributions:** S.J. designed and supervised the experiments and edited the paper; Y.S. performed the experiments and wrote the paper; D.K. performed the experiments and wrote the initial manuscript; Y.H. and Y.K. performed the methodology; I.K.H. and M.S.K. validated and re-**Author Contributions:** S.J. designed and supervised the experiments and edited the paper; Y.S. performed the experiments and wrote the paper; D.K. performed the experiments and wrote the initial manuscript; Y.H. and Y.K. performed the methodology; I.K.H. and M.S.K. validated and reviewed the paper. All authors have read and agreed to the published version of the manuscript.

viewed the paper. All authors have read and agreed to the published version of the manuscript. **Funding:** This work was supported by the Technology Innovation Program (20016324, Development of biomaterials based on novel microbial exopolysacchrides) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and partially supported by the National Research Foundation **Funding:** This work was supported by the Technology Innovation Program (20016324, Development of biomaterials based on novel microbial exopolysacchrides) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and partially supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF-2021R1A2C1013120), SDG.

of Korea (NRF) grants funded by the Korea government (MSIT) (NRF-2021R1A2C1013120), SDG. **Institutional Review Board Statement:** Not applicable.

**Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable.

**Informed Consent Statement:** Not applicable. **Data Availability Statement:** Not applicable.

**Data Availability Statement:** Not applicable. **Conflicts of Interest:** The authors declare no conflict of interest. **Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

review. *Biomacromolecules* **2011**, *12*, 1387–1408.


**4. Methodology of self-study**

As previously mentioned, Bullough and Pinnegar [20] point out that self-study is not a recipe or a procedure, but rather a methodology. Pinnegar [42] highlights the fact that self-study is a methodology for studying the professional practices of teacher educators, and that there is no particular method or correct way to conduct self-study. Rather, what determines how a self-study is conducted depends on which focus area of practice that is wanted to be better understood and developed. Pinnegar [42] offers a way to understand the methodological when she states:

*"while the methods and methodologies of self-study are not much different from other research methods, self-study is methodologically unique … self-studies involves a different philosophical and political stance … researchers who embrace self-study through the simple act of choosing to study their own practice, present an alternative representation of the relationship of the researcher and the researched … as they explore the development of understanding practice" (p. 31–32).*

Therefore, it is crucial when considering how to approach the self-study to be aware of the continuous interplay between research and practice in the practice context [31]. This means i.e., as the research unfolds, learning and development through research will affect the practice, and because the practitioner is the researcher, the practice will inevitably change as a result of this interaction and thus affect what is being researched. According to Feldman [43] and Loughran [44], a persistent criticism related to self-study is the lack of methodological rigor and transparency. LaBoskey [45] presents insightful perspectives into the methodology of self-study, outlining what she sees as four integrated aspects.

The first aspect is that self-study aims to create changes and improvements, both at the individual, the collective and at the institutional level. In addition, one of the purposes of self-study is to bring in new knowledge and development related to the education of teachers. Therefore, it is necessary that self-study thoroughly elucidates and justifies choices made, and studies should point to changes or new structures in thinking, learning- and development processes and actions. It is also important in the studies to highlight how and why changes and improvements occurred. The second aspect is about the interactivity in self-study. Interactivity is connected to interaction with colleagues, with students, with literature and research in the field and the interaction with our own previous practice. According to Zeichner [46], there is an especially important link between the researcher and the students. Students are important participants and must be actively involved in self-studies. Students will be the researcher's "mirror" of what happens in the processes of information, feedback and advice related to teaching and processes, development, learning and their understanding. Pinnegar and Quiles-Fernández [47] point out the importance of building good relationships between researchers in self-studies that takes place in a research collaboration with other researchers. Hauge [35] emphasize that relationships between researchers and teachers based on trust, respect and equality contribute to; delve deeper into the topics being investigated, dare to ask the difficult questions and being critical, creates a lower threshold for trying out and experimenting with new approaches, and this enables them to have a deeper understanding of what is being studied.

The third aspect is that self-study uses several, primarily qualitative methods. Some of these methods are often used in educational research and intervention research, while some methods may have a more innovative character. An essential purpose of methods of an innovative character, or what Engeström and Sannino describe as expansive learning, is about thinking new, experimenting, trying new approaches, and, it is an opportunity to develop "something that is not yet there" ([48], p. 2). The methodological aspect is, as mentioned, a controversial area within self-study (both by researchers who have completed self-study and others who are more critical), and although there has been development, refinement and demarcation in the field, the one correct way or template for self-studies has not emerged. Rather, self-study tends to be methodologically framed through questions, challenges, problems and desires related to areas in practice which should be changed and developed, and where the method most appropriate for examining and collecting data is used according to the study's intent. The fourth aspect is about the need to formalize the work and make it available to the professional community (i.e other researchers in self-study and teacher education research). For self-study to be examples of the development of knowledge and practice, they must be intelligible to other researchers in self-study / teacher education research and to teacher educators, reflective over the human actions, and they must be socially and contextually situated. In addition, the researcher who completed the study must engage and guide the reader into examining aspects of the study related to teaching and learning by elucidating experiences and situations, and the research (text)

**145**

revealed in the study.

*Self-Study Research: Challenges and Opportunities in Teacher Education*

must involve the researcher, the author's "self". This means that the researcher must show who he / she is in this landscape by illuminating thoughts, opinions, interpretations, actions, development, and it must involve the construction of meaning and knowledge. In other words: a self-study should not only describe the development of meaning and knowledge, but the moral dimension that involves the researcher's "self" must be brought into the research. Together, these four aspects demonstrate an expectation that learning and knowledge development from self-studies will not only be informative to the individual who completed the study, but also meaningful

A basic starting point in all empirical research is to choose the research design and the methods that are best suited to illuminate and answer the research questions [49]. It is thus the research questions that lay the foundation for the choice of design and methods for the research. To map, reconstruct, explore, analyze, develop and represent the study to a wider audience, self-study researchers use "whatever methods will provide the needed evidence and context for understanding their practice" ([50], p. 240). To show that self-studies borrows its repertoire of research methods and strategies from empirical-analytical and/or qualitativeinterpretive research, some examples of self-study is presented below. The method-

There are many teacher educators who are concerned about the impact of specific pedagogical interventions have on teacher students´ learning and development, and there can be various reasons to investigate one's own practice. To initiate discussions and reflections among students related to her methods in mathematics teaching, Brandenburg [51] organized short weekly *Round Table Reflection* sessions. The intention was to improve the students´ reflective thinking and to investigate whether her teaching created meaning and understanding, and whether the methods were good approaches for these students. A study conducted by Dourneen and Matthewman [52] analyzed how two student teachers´ transformed an education course on ICT in their internship lessons in a school through *videotaping the lessons*. The purpose was to identify what the students emphasized in their teaching, and how the teacher educators´ didactic approach affected the students and to create a focus for their ongoing improvements in practice with teacher students. In a study of Dinkelman [53], the starting point was an incident in his classroom where a student stated that she did not feel safe or could speak freely in this classroom. To investigate what this was about, a number of *individual interviews* with students´ in the class were conducted. In the interviews, several students appeared who felt insecure and lacked openness to speak freely in class, and the students pointed out that the reason was largely about the teacher's facial expression was perceived by some as judgmental. Although open classroom discourse were an important goal and a personal value in his teaching, there was something in his demeanor and behavior that placed restrictions on its implementation. This is what Whitehaed [27] describes as perceiving oneself as a "living contradiction", which in this case is about valuing one thing but showing something else. Dinkleman's study also show that those who complete a self-study must be able to cope with and handle what is

Berry and Forgasz [40] have conducted a study based on Clandinin and Connelly's [54] metaphor of "professional knowledge landscape" as a support for conceptualizing teachers' professional knowledge. This "landscape" consists of two fundamentally different places: one is in the classroom with students, and the other is in professional places with others (colleagues, administrators, etc.). At the

and useful for other researchers and teacher educators in their practice.

*DOI: http://dx.doi.org/10.5772/intechopen.96252*

**4.1 Methods for self-study in practice**

ological approaches are highlighted in italics.

#### *Self-Study Research: Challenges and Opportunities in Teacher Education DOI: http://dx.doi.org/10.5772/intechopen.96252*

must involve the researcher, the author's "self". This means that the researcher must show who he / she is in this landscape by illuminating thoughts, opinions, interpretations, actions, development, and it must involve the construction of meaning and knowledge. In other words: a self-study should not only describe the development of meaning and knowledge, but the moral dimension that involves the researcher's "self" must be brought into the research. Together, these four aspects demonstrate an expectation that learning and knowledge development from self-studies will not only be informative to the individual who completed the study, but also meaningful and useful for other researchers and teacher educators in their practice.

#### **4.1 Methods for self-study in practice**

A basic starting point in all empirical research is to choose the research design and the methods that are best suited to illuminate and answer the research questions [49]. It is thus the research questions that lay the foundation for the choice of design and methods for the research. To map, reconstruct, explore, analyze, develop and represent the study to a wider audience, self-study researchers use "whatever methods will provide the needed evidence and context for understanding their practice" ([50], p. 240). To show that self-studies borrows its repertoire of research methods and strategies from empirical-analytical and/or qualitativeinterpretive research, some examples of self-study is presented below. The methodological approaches are highlighted in italics.

There are many teacher educators who are concerned about the impact of specific pedagogical interventions have on teacher students´ learning and development, and there can be various reasons to investigate one's own practice. To initiate discussions and reflections among students related to her methods in mathematics teaching, Brandenburg [51] organized short weekly *Round Table Reflection* sessions. The intention was to improve the students´ reflective thinking and to investigate whether her teaching created meaning and understanding, and whether the methods were good approaches for these students. A study conducted by Dourneen and Matthewman [52] analyzed how two student teachers´ transformed an education course on ICT in their internship lessons in a school through *videotaping the lessons*. The purpose was to identify what the students emphasized in their teaching, and how the teacher educators´ didactic approach affected the students and to create a focus for their ongoing improvements in practice with teacher students. In a study of Dinkelman [53], the starting point was an incident in his classroom where a student stated that she did not feel safe or could speak freely in this classroom. To investigate what this was about, a number of *individual interviews* with students´ in the class were conducted. In the interviews, several students appeared who felt insecure and lacked openness to speak freely in class, and the students pointed out that the reason was largely about the teacher's facial expression was perceived by some as judgmental. Although open classroom discourse were an important goal and a personal value in his teaching, there was something in his demeanor and behavior that placed restrictions on its implementation. This is what Whitehaed [27] describes as perceiving oneself as a "living contradiction", which in this case is about valuing one thing but showing something else. Dinkleman's study also show that those who complete a self-study must be able to cope with and handle what is revealed in the study.

Berry and Forgasz [40] have conducted a study based on Clandinin and Connelly's [54] metaphor of "professional knowledge landscape" as a support for conceptualizing teachers' professional knowledge. This "landscape" consists of two fundamentally different places: one is in the classroom with students, and the other is in professional places with others (colleagues, administrators, etc.). At the intersection of these rooms, *the secret lived stories of practice*, teachers can learn to both tell and live their professional knowledge, while at the same time having the opportunity to reconstruct and adjust their professional knowledge. The broader landscape of teachers' professional knowledge, on the other hand, is full of imposed prescriptions on teaching, *the sacred stories*. Sacred stories are "pulled down" to teachers by a number of seemingly undeniable external authorities, including researchers and policy makers. By disseminating their *secret lived stories*, it was a goal for Berry and Forgasz [40] to find out how articulation and disclosure of own *secret stories* of teaching to colleagues could contribute to a broader and more pedagogical understanding of teacher education practices and at the same time be critical of the *sacred stories* of teaching.

In a study by Vanassche and Keltchermans [24], six teacher educators worked with their professional development in a two-year period where they had monthly meetings. Vanassche and Keltchermans facilitated the study and the monthly meetings, and the six participants made their own research questions related to focus in their professional development. The study was a formative intervention study based on a research collaboration between researchers and participants, where the goal was to create development and understanding of the teacher educators´ practice for both parties, both the facilitators and the teacher educators. The intention of formative intervention research is to carry out interventions to promote change and improvement, and development and research take place in parallel [48], which means that research is carried out on the ongoing development processes while the study is in progress. In order to bring out tensions and contradictions between the teacher educators´ practices and their normative beliefs, *"mirror data"* was collected as a basis for systematic discussions and reflections. According to Cole and Engeström [55], "mirror data" will act as a collective mirror on the participants practice, and the data literally "hold up the mirror". The mirror data included: *videorecordings of lessons with student-teachers´, teacher educators´ staff meetings, observations of teaching and internship evaluation reports.*

Smith and Krumsvik [56] conducted a self-study in teacher education where they searched for weak points in their own pedagogy teaching. An essential part of the study was the teacher educators´ reflection processes. The methods used to collect data for the reflection processes were video recordings of lectures with many students present and seminars with a small group of students. In addition, the teacher educators´ log notes were part of the data base. The video recording became the empirical basis for searching for weak points in the teaching. Based on the video recordings and log notes, the reflection processes were first carried out on an individual level, then they reflected together, before showing the video recordings and discussing the reflections with all the other colleagues in the department of pedagogy. In this study, *video recordings* and *log notes* are used for thorough *individual reflections, reflections in pairs*, and to invite an ever-expanding audience to participate in the reflection processes related to the two teacher educators´ learning process.

An important point that large parts of the literature in self-study agree on is the need for self-study to be a collaborative process (e.g. multiple chapters in the *International handbook of self-study of teaching and teacher education practices*: [10, 20, 45]). Freese [57] conducted a two years self-study collaborating with one student teacher aiming at a better understanding of the complexities of learning to teach, and which specific tasks and activities could contribute to teacher students´ growth and development. Another goal of this study was to discover "oneself" as a teacher through reflection and an inquiry approach in collaboration. In this study, *discussions and reflections* (*audio-recorded*) related to growth and development for both parties, in addition to finding themselves as teachers` were essential methods.

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*Self-Study Research: Challenges and Opportunities in Teacher Education*

Koster and van den Berg [58] have conducted a study that focuses on how teachers who participated in a Master's degree study of learning and innovation can explore their professional identity. One of the methods used in this study was a *biographical* approach [59]. There are different approaches to a biographical approach, but here a constructivist approach was used where the students actively constructed

What emerges in these different examples from different self-studies is that it does not involve following a recipe or a fixed procedure, but that there are many different methodological approaches. What determines which methodological measures self-study the researcher should take depends on the focus of the study, what is to be investigated and what is the goal and purpose of the study [42]. Until now, it has been tried to illuminate and describe what self-studies is and what makes self-studies special in terms of methodological approaches. In the following, some challenges and opportunities related to self-study will be highlighted in order to

The discussion related to challenges in self-study research could have been an article in itself, but here three challenges will be highlighted: 1) the challenge of the self-critical nature of one's own work and practice, 2) the requirement for quality,

Being able to ask "serious" critical questions about own actions and existing interpretations and assumptions is difficult when exploring and investigating own practice and experiences [31, 60]. There is a risk that the learning that comes out of a self-study just becomes a pseudonym for rationalization or self-justification. One of the core elements of self-study is about being informed to a greater extent about and developing own practice through adopting a reflective attitude towards own teaching. A vital feature of reflective teaching involves having a critical view on practice and a critical look at existing assumed assumptions related to various aspects of the educational process. In the field of teacher education there is a widespread agreement that there is a strong relationship between what a teacher believes and how teaching occurs in the classroom [45]. Thus, in order to influence practice, teachers´ thinking about teaching must be transformed and critical questions must be asked about what is being done [45]. But, for various reasons this is easier said than done. Firstly, this is due to the fact that our beliefs, values and knowledge about teaching are derived from our experiences and our personal history which is necessarily limited. Secondly, many of these assumptions are implicit, which means they have never been articulated, not even for us self [61]. It is also a fact that some of these ideas and beliefs about teaching are deeply ingrained in us and intimately connected to our identities as teachers and teacher educators [45]. For these reasons, it can therefore be challenging to ask critical and constructive questions

Another challenge, according to Vanassche and Keltchermans [24] is related to the tensions between the relevance of self-study and methodological rigor. In other words, a question of quality and what requirements are placed on quality for a self-study. Self-studies are inevitable in this field of tensions and it is a direct consequence of its dual research agenda. One the one hand, the requirements to contribute to professional development and improvements in practice, and on the other hand the development of a public and accessible knowledge base in teacher

and 3) the tensions between efficiency and understanding.

their career experiences into a story that became meaningful to them.

*DOI: http://dx.doi.org/10.5772/intechopen.96252*

study and develop own practice.

**5.1 Challenges**

**5. Challenges and opportunities**

related to one's own teaching and practice.

#### *Self-Study Research: Challenges and Opportunities in Teacher Education DOI: http://dx.doi.org/10.5772/intechopen.96252*

Koster and van den Berg [58] have conducted a study that focuses on how teachers who participated in a Master's degree study of learning and innovation can explore their professional identity. One of the methods used in this study was a *biographical* approach [59]. There are different approaches to a biographical approach, but here a constructivist approach was used where the students actively constructed their career experiences into a story that became meaningful to them.

What emerges in these different examples from different self-studies is that it does not involve following a recipe or a fixed procedure, but that there are many different methodological approaches. What determines which methodological measures self-study the researcher should take depends on the focus of the study, what is to be investigated and what is the goal and purpose of the study [42]. Until now, it has been tried to illuminate and describe what self-studies is and what makes self-studies special in terms of methodological approaches. In the following, some challenges and opportunities related to self-study will be highlighted in order to study and develop own practice.


*4.1.4 Glucagon like peptide receptor agonists*

Glucagon like peptide receptor agonist (GLP-1 RA) in animal studies has shown to activate ACE-2 expression and there have been speculations if this accelerated virus entrance into host cells but also if this expression neutralizes the virus limiting infection [25]. There is support for the hypothesis that GLP-1 RA may mitigate a more adverse clinical course in PWD and COVID-19 infection [26]. GLP-1 RA also are beneficial with weight loss. There are a few studies on GLP1RA and COVID-19 infection and even the final report of the CORONADO study did not find any benefit or harm with its use [27]. Dehydration is likely to lead to serious illness so patients on GLP1RA with COVID-19 should be monitored [15]. Regular meals and adequate hydration should be encouraged [15].


**Author details**

D. Tran, N. Haider, S. E. Valavan, O. Yarovyi and L. P. Ligthart *International Research Centre for Telecommunication and Radar(IRCTR), Netherlands*

I. E. Lager

*Laboratory of Applied Mathematics, Delft University of Technology, Netherlands*

A. Szilagyi *International Research Centre for Telecommunication and Radar(IRCTR), Netherlands Military Equipment and Technologies Research Agency, Romania*


*5.3.5. Hypoglycemia unawareness and associated autonomic failure*

Autonomic neuropathy is associated with more severe hypoglycemic events [126, 127] and the loss of symptoms prompting awareness of hypoglycemia [128, 129]. However, the jury is still out on whether autonomic neuropathy actually causes a loss of counter-regulatory responses to hypoglycemia [130]. Hypoglycemia associated autonomic failure (HAAF) can occur in the absence of autonomic neuropathy [126, 131, 132]. Conversely, diabetic autonomic neuropathy is observed in the absence of hypoglycemia symptom loss [131, 133]. Additionally, reversal of hypoglycemia symptom awareness is observed after strict avoidance of hypoglycemia for a relative short period of times, i.e. several weeks to months [134-136]. This would all appear to suggest that autonomic neuropathy is not a causative factor in hypoglycemia unawareness, i.e. the loss of the counter-regulatory responses to hypoglycemia. However, in the presence of autonomic neuropathy and in combination with HAAF a greater reduction in counterregula‐ tory response hypoglycemia is observed than in hypoglycemia unawareness without auto‐ nomic neuropathy [130]. Furthermore, the recovery of hypoglycemia awareness symptoms with strict avoidance of hypoglycemia is not as complete in those with autonomic neuropathy [128]. Even with the recovery of awareness symptoms, the epinephrine response to hypogly‐ cemia is only partially recovered and even less so in those with autonomic neuropathy [130]. In aggregate, this suggests that autonomic neuropathy is not the predominate cause of hypoglycemia unawareness but does enhance its severity and may play a partial etiologic role. standing position and the blood pressure response to a sustained hand grip reflects sympa‐

Diabetic Neuropathy

345

http://dx.doi.org/10.5772/55372

Assessment of autonomic neuropathy affecting the gastrointestinal track can be done by endoscopy and scintographic measurement of esophageal bolus transit time for esophageal dysfunction; scintography, isotope breath tests, and ultrasonography for gastroparesis; hydrogen breath test for diabetic diarrhea; barium enema for constipation; and anorectal manometry, endoanal ultrasonography, colon transit tests, digital examination of the rectum,

Erectile dysfunction can be assessed by taking a case history, such as with the International Index of Erectile Dysfunction, by physical examination including examining of external genitalia, blood tests including measurement of testosterone levels, measurement of nocturnal penile tumescence, and Doppler studies [2, 117, 140]. The Female Sexual Function Index has been used to evaluate sexual dysfunction in women with type 1 diabetes [124]. Vaginal plethysmography has also been used to directly assess vaginal lubrication in women with

Post void residual volume can be assessed by transurethral catherization or non-invasively via ultrasound [142]. Bladder sensation and upper urinary tract dilation can be assessed with cystometry and voiding cystometrogram. Uroflometry can be used to assess urinary flow rate and sphincter function. A urine culture should also be done to assess bacteria cystitis. In women, a urogynecological examination should be done in order to exclude pelvic prolapse.

Assessment of sudomotor function can be done with the Quantitative Sudomotor Axon Reflex Test (QSART), thermoregulatory sweat test, or the sympathetic skin response. The thermo‐ regulatory sweat test can be used to assess the pattern and distribution of anhydrosis [143]. The QSART is used to assess postganglionic sudomotor nerve function [119, 143]. The sympathetic skin response assesses postganglionic sudomotor sympathetic nerve fibers [144].

Treatment and management of diabetic autonomic neuropathy includes tight glycemic control [4]; however, the primary focus is on alleviation of symptoms [4, 101]. Manage‐ ment of orthostatic hypotension consists of educating the patient in strategies to avoid or address reversible causes of hypotension, increased fluid and salt consumption supplement‐ ed with mineralocorticoid therapy, pharmacotherapy with sympathomimetic agents, and wearing clothing such as compression stockings that increase venous return [2, 101, 118]. Antioxidants and cardioselective beta-blockers may be beneficial in cardiac autonomic

For patients with esophageal dysmotility, proton pump therapy is conventionally used [119]. Fluid consumption immediately after consumption of medications should be advised in order to avoid pill-induced esophagitis in these patients [119]. Diets low in fat and soluble fiber may be beneficial in patients with gastroparesis [2, 119], although pharmaco‐ therapy with prokinetic agents is the mainstay of therapy [119]. Insulin pump therapy may

thetic nervous system function [2].

diabetes [141].

**5.5. Management**

neuropathy [2].

protoscopy and sigmoidoscopy for fecal incontinence [2, 139].

#### *5.3.6. Sudomotor autonomic neuropathy*

Abnormalities in thermoregulation are common in type 1 diabetes [137, 138]. The sweat glands are innervated by sudomotor postganglionic unmyelinated sympathetic c-fibers. Autonomic neuropathy affecting sudomotor nerve function results in both anhidrosis and hyperhidrosis. Sudomotor dysfunction manifests symptomatically as dry scaly skin of the limbs and appen‐ dages, heat intolerance, and gustatory sweating. With increasing duration of diabetes, anhidrosis becomes more severe, progressing in a distal to proximal direction [118]. Gustatory sweating, in which there is excessive sweating in the face and trunk in response to eating is thought to result from imperfect reinnervation of postganglionic sudomotor C-fibers following denervation [118].

#### **5.4. Assessment**

Assessment of cardiovascular autonomic nervous system function can be done by measuring heart rate variability, the heart rate response in postural change from lying or sitting to standing, the blood pressure change from lying or sitting to standing, and the diastolic blood pressure response to a sustained hand grip. Heart rate variability can be assessed my meas‐ uring the heart rate response to paced deep breathing, the Valsava maneuver, and spectral analysis. The heart rate response to deep breathing and the heart rate response to a change in posture to the standing position predominately reflect parasympathetic function [2]. The heart rate response to the Valsalva maneuver reflects both parasympathetic and sympathetic function fairly equally [2]. The change in blood pressure from a lying or sitting position to a standing position and the blood pressure response to a sustained hand grip reflects sympa‐ thetic nervous system function [2].

Assessment of autonomic neuropathy affecting the gastrointestinal track can be done by endoscopy and scintographic measurement of esophageal bolus transit time for esophageal dysfunction; scintography, isotope breath tests, and ultrasonography for gastroparesis; hydrogen breath test for diabetic diarrhea; barium enema for constipation; and anorectal manometry, endoanal ultrasonography, colon transit tests, digital examination of the rectum, protoscopy and sigmoidoscopy for fecal incontinence [2, 139].

Erectile dysfunction can be assessed by taking a case history, such as with the International Index of Erectile Dysfunction, by physical examination including examining of external genitalia, blood tests including measurement of testosterone levels, measurement of nocturnal penile tumescence, and Doppler studies [2, 117, 140]. The Female Sexual Function Index has been used to evaluate sexual dysfunction in women with type 1 diabetes [124]. Vaginal plethysmography has also been used to directly assess vaginal lubrication in women with diabetes [141].

Post void residual volume can be assessed by transurethral catherization or non-invasively via ultrasound [142]. Bladder sensation and upper urinary tract dilation can be assessed with cystometry and voiding cystometrogram. Uroflometry can be used to assess urinary flow rate and sphincter function. A urine culture should also be done to assess bacteria cystitis. In women, a urogynecological examination should be done in order to exclude pelvic prolapse.

Assessment of sudomotor function can be done with the Quantitative Sudomotor Axon Reflex Test (QSART), thermoregulatory sweat test, or the sympathetic skin response. The thermo‐ regulatory sweat test can be used to assess the pattern and distribution of anhydrosis [143]. The QSART is used to assess postganglionic sudomotor nerve function [119, 143]. The sympathetic skin response assesses postganglionic sudomotor sympathetic nerve fibers [144].

### **5.5. Management**

*5.3.5. Hypoglycemia unawareness and associated autonomic failure*

*5.3.6. Sudomotor autonomic neuropathy*

denervation [118].

344 Type 1 Diabetes

**5.4. Assessment**

Autonomic neuropathy is associated with more severe hypoglycemic events [126, 127] and the loss of symptoms prompting awareness of hypoglycemia [128, 129]. However, the jury is still out on whether autonomic neuropathy actually causes a loss of counter-regulatory responses to hypoglycemia [130]. Hypoglycemia associated autonomic failure (HAAF) can occur in the absence of autonomic neuropathy [126, 131, 132]. Conversely, diabetic autonomic neuropathy is observed in the absence of hypoglycemia symptom loss [131, 133]. Additionally, reversal of hypoglycemia symptom awareness is observed after strict avoidance of hypoglycemia for a relative short period of times, i.e. several weeks to months [134-136]. This would all appear to suggest that autonomic neuropathy is not a causative factor in hypoglycemia unawareness, i.e. the loss of the counter-regulatory responses to hypoglycemia. However, in the presence of autonomic neuropathy and in combination with HAAF a greater reduction in counterregula‐ tory response hypoglycemia is observed than in hypoglycemia unawareness without auto‐ nomic neuropathy [130]. Furthermore, the recovery of hypoglycemia awareness symptoms with strict avoidance of hypoglycemia is not as complete in those with autonomic neuropathy [128]. Even with the recovery of awareness symptoms, the epinephrine response to hypogly‐ cemia is only partially recovered and even less so in those with autonomic neuropathy [130]. In aggregate, this suggests that autonomic neuropathy is not the predominate cause of hypoglycemia unawareness but does enhance its severity and may play a partial etiologic role.

Abnormalities in thermoregulation are common in type 1 diabetes [137, 138]. The sweat glands are innervated by sudomotor postganglionic unmyelinated sympathetic c-fibers. Autonomic neuropathy affecting sudomotor nerve function results in both anhidrosis and hyperhidrosis. Sudomotor dysfunction manifests symptomatically as dry scaly skin of the limbs and appen‐ dages, heat intolerance, and gustatory sweating. With increasing duration of diabetes, anhidrosis becomes more severe, progressing in a distal to proximal direction [118]. Gustatory sweating, in which there is excessive sweating in the face and trunk in response to eating is thought to result from imperfect reinnervation of postganglionic sudomotor C-fibers following

Assessment of cardiovascular autonomic nervous system function can be done by measuring heart rate variability, the heart rate response in postural change from lying or sitting to standing, the blood pressure change from lying or sitting to standing, and the diastolic blood pressure response to a sustained hand grip. Heart rate variability can be assessed my meas‐ uring the heart rate response to paced deep breathing, the Valsava maneuver, and spectral analysis. The heart rate response to deep breathing and the heart rate response to a change in posture to the standing position predominately reflect parasympathetic function [2]. The heart rate response to the Valsalva maneuver reflects both parasympathetic and sympathetic function fairly equally [2]. The change in blood pressure from a lying or sitting position to a Treatment and management of diabetic autonomic neuropathy includes tight glycemic control [4]; however, the primary focus is on alleviation of symptoms [4, 101]. Manage‐ ment of orthostatic hypotension consists of educating the patient in strategies to avoid or address reversible causes of hypotension, increased fluid and salt consumption supplement‐ ed with mineralocorticoid therapy, pharmacotherapy with sympathomimetic agents, and wearing clothing such as compression stockings that increase venous return [2, 101, 118]. Antioxidants and cardioselective beta-blockers may be beneficial in cardiac autonomic neuropathy [2].

For patients with esophageal dysmotility, proton pump therapy is conventionally used [119]. Fluid consumption immediately after consumption of medications should be advised in order to avoid pill-induced esophagitis in these patients [119]. Diets low in fat and soluble fiber may be beneficial in patients with gastroparesis [2, 119], although pharmaco‐ therapy with prokinetic agents is the mainstay of therapy [119]. Insulin pump therapy may also help to improve symptoms in type 1 diabetic patients with gastroparesis [119]. Antibiotic therapy is beneficial in the treatment of diarrhea [2, 119].

**References**

[1] Dyck PJ, Albers JW, Anderson H, Arezzo JC, Biessels GJ, Bril V, et al. Diabetic Poly‐ neuropathies: Update on Research Definition, Diagnostic Criteria and Estimation of

Diabetic Neuropathy

347

http://dx.doi.org/10.5772/55372

[2] Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic Autonomic Neuropathy. Dia‐

[3] Coppini DV, Bowtell PA, Weng C, Young PJ, Sonksen PH. Showing neuropathy is related to increased mortality in diabetic patients-a survival analysis using an accel‐

[4] Boulton AJ, Vinik AI, Arezzo JC, Bril V, Feldman EL, Freeman R, et al. Diabetic Neu‐ ropathies: a statement by the American Diabetes Association. Diabetes Care.

[5] Tesfaye S, Stevens LK, Stephenson JM, Fuller JH, Plater M, Ionescu-Tirgoviste C, et al. Prevalence of diabetic peripheral neuropathy and its relation to glycaemic control and potential risk factors: the EURODIAB IDDM Complications Study. Diabetologia.

[6] Fagerberg SE. Studies on the pathogenesis of diabetic neuropathy. III. Diabetic neu‐ ropathy in relation to diabetic vessel complications. Acta Med Scand. 1957;157(5):

[7] Albers JW, Herman WH, Pop-Busui R, Martin CL, Cleary P, Waberski B. Subclinical neuropathy among Diabetes Control and Complications Trial participants without diagnosable neuropathy at trial completion: possible predictors of incident neuropa‐

[8] England JD, Gronseth GS, Franklin G, Miller RG, Asbury AK, Carter GT, et al. Distal symmetric polyneuropathy: a definition for clinical research: report of the American Academy if Neurology, the Amerian Association of Electrodiagnostic Medicine, and the American Academy of Physical Medicine and Rehabilitation. Neurology.

[9] Dyck PJ, Kratz KM, Karnes JL, Litchy WJ, Klein R, Pach JM, et al. The prevalence by staged severity of various types of diabetic neuropathy, retinopathu, and nephrop‐ athy in a population-based cohort: the Rochester Diabetic Neuropathy Study. Neu‐

[10] Pop-Busui R, Herman WH, Feldman EL, Low PA, Martin CL, Cleary PA, et al. DCCT and EDIC Studies in Type 1 Diabetes: Lessons for Diabetic Neuropthy Regarding

Metabolic Memory and Natural History. Curr Diab Rep. 2010;10:276-82.

Severity. Diabetes Metab Res Rev. 2011. Epub Jun 21, 2011.

erated failure time model. J Clin Epidemiol. 2000;53(5):519-23.

betes Care. 2003;26:1553-79.

2005;28(4):956-62.

1996;39(11):1377-84.

2005;65(2):199-207.

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401-6.

Treatment of bladder dysfunction may be behavioral, pharmacological, or surgical. Behavioral management includes pelvic floor exercises to strengthen the muscles of the pelvic floor that support the bladder and urethretha. It also includes a program of scheduled fluid intake and micturition, manual procedures such as the Crede's maneuver, pelvic tapping, the Valsava maneuver, and clean intermittent self-catheterization. Pharmacotherapy includes the use of antimuscarinic agents, cholinergic agents, and tricyclic antidepressants. In cases refractory to non-pharmacological and pharmacological treatment, surgical procedures such as vesicle neck resection, selective pudendal nerve block, unilateral pudendal neurectomy, and sacral neuromodulation may be beneficial [2, 119, 142, 145].

Treatment and management of erectile dysfunction should include psychological counseling; however, pharmacotherapy with the PDE5 inhibitors (sildenafil, vardenafil, tadalafil) is the mainstay of therapy. Intracavernous or intraurethral injections with vasoactive medication, vacuum constriction devices, and penile prosthesis implantation are also options [2, 119]. Vaginal estrogen creams in has been shown to be beneficial in diabetic women with female sexual dysfunction [118].


**4. Steganography hides information**

Take a look at a few of the most basic methods for hiding text in a digital image. One straightforward method is to simply insert the material into the file at the end. These works do not prohibit the photograph from being displayed regularly, nor do they alter its esthetic look. We merely put "hello world" to the file's conclusion. The hex dump output shows us putting the extra bytes.

A program can easily read or discard the plain text string. In this scenario, we'll invert the hexadecimal number and output it in plain English using a software. For example, a received image displays a picture in a photograph viewer application ordinarily, but when examined with the WinRaR archiving utility, we can find that the unpacked.jpg file contains a concealed 28-byte text file.

These types of basic approaches can be helpful in collecting user data, but they do have some disadvantages. First, they inflate the file size, and second, they change the file hash. It's still very convenient for security tools to spot because of its unexpected format.

The best way is to enter into the code at the binary stage and deal with the least important bits (LSBs) of each pixel. Pixels can be represented in a 3-byte color image, one per RGB each (red, green, blue). Suppose we have three bytes

#### **Figure 1.**

representing a particular color as seen in **Figure 1**. You should swap the last four bits of the orange code with the first four bits of the turquoise code, to produce the composite RGB [5].

If we write software to read and extract these last four bits separately, we have effectively hidden the purple signal within the orange color software. Two pixels for the price of one, as there is no increase in file size. We can send our cryptic information without doubling the bandwidth of the actual message or modifying the file format, thus simple detection approaches that rely on examining files to find them are rendered obsolete. In actuality, the code is extremely cluttered before the attacker reassembles it.

In a sense, this ensures that an intrusion will utilize the last four bits of encoding RGB values to write extra information without interfering with the image's graphic display or increasing the file size. Another program will then read the hidden data and use it to reassemble a malicious script or sort customer data.

LSB processing is one of several steganography processes. There are numerous other instances in which photographs and other file formats might be modified to conceal a concealed message. To relay secret communications, the attackers employed information buried in network protocols, a technique known as "network hiding." The approach is the same in both cases: hide in clear view by downloading an invisible message to the accessible carrier.

Steganography for shielding information has affected both Windows and Mac OS operating systems. An intruder has been found to use cryptography to conceal portions of the ransomware attack code, add malicious JavaScript, and even download encryption software.