*3.3. Phase Transition Mechanism of PNIPAm-co-PAAm-Mela HG System*

The relative turbidity of PNIPAm-co-PAAm-Mela HG polymer (25 mg/mL) was investigated from 25 to 80 ◦C (Figure 4a). At a medium temperature of less than 40 ◦C, the sample displayed a transparent and homogenous clear solution, as illustrated in the graph (Figure 4a) and the sample vial. The PNIPAm-co-PAAm-Mela HG polymer absorbs water and becomes hydrated, swelling at this low LCST (linear structure). At temperatures above 40 ◦C, however, the clear solution became turbid, and the relative turbidity in the solution medium increased with increasing temperature to above 45 ◦C, indicating that the linear copolymer chain collapses into a hydrophobic globule micelle structure [31]. UV-Vis absorption and relative turbidity were used to study the swelling and deswelling properties of the prepared PNIPAm-co-PAAm-Mela HG at various solution temperatures. At 25 ◦C and 45 ◦C, UV-Vis absorbance of the PNIPAm-co-PAAm-Mela HG sample (25 mg/mL) was determined. As shown in Figure 4b, a significant weak absorption peak was seen at 25 ◦C, which might be attributed to the linear polymer structure and clear solution medium. At 45 ◦C, however, the solution medium becomes turbid due to temperature-induced micelle formation caused by the transition of the PNIPAm-co-PAAm-Mela HG's hydrophilic linear to hydrophobic globule structure above LCST (Figure 4b).

**Figure 3.** (**a**) Zeta potential and (**b**) particle size of PNIPAm−co−PAAm-Mela HG sample. Mean with error bar *n* = 3. (**c**) DLS analysis of PNIPAm−co−PAAm-Mela HG at below and above LCST.

**Figure 4.** (**a**) Relative turbidity of PNIPAm−co−PAAm-Mela HG at 25 ◦C to 80 ◦C. (**b**) UV-vis absorption of PNIPAm-co-PAAm-Mela HG sample at 25◦C and 45◦C. (**c**) Illustrates the phase transition of PNIPAm−co−PAAm−Mela HG below and above LCST. The violet color indicates the hydrophobic PNIPAm domain, and the brown color indicates the hydrophilic PAAm segments in the presence of water.

The unique properties of dual-stimuli-responsive hydrogels include the ability to undergo noticeable phase transitions in response to physical stimuli rather than chemical or mechanical stimuli. At 25 ◦C, the PNIPAm-co-PAAm-Mela HG polymer dissolves readily in water and forms a non-cross-linked homogenous solution. The copolymer segments of PNIPAm-co-PAAm-Mela HG comprise hydrophilic amide (-CO-NH-) groups and hydrophobic isopropyl (-CH(CH3)2) groups. In deionized water, the PNIPAm-co-PAAm-Mela HG polymer undergoes a sharp phase transition and exists in solution as a linear hydrophilic polymer chain below LCST. In contrast, above LCST, the PNIPAm-co-PAAm-Mela HG transformed into a hydrophobic globule coil shape (Figure 4c).

#### *3.4. Cur Loading and pH-Responsive Delivery from PNIPAm-co-PAAm-Mela/Cur HG System*

Because the PNIPAm-co-PAAm-Mela HG has a higher LCST than human body temperature, fast micelle formation may be prevented when it is injected into the body. By fine-tuning the temperature stimuli, it is possible to maintain selective and controlled drug release to the target sites. Cur, an anticancer drug, may be encapsulated into the PNIPAmco-PAAm-Mela HG by mixing them with a polymer solution at low temperatures. At low temperatures, the loaded Cur in the PNIPAm-co-PAAm-Mela HG can be protected against denaturation. The drug molecules can be interacted with the PNIPAm-co-PAAm-Mela HG by hydrogen bonding or electrostatic interactions (Scheme 2). The amide, imine, and carbonyl functional groups in the PNIPAm-co-PAAm-Mela HG system act as drug binding sites as well as protonation centers, ionize in low pH conditions and promote the release of loaded drugs from the PNIPAm-co-PAAm-Mela HG system (Scheme 2).

The pH and temperature-responsive drug release behavior of the prepared Cur loaded PNIPAm-co-PAAm-Mela/Cur HG has been evaluated at different pH and temperature conditions, specifically at different pH (pH 7.4 and 5.0); at different temperatures (25 ◦C and 45 ◦C); and the combined pH and temperature (pH 7.4/45 ◦C, pH 7.4/45 ◦C, respectively). First, the pH-responsive Cur release behavior from the PNIPAm-co-PAAm-Mela/Cur HG was studied.

Figure 5a demonstrated the Cur release behavior at various pHs, with approximately ~30% and ~82% of Cur released in 12 h at pH 7.4 and 5.0, respectively. The enhanced Cur release observed at pH 5.0 might be attributed to acid-induced protonation of the nitrogen part of PNIPAm and cross-linked Mela groups, as well as the loaded Cur molecules (Figure 5a). Second, Figure 5b depicted the temperature-responsive release behavior, which revealed that around ~26% and ~68% of Cur release was observed at 25 ◦C and 45 ◦C, respectively, throughout a 12 h release period. The release of physisorbed Cur molecules was responsible for the increase in release at 45 ◦C (Figure 5b). Third, at pH 7.4/45 ◦C and pH 5.0/45 ◦C, respectively, the combined pH and temperature-stimuliresponsive Cur release were determined. As seen in Figure 5c, a gradual release of Cur was detected, with about ~65% released at pH 7.4/45 ◦C; an almost complete release of Cur was observed at pH 5.0/45 ◦C, respectively, in a 12 h release period. Under the combined pH and temperature conditions, enhanced Cur release was observed due to the temperature-induced phase transition and pH-induced protonation of the functional groups and Cur molecules, which induce an electrostatic repulsive force. Therefore, the PNIPAm-co-PAAm-Mel HG system showed considerably enhanced Cur release under the combined pH and temperature stimuli conditions.

The drug loading mechanisms could be described as follows. As shown in Scheme 2, the loaded Cur molecules are strongly associated with the amine, imine, and amide groups via H-bonding/electrostatic interactions at pH 7.4. As a result, only a negligible amount of Cur was released from the PNIPAm-co-PAAm-Mela/Cur HG system at pH 7.4. The enhanced Cur release observed at the combined acidic pH and temperature (pH 5.0/45 ◦C) might be attributed to the temperature-induced phase change and acid-induced protonation of drug-binding functional sites and drug molecules, both of which force out the Cur molecules from the PNIPAm-co-PAAm-Mela/Cur HG system (Scheme 2).

The experiment results showed that combining pH and temperature stimuli resulted in greater Cur release efficiency from the PNIPAm-co-PAAm-Mela/Cur HG system than single stimuli, such as only pH or temperature stimuli (Tables 1 and 2). The majority of thermoresponsive polymers reported in the literature [23,32–34] primarily focused on temperature (Table 3), but in this work, our proposed melamine cross-linked PNIPAm-

co-PAAm-Mela/Cur HG system has advantages such as enhanced drug loading and dual-stimuli-responsive drug release to the target sites.

**Figure 5.** In vitro Cur delivery of PNIPAm-co-PAAm-Mela/Cur HG system. (**a**) Cur release at different pH, (**b**) Cur release at different temperature stimuli, and (**c**) Cur release with the combined pH and temperature conditions, respectively. Mean with error bar *n* = 3.

**Table 1.** Cur release efficiency from PNIPAm-co-PAAm-Mela HG system at different temperatures.


**Table 2.** Cur release efficiency from PNIPAm-co-PAAm-Mel HG system at pH/temperatures.


**Table 3.** Various PNIPAM-based copolymer hydrogels for Cur delivery.


*3.5. Cytocompatibility*

The cytocompatibility of the prepared PNIPAm-co-PAAm copolymer, PNIPAm-co-PAAm-Mela HG, Cur-loaded PNIPAm-co-PAAm-Mela/Cur HG system, and pure Cur was

tested in vitro at 37 ◦C using the HepG2 cell line. The cytocompatibility of control HepG2 cells and different concentrations of synthesized PNIPAm-co-PAAm copolymer samples are shown in Figure 6A. As shown in Figure 6A, the synthesized PNIPAm-co-PAAm copolymer exhibits about ~90% cell viability even at a sample concentration of 200 μg/mL, indicating that the PNIPAm-co-PAAm copolymer is biocompatible in nature [35,36]. On the other hand, Figure 6B shows that the PNIPAm-co-PAAm-Mela HG system without Cur loading demonstrated ~90% cell survival in all investigated sample concentrations, demonstrating that the prepared PNIPAm-co-PAAm-Mela HG also shows excellent biocompatibility to the HepG2 cells. In contrast, the Cur-loaded PNIPAm-co-PAAm-Mela/Cur HG system was shown to be toxic to HepG2 cells at all sample concentrations. It was observed that cells treated with a sample concentration of 200 μg/mL demonstrated nearly complete cell killing efficiency, implying that a concentration of 200 μg/mL of PNIPAm-co-PAAm-Mela HG is sufficient for complete cell killing [37]. This in vitro study indicates that the prepared PNIPAm-co-PAAm-Mela HG might be used for drug loading and pH stimuli-responsive drug release to specific sites.

**Figure 6.** (**A**) In vitro cytocompatibility of (**a**) control HepG2 cells and (**b**) PNIPAm-co-PAAm copolymer; (**B**) In vitro cytocompatibility of (**a**)PNIPAm-co-PAAm-Mela HG; (**b**) PNIPAm-co-PAAm-Mela/Cur HG; and (**c**) pure Cur drug, respectively, at different concentrations. Statistical significance to the cell toxicity with different samples (\*, significant *p* < 0.05; \*\*, highly significant *p* < 0.01). (**C**) Fluorescence microscopy images of HepG2 cells represent (**a**) dark-field image; (**b**) PNIPAmco-PAAm-Mela HG treated cells; and (**c**) Cur loaded PNIPAm-co-PAAm-Mela/Cur HG system, respectively. (**D**) Cell viability of (**a**) PNIPAm-co-PAAm-Mela HG treated cells; and (**b**) Cur loaded PNIPAm-co-PAAm-Mela/Cur HG system, respectively, at different sample concentrations. Mean with error bar *n* = 3.
