*2.4. PNIPAm-co-PAAm Copolymer Cross-Linked with Mela-Glu Precursor*

The PNIPAm-co-PAAm copolymer was cross-linked with Mela-Glu precursor as follows. Approximately 1.0 g of PNIPAm-co-PAAm copolymer was dissolved in 25 mL ethanol. Next, 0.3 g of Mela-Glu precursor in ethanol (5 mL) was slowly introduced, allowing for a cross-linking reaction between the amine groups of PAAm segments and the aldehyde groups of Mela-Glu units through Schiff base reaction in the presence of an acetic acid catalyst [27]. The obtained hydrogel sample was placed in a dialysis membrane tubing with molecular weight cutoff (MWCO) at 3.5 kDa and placed in a beaker containing 200 mL of ethanol-water (1:1 *v*/*v*) mixture. The surrounding solvent was exchanged every 6 h, and a fresh 200 mL of ethanol-water was used. Finally, the purified sample was separated and dried at room temperature under vacuum for 12 h. The Mela-Glu precursor cross-linked hydrogel obtained was designated as PNIPAm-co-PAAm-Mela HG (Scheme 1).

#### *2.5. Characterization*

1H-NMR analysis of the PNIPAm-co-PAAm copolymer, Mela-Glu precursor, and PNIPAm-co-PAAm-Mela HG sample was carried out using the OXFORD instrument (600 MHz). Scanning electron microscopy (SEM, JEOL 6400 instrument) at 10 kV was applied to measure the surface structure of the prepared copolymer sample. X-ray photoelectron spectroscopy (XPS) analysis was performed on the XPS instrument (Tucson, AZ, USA 85706). Fourier-transform infrared (FTIR) analysis was carried out using JASCO FTIR 4100 instrument. Particle size and the zeta potential measurements were performed on the Malvern Zetasizer Nano-ZS. UV-vis spectral analysis was performed using an Agilent Inc. UV-Vis spectrophotometer.

## *2.6. Turbidity Measurement*

For this experiment, about 25 mg/mL of the PNIPAm-co-PAAm-Mela HG sample was dissolved in 5 mL distilled water to measure the turbidity of the synthesized PNIPAm-co-PAAm-Mela HG sample. The absorbance of the obtained polymer solution was measured at various temperatures ranging from 25 to 80 ◦C. The absorbance of the sample at each temperature was measured.

#### *2.7. Drug Loading into PNIPAm-co-PAAm-Mela HG*

A model drug, Cur, was used for drug loading and release experiments in vitro. The swelling diffusion method was used to load Cur into the PNIPAm-co-PAAm-Mela HG system at a polymer: drug *w*/*w* ratio of (10:3) [28]. In 3 mL of water, 0.1 g of PNIPAm-co-PAAm-Mela HG sample was dissolved, and 30 mg of Cur drug was combined. For 24 h, the suspension mixture was stirred at room temperature. The drug loading content into the PNIPAm-co-PAAm-Mela HG was determined using a UV-Vis spectrophotometer at 425 nm after the Cur encapsulated copolymer was separated by centrifugation at 50 ◦C. The drug-loaded sample was labeled as PNIPAm-co-PAAm-Mela@Cur HG (Scheme 2). The drug loading percentage was calculated to be around 78% using the following equation.

$$\text{Drug loading (\%)} = \text{(Wt. of the drug in sample/Wt. of the sample)} \times 100\tag{1}$$

**Scheme 2.** A proposed schematic representation of drug loading into the PNIPAm-co-PAAm-Mela HG system. The pH stimuli and the combined pH and temperature-stimuli-responsive release behavior of the PNIPAm-co-PAAm-Mela HG system.

#### *2.8. pH and Temperature-Responsive Drug Release Experiments*

The PNIPAm-co-PAAm-Mela/Cur HG was evaluated in vitro under various circumstances, including (i) varied pH (pH 7.4 and 5.0); (ii) different temperatures (25 ◦C and 45 ◦C); and (iii) the combination of pH + temperature (pH 7.4/45 ◦C and pH 5/45 ◦C, respectively). For these experiments, approximately 25 mg/mL of drug-loaded sample PNIPAm-co-PAAm-Mela@Cur HG was placed in a dialysis bag (Mol. wt. cut off 5000 kDa), and the bag was immersed in a beaker containing 25 mL of phosphate-buffered saline (PBS) solution at different pH and temperature under gentle magnetic stirring. At the set time, about 1 mL of release media was removed, and the released Cur was measured using a UV-Vis spectrophotometer at 425 nm. The calibration curve plot was used to calculate the released Cur. The cumulative drug release was calculated using the equation below. Drug release (%) = (Amount of Cur released at time t/Total amount of Cur in the HG sample) × 100.

#### *2.9. Cytocompatibility Study*

The synthesized PNIPAm-co-PAAm-Mela HG, Cur loaded PNIPAm-co-PAAm-Mela@Cur HG, and pure Cur samples were tested for in vitro cytocompatibility utilizing the 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. HepG2 cells (2 × <sup>10</sup><sup>4</sup> cells/well) were grown in a 96-well plate for 24 h at 37 ◦C for this experiment. The existing medium was replaced with new media containing varying concentrations of PNIPAm-co-PAAm-Mela HG, PNIPAm-co-PAAm-Mela@Cur HG, and pure Cur. After 4 h, the MTT solution was added to each well and incubated for another 4 h. Following that, about 20 μL of DMSO was added to dissolve the existing formazan crystals, and the absorbance at 592 nm was measured using a microplate reader.
