*3.2. Physicochemical Properties of PNIPAm-co-PAAm-Mela HG System*

The surface charge of the synthesized PNIPAm-co-PAAm-Mela HG was evaluated using zeta potential analysis. The presence of amide (-N-H), imine (-C=N-), and carbonyl (-C=O) groups in the copolymer segments increased the pH sensitivity of the PNIPAmco-PAAm-Mela HG. The zeta potential of the PNIPAm-co-PAAm-Mela HG samples was measured at 25 ◦C and 45 ◦C, respectively. As shown in Figure 3a, the zeta potential value decreased from +19 mV to −13 mV and from +16 mV to −9 mV for samples evaluated at 25 ◦C and 45 ◦C, respectively, as the pH increased from 2 to 10. At low pH and higher temperature (45 ◦C), the hydrophobic PNIPAm segments combine to form the hydrophobic micelle core, whereas the hydrophilic PAAm forms the outer shell structure. The PNIPAm-co-PAAm-Mela HG, on the other hand, enhances hydrophilicity at higher pH, allowing them to transit the sol phase. The zeta potential values are slightly lower at higher temperatures (45 ◦C) than at lower temperatures (25 ◦C), which may be attributed to the development of aggregated micelles above LCST. The dynamic light scattering (DLS) technique was used to determine the linear to globule phase transition at different temperatures, such as 25 ◦C and 45 ◦C. As seen in Figure 3b, the DLS intensity remained consistent at 25 ◦C for the sample concentration of 25 mg/mL. Moreover, the solution temperature above LCST, the PNIPAm-co-PAAm-Mela HG showed increased turbidity (gel phase) and showed appropriate particle size when the solution temperature increased from 25 ◦C

to 45 ◦C. On the other hand, when cooling the sample to about 25 ◦C, the sample did not show any considerable particle size and appeared to be a clear transparent solution. The DLS measurement under repeated heating and cooling conditions was examined at 45 ◦C and 25 ◦C, respectively (Figure 3b). DLS methods were also used to investigate the temperature responsiveness of the PNIPAm-co-PAAm-Mela HG polymer. Figure 3c shows that at 25 ◦C, no significant particles are formed. However, tiny particles are repeatedly formed at 45 ◦C. This work demonstrated that the PNIPAm-co-PAAm-Mela HG polymer undergoes a significant phase transition at temperatures above the LCST.

**Figure 2.** XPS analysis of PNIPAm-co-PAAm-Mela HG. The core level spectra of (**a**) C 1s; (**b**) N 1s; and (**c**) O 1s, respectively.
