*3.6. Fluorescence Image Analysis*

The cell-killing efficacy of the released Cur was determined by staining HepG2 cells with propidium iodide and acridine orange to distinguish between living and dead cells. As seen in Figure 6C, the cells treated with PNIPAm-co-PAAm-Mela HG fluoresced green, indicating that the majority of the cells were alive. The Cur-loaded PNIPAm-co-PAAm-Mela/Cur HG sample treated cells, on the other hand, exhibit red fluorescence, indicating that around >95% of cells were killed [38,39]. This might be due to the released Cur drugs from the PNIPAm-co-PAAm-Mela/Cur HG system, which induced cell death. As seen in Figure 6D, the cell killing efficiency is a function of sample concentration. The cell killing efficiency rose when the PNIPAm-co-PAAm-Mela/Cur HG sample concentration increased, which may be attributed to an increase in released Cur from the PNIPAm-co-PAAm-Mela/Cur HG system, and the released Cur induced more cell death.

#### **4. Conclusions**

In this study, we developed a dual-stimulus PNIPAm-co-PAAm-Mela/Cur HG copolymer system for temperature-responsive and pH-induced drug delivery applications. To evaluate the drug delivery behavior of the PNIPAm-co-PAAm-Mela HG copolymer, we employed Cur as a model drug. The PNIPAm-co-PAAm-Mela HG system had much higher Cur loading efficiency (73%) due to the existence of more drug-binding sites of amide, amine, and imine sites in the PNIPAm and Mel cross-linked PAAm segments. The drug release investigation revealed that a nearly complete release of Cur was accomplished in the presence of the combined pH and temperature stimulation conditions (pH 5.0/45 ◦C Furthermore, the results of the cytocompatibility investigation show that the prepared PNIPAm-co-PAAm-Mela HG system is cytocompatible and that the loaded drug may be released in the intracellular microenvironment. The overall study findings demonstrated that the PNIPAm-co-PAAm-Mela HG system might be used for controlled drug release to specific sites in chemotherapeutic applications.

**Author Contributions:** Conceptualization, methodology, formal analysis, K.T. and T.T.V.P.; validation, M.S. and V.R.; data curation, K.T.; writing—original draft preparation, T.T.V.P., M.S. and V.R.; writing—review and editing; supervision, project administration, funding acquisition, S.-C.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2020R1I1A3052258). In addition, the work was also supported by the Technology Development Program (S3060516), funded by the Ministry of SMEs and Startups (MSS, Republic of Korea), in 2021.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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