**1. Introduction**

The controlled delivery of therapeutic drugs has been considered an effective technique for maintaining therapeutic effectiveness [1–3]. Controlled drug delivery carriers enable therapeutic concentrations to be maintained while also protecting drugs from enzymatic degradation, improving drug solubility, decreasing adverse effects, and extending release time [4]. Although drug delivery carriers are advantageous, they do have significant clinical limits [5]. As a result, self-regulating drug delivery systems that deliver drugs based on changes in physiological parameters are required [6]. As drug carrier systems, a variety of drug delivery methods, such as nanoparticles, polymeric materials, and lipids, have been employed [7–9].

Hydrogels are cross-linked polymeric networks having the ability to expand in an aqueous medium or biological fluid and hold a considerable amount of water [10,11]. The hydrogels mimic adjacent tissues due to their biocompatibility and high-water content.

**Citation:** Thirupathi, K.; Phan, T.T.V.; Santhamoorthy, M.; Ramkumar, V.; Kim, S.-C. pH and Thermoresponsive PNIPAm-co-Polyacrylamide Hydrogel for Dual Stimuli-Responsive Controlled Drug Delivery. *Polymers* **2023**, *15*, 167. https:// doi.org/10.3390/polym15010167

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 18 November 2022 Revised: 21 December 2022 Accepted: 23 December 2022 Published: 29 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Because of their swelling–deswelling capabilities, hydrogels are widely used in biotechnological and biomedical applications such as tissue engineering, regenerative medicine, and diagnostic biosensor fields [12,13]. Chemically cross-linked hydrogel polymers are currently attracting much research attention as prospective drug delivery matrices. The drug release behavior might be influenced by internal or external stimuli such as pH, temperature, light, and ultrasound [14–19]. Dual-stimuli materials, such as temperature and pH-sensitive hydrogel materials, are frequently used in biomedical fields because these parameters may be easily controlled in vitro and in vivo [20].

However, for practical applications, homogenous dispersion of therapeutic molecules in hydrogels is essential, which is one of the key disadvantages of present hydrogel materials. Because of the enhanced swelling capacity of hydrogels, the drug is released quickly. The selective and controlled release of a loaded drug is considered to be an efficient strategy for minimizing undesirable side effects on normal cells and tissues [21]. As a result, the development of dual-stimuli controlled delivery systems, such as pH and temperatureresponsive systems, is thought to be important for regulated and selective drug delivery in target sites. Because of their ability to respond to temperature changes in the presence of biological fluid, thermosensitive hydrogels are gaining popularity in drug delivery and tissue encapsulation [22]. Among them, thermoresponsive poly(N-isopropyl acrylamide) (PNIPAm) is the most well-investigated polymer due to its distinctive phase transition from an extended hydrophilic state to a collapsed hydrophobic state in water at around 32 ◦C. The PNIPAm-based hydrogels undergo an abrupt phase transition both below and above LCST (about 32 ◦C). In the presence of temperature stimuli, the drug-loaded PNI-PAm hydrogels undergo globular structural change, releasing the loaded drugs from the hydrogel network [23]. At swelling temperatures below LCST, the cross-linked hydrogel formed with PNIPAm polymer is hydrophilic. They, on the other hand, acquire a collapsed state whenever the temperature in the aqueous medium is above the LSCT.

Aside from thermoresponsive hydrogels, dual-stimuli responsive hydrogels are produced by copolymerizing two or more appropriate monomers. These pH-responsive hydrogels are a type of stimuli-responsive material used in biological applications. In response to pH variations, such hydrogel systems regulate drug release from the drugloaded hydrogel. These pH-responsive hydrogels are often synthesized from polymers with weakly basic (-NH2) or weakly acidic (-COOH) functional units [16,24]. The drug release can be accomplished by the protonation and deprotonation of these functional groups present in hydrogels. Both pH and temperature-responsive hydrogel systems are required for specific biomedical applications such as synergistic chemo-photothermal therapy and magnetic hyperthermia-induced drug delivery applications. Copolymerization of NIPAm with appropriate pH-sensitive monomers bearing basic functional groups in the presence of a limited number of cross-linkers results in dual pH and temperature-stimuli-responsive hydrogels [25].

We developed a thermo- and pH-responsive PNIPAm-based copolymer by combining polyacrylamide (PAAm) units cross-linked with melamine (Mel) units to produce PNIPAm-co-PAAm-Mel copolymer hydrogel (HG). The thermoresponsive PNIPAm has used swelling–deswelling behavior under temperature changes to achieve its sharp phase transition. Because of its basic amine functional groups, PAAm has a pH-responsive segment that can act as a drug-binding site. Additionally, Mel units have been employed to increase the cross-linkage of the copolymer network as well as the number of drug-binding sites. In order to characterize the synthesized PNIPAm-co-PAAm-Mel HG material, various experimental methods such as 1H NMR, FTIR, SEM, and zeta potential were used. Curcumin (Cur) was used as a model cargo to assess in vitro drug loading and pH and temperature-responsive release characteristics. The in vitro drug release study performance at 8 h revealed that under combined pH and temperature stimuli, nearly complete release of Cur occurs at 45 ◦C and pH 5.0, compared to approximately 75% release only at pH stimuli (pH 5.0) or approximately 58% release only at temperature stimuli (45 ◦C), respectively. As

a result, the prepared dual-stimuli responsive PNIPAm-co-PAAm-Mel HG system may be used for selective drug delivery to the target region.

#### **2. Materials and Methods**

#### *2.1. Reagents and Chemicals*

Acrylamide (AAm, 99%), melamine (Mel, 99%), 2,2-azobisisobutyronitrile (AIBN, 12 wt.% in acetone), N-Isopropylacrylamide (NIPAm, 97%), ethanol (99%), tetrahydrofuran (THF, 99.9%), diethyl ether (99.7%) and curcumin (98%), were purchased from Sigma Aldrich Chemical Co., Saint Louis, MO, USA, and used as received.

#### *2.2. Synthesis of PNIPAm-co-PAAm Copolymer*

The PNIPAm-co-PAAm copolymer was synthesized via a free radical polymerization process using AIBN as the initiator [26]. 2.0 g (17.5 mmol) NIPAm and 1.38 g (17.7 mmol) AAm were solubilized in a 50 mL two-necked round bottom flask containing 15 mL dry THF solvent for this experiment. The reaction flask was then continuously purged with nitrogen gas for 45 min before adding around 0.05 g of AIBN in THF (0.5 mL) and performing the reaction at 68 ◦C for 24 h under inert conditions. The resulting viscous mass was then precipitated in hexane (100 mL). This precipitation procedure was repeated five times to remove the unreacted monomer, and the white precipitate that formed was vacuum dried at room temperature. The copolymer was named PNIPAm-co-PAAm copolymer (Scheme 1A).

**Scheme 1.** The scheme represents the (**A**) preparation of PNIPAm-co-PAAm copolymer; (**B**) Mela-Glu cross-linked PNIPAm-co-PAAm HG system.

#### *2.3. Synthesis of Glutaraldehyde-Modified Melamine Precursor*

Approximately 1.0 g (7.9 mmol) melamine was mixed in 25 mL of water: ethanol (40:60 vol/vol) mixture for this reaction. In the presence of an acetic acid catalyst, 2.38 mL (2.3 mmol) of glutaraldehyde was added. The reaction was carried out at 90 ◦C for 48 h. The product was purified and recrystallized from hot methanol after the reaction mixture was concentrated using a rotary evaporator. The final product was designated as Mela-Glu precursor (Scheme 1B).
