2.2.1. Synthesis of Graphene Oxide

GO was prepared by a well-known Hummer method [22]. Graphite powder was used as a precursor for the synthesis of GO. Briefly, 3 g of graphite powder was added to the mixture of H3PO4/H2SO<sup>4</sup> (1:9). Then, 18 g of KMnO<sup>4</sup> was added slowly to the mixture under continuous stirring for 12 h using a hotplate. When the reaction is completed, deionized water (400 mL) was added to the mixture, and the mixture was titrated with 3 mL (30% H2O2). Finally, the solution was filtered using filter paper, and the filtrated residues were washed with 30% HCl, ethanol, and deionized water repeatedly until neutral pH 7. The resultant residues were dried at 60 ◦C for 12 h in a vacuum oven and stored in sample containers [23].

#### 2.2.2. Synthesis of Hydrogels

A series of CS/PVA blended films were fabricated via solution casting technique. CS (0.6 g) was dissolved in 2% acetic acid, and PVA (0.4 g) was dissolved in 50 mL deionized water. CS and PVA solutions were mixed under continuous stirring at 55 ◦C for 2 h. TEOS (100 µL) dissolved in10 mL ethanol was added dropwise into the stirring homogeneous solution at 55 ◦C. The stirring was continued at 55 ◦C for another 3 h. After that, the solution was poured carefully into well-cleaned and dried Petri dishes. The Petri dishes were kept in the oven at 50 ◦C for 3 days. The films with 100 and 150 µL TEOS were prepared via repeating the same procedure. The 100 and 150 µL were coded as CP-1, CP-2. The controlled film sample was synthesized using the same procedure but without the addition of TEOS. The controlled sample was coded as CP. The formulation is tabulated in Table 1.

films.


**Table 1.** Formulations used for the preparation of hydrogels.

Formulation of CS/PVA/GO Hydrogels 0.1 % and 0.5% of the prepared GO was dispersed via ultrasonication in deionized

Formulation of CS/PVA/GO hydrogels

0.1 % and 0.5% of the prepared GO was dispersed via ultrasonication in deionized water (DI) until it was completely dispersed. The homogeneously dispersed solution of GO had light brown and dark brown colors (Figure 2b,c). Composite films with a varying weight ratio of GO (0, 0.1% and 0.5%) were fabricated via the solution casting technique. The proposed chemical mechanism has been presented in the schematic diagram. The samples were coded as CP-1, CPGO-0.1% and CPGO-0.5%. The proposed mechanism of the fabrication of the hydrogels and GO/ hydrogel composite is depicted in Figure 1, and the digital photographs of composite films are shown in Figure 2g–i. The formulation is tabulated in Table 1. water (DI) until it was completely dispersed. The homogeneously dispersed solution of GO had light brown and dark brown colors (Figure 2b,c). Composite films with a varying weight ratio of GO (0, 0.1% and 0.5%) were fabricated via the solution casting technique. The proposed chemical mechanism has been presented in the schematic diagram. The samples were coded as CP-1, CPGO-0.1% and CPGO-0.5%. The proposed mechanism of the fabrication of the hydrogels and GO/ hydrogel composite is depicted in Figure 1, and the digital photographs of composite films are shown in Figure 2g–i. The formulation is tabulated in Table 1.

**Figure 1.** Proposed chemical mechanism of chitosan/polyvinyl alcohol/Graphene oxide composite **Figure 1.** Proposed chemical mechanism of chitosan/polyvinyl alcohol/Graphene oxide composite films.

#### **Table 1.** Formulations used for the preparation of hydrogels. 2.2.3. Drug-Loading into CS/PVA/GO Hydrogels

**Samples Formulation CS (%) PVA (%) GO (%) TEOS (µL) CP 1** CS/PVA 80 20 0.00 100 **CP 2** CS/PVA 60 40 0.00 100 **CP 3** CS/PVA 20 80 0.00 100 **CP 4** CS/PVA 40 60 0.00 100 **CP 5** CS/PVA 60 40 0.00 0 **CP 6** CS/PVA 60 40 0.00 150 CS, PVA and GO solutions were prepared and then blended as mentioned in Section 2.2.2. After 1 h of blending, 20 mg of paracetamol dissolved in 10 mL deionized water was added dropwise to the blended solution by a dropper. The solution was further stirred for 1 h until all the drugs become dissolved. 100 µL of TEOS was dissolved in 10 mL of ethanol and was added dropwise into the drug-loaded blend. The final blend was further stirred for 3 h and poured carefully into the well cleaned and dried Petri dish. The casted films were kept in a vacuum drying oven at 55 ◦C for two or three days.

#### **CP 7** CS/PVA/GO-0.1% 60 40 0.1 100 **3. Characterization**

#### **CP 8** CS/PVA/GO-0.5% 60 40 0.5 100 *3.1. Fourier Transform Infrared Spectroscopy (FTIR)*

2.2.3. Drug-Loading into CS/PVA/GO Hydrogels CS, PVA and GO solutions were prepared and then blended as mentioned in section 2.2.2. After 1 h of blending, 20 mg of paracetamol dissolved in 10 mL deionized water was The functional group and interactions among all of the components of the prepared hydrogels were investigated using FTIR spectroscopy in ATR mode (Agilent Cary 630). The FTIR study was carried out at a scan rate of 60 scans and wavenumber ranging from 4000 to 400 cm−<sup>1</sup> , with 4 cm−<sup>1</sup> resolutions.

#### added dropwise to the blended solution by a dropper. The solution was further stirred for 1 h until all the drugs become dissolved. 100 µL of TEOS was dissolved in 10 mL of ethanol *3.2. SEM Morphology*

and was added dropwise into the drug-loaded blend. The final blend was further stirred for 3 h and poured carefully into the well cleaned and dried Petri dish. The casted films were kept in a vacuum drying oven at 55 °C for two or three days. Scanning electron microscopy (SEM, JEOL-JSM-6480, Akishima, Japan) was used to examine the morphologies of the well-dried hydrogels, and hydrogel films were sliced and gold-sputtered. These were placed on a stub before being placed in the vacuum chamber to observe surface morphology.

#### **3. Characterization**  *3.3. Swelling and Degradation Analysis*

*3.1. Fourier Transform Infrared Spectroscopy (FTIR)*  The functional group and interactions among all of the components of the prepared hydrogels were investigated using FTIR spectroscopy in ATR mode (Agilent Cary 630). The FTIR study was carried out at a scan rate of 60 scans and wavenumber ranging from 4000 to 400 cm−1, with 4 cm−1 resolutions. The swelling experiments of the hydrogel (CS/PVA) and the composite (CS/PVA/G0- 0.1% and CS/PVA/GO-0.5%) were conducted in DI water, buffer and salt solutions. The blended dried films were first to cut into small pieces, dried and weighed. Then, the samples were placed in DI water in separate containers and allowed to swell. The samples were removed from the containers at pre-decided intervals, adequately cleaned with filter paper to remove the surface water, and weighed again. The swelling experiments were conducted in DI, Buffer solution (at pH 2, 4, 7 and 10) and electrolyte solution (NaCl and

CaCl<sup>2</sup> (0.1 M, 0.3 M, 0.7 M and 0.9 M). Swelling (DS) was calculated using Equation (1) [24]. The swelling analysis was performed in triplicated, and average values were taken to calculate the swelling analysis.

$$\text{Swelling index} = \frac{\text{Ws} - \text{Wd}}{\text{Wd}} \tag{1}$$

where *Ws* = swollen weight of films, and *Wd* = the dried weight of the films.

The well-dried hydrogels films were cut into square form and weighted (45 mg) carefully. The degradation analysis was performed in PBS solution with pH = 7.4, and these samples were incubated at 37 ◦C for different periods (1, 2, 3, 5 and 7 days). The degradation of hydrogels was calculated using Equation (2). [24]. The degradation analysis was performed in triplicates, and average values were taken to calculate degradation analysis.

$$\text{Weight loss } (\%) = \frac{W\_0 - W\_t}{W\_0} \times 100 \tag{2}$$

where *W*<sup>0</sup> = initial hydrogel weight, and *W<sup>t</sup>* = hydrogel weight at the time "*t*".

#### *3.4. Atomic Force Microscopy (AFM)*

AFM (Nano-Solver, NT-MDT) equipped with a silicon nitride tip was used to investigate the surface roughness of the hydrogel samples at ambient conditions. AFM was performed using Nova-Px software over an area of 5 µm × 5 µm in the semi-contact mode. Dried samples (thin films) were used and stuck to the sample holder for AFM analysis.
