Response Surface Optimization of Curcumin Oil-Loaded Dual-Crosslinked PVOH/CMC/Gellan Gum Hydrogels with Controlled Release and Anti-Inflammatory Activity

Abstract

Wound-related inflammatory pain is a major contributor to wound healing success and requires wound-specific therapeutic platforms with minimal systemic adverse effects. This study builds a dual-crosslinked polyvinyl alcohol (PVOH)/carboxymethyl cellulose (CMC)/gellan gum hydrogel system with optimized mechanical strength and sustained anti-inflammatory drug delivery by developing predictive mathematical models using response surface methodology with central composite design. The effects of citric acid (5–15% w/w) and dialdehyde carboxymethyl cellulose (DCMC, 0.0125–0.0375% w/w) on mechanical properties were systematically evaluated. The optimal formulation (2.23 g low-acyl gellan gum, 1.00 g high-acyl gellan gum, 0.02% DCMC, 10.21% citric acid) achieved firmness of 1.27 ± 0.06 N, rupture strength of 24.24 ± 0.52 N, and compressive strength of 41.91 ± 0.62 kPa. Curcumin oil incorporation yielded 82% cumulative release over 360 min following Korsmeyer–Peppas kinetics (R² = 0.9887, n = 0.8773). Cell viability exceeded 70% throughout the release period, confirming biocompatibility. The hydrogel strongly inhibited reactive oxygen species (ROS) and nitric oxide (NO) production in lipopolysaccharide-stimulated macrophages (p < 0.001) and enhanced macrophage migration, increasing wound closure from 40–80% (p < 0.001). This dual-crosslinked hydrogel shows great potential for localized inflammatory pain relief.

1. Introduction

Inflammatory pain management of wounds and tissue injuries is an urgent issue in clinical practice, which greatly affects the quality of life of patients, treatment compliance, and healing. The interaction of tissue damage, activation of immune cells, release of pro-inflammatory mediators such as reactive oxygen species (ROS), nitric oxide (NO), prostaglandins, and pro-inflammatory cytokines is a complex phenomenon leading to inflammatory pain by sensitizing pro-inflammatory cytokines and enhancing pain signaling pathways [1]. Conventional approaches to pain management that use systemic analgesics tend to cause poor local analgesia and systemic adverse reactions. The clinical requirement is, therefore, local, non-systemic therapeutic platforms that are able to provide long-term pain relief by targeting the inflammatory cascade directly at the wound site [2,3,4]. Hydrogel dressing has become a promising alternative to this, as it can provide structural support along with moisture control as well as a controlled-release system of anti-inflammatory and analgesic bioactive compounds [4,5,6]. Natural biopolymers, including gellan gum and carboxymethyl cellulose (CMC), are becoming increasingly popular in recent years as starting materials in the hydrogel fabrication process since they are inherently biocompatible, biodegradable, have a low level of immunogenicity, and possess versatile functional groups allowing the customization of mechanical and biological properties [7,8,9,10,11].

Although hydrogel-based pain management systems have therapeutic potential, the conventional single-polymer hydrogel has severe limitations preventing its clinical translation. One of them is a lack of mechanical stability during physiological conditions, resulting in early degradation, loss of structural integrity, and poor retention at the wound location-factors, which affect its ability to deliver drugs and provide effective pain relief over time [2,12]. Also, efficient delivery of hydrophobic anti-inflammatory agents, e.g., curcumin, is becoming an ongoing challenge [13,14]. Curcumin is a polyphenolic derivative of Curcuma longa that was widely examined regarding its potential in inhibiting essential inflammatory pathways, such as cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-kB), and inflammasome activation [15,16,17]. Curcumin directly suppresses the biochemical mediators of peripheral and central sensitization of inflammatory pain states by suppressing ROS production, NO synthesis, and release of pro-inflammatory cytokines [16,17]. Nevertheless, poor water solubility, high sensitivity to physiological pH and light environment, low bioavailability, and difficulties in obtaining long-term therapeutic levels at target locations severely limit the clinical usefulness of curcumin [18,19]. The use of encapsulation in hydrogel matrices generates further technical issues such as phase separation, uncontrolled burst-release, and bioactivity loss [15,20]. Hence, the new hydrogel formulations will be capable of not only showing good mechanical behavior, but also be able to maintain controlled release of hydrophobic anti-inflammatory agents as well as manage the inflammatory pain pathways [6,13].

This study uses a strategic combination of polyvinyl alcohol (PVOH), CMC, and gellan gum to develop a multi-component hydrogel system that is best suited to applications in the field of pain-relief due to the ability to deliver the anti-inflammatory drugs over time [21]. PVOH also adds to good film-forming qualities, mechanical strength, and moisture retention, which are critical in keeping it in contact with the wound site and providing uniform drug release [22]. CMC improves viscosity, muco-adhesion, and biocompatibility to enable direct contact with the wound tissue and high residence time [21,23]. Gellan gum is a microbial exopolysaccharide, which is in low-acyl and high-acyl forms and acts as the major gelling ingredient [7,24]. Low-acyl gellan gum is used to produce rigid and brittle gels as a result of strong ionic interaction, and high-acyl gellan gum is used to produce soft and elastic gels as a result of weaker hydrogen bonding and hydrophobic interactions [10,12]. The two forms can be combined to form a modulated gel texture, elasticity, and mechanical resilience to produce a comfortable, conformable dressing that prevents mechanical irritation and secondary pain with structural integrity [8,25]. Importantly, critically, the strategy of the studies is dual-crosslinking to improve the structural and functional performance of the hydrogel [26]. It is chemically crosslinked using dialdehyde carboxymethyl cellulose (DCMC), prepared by periodate oxidation of CMC, which introduces aldehyde groups that form covalent acetal/hemiacetal linkages with hydroxyl groups on the polymer chains [12,21,27]. Simultaneously, a secondary chemical crosslinking is triggered by citric acid, a biocompatible polycarboxylic acid that esterifies with hydroxyl groups on the polymer backbone under controlled thermal conditions [11,28,29]. This two-fold crosslinking technique not only enhances the three-dimensional polymer network, but also allows one to control the kinetic characteristics of drug release with great precision—a necessary criterion towards sustained pain relief through the ability to sustain pharmacologically active anti-inflammatory concentrations over prolonged intervals [10,26,28].

In addition, this hydrogel system includes curcumin oil, as the main anti-inflammatory and analgesic bioactive ingredient [15,30]. Curcumin oil, a lipophilic curcumin extract that includes curcuminoids and synergistic essential oils, is more stable, has a higher encapsulation efficiency, and may have a better analgesic effect than pure curcumin powder because of the presence of complementary bioactive substances [18,30]. Particularly in the management of pain, the multi-targeted anti-inflammatory activity of curcumin is applicable. Curcumin directly prevents COX-2 and inducible nitric oxide synthase (iNOS), thus decreasing the formation of prostaglandin E2 (PGE2) and NO, which are key agents that sensitize nociceptors and amplify transmissions of pain [16,17]. Moreover, curcumin inhibits the formation of ROS, which, in addition to causing tissue damage directly, opens transient receptor potential (TRP) channels and other pain-sensitive structures [17,19]. Curcumin also reduces the production of Interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), which are cytokines that are central to the process of inflammatory hyperalgesia because of their modulation of macrophage polarization and inhibition of inflammasome activation [4,16]. Nonetheless, the clinically significant levels of pain reduction require the sustained therapeutic levels of curcumin at the wound bed during the inflammatory period of the healing [20,31]. Burst release results in short-lasting effects and possible cytotoxicity, and a lack of sufficient release cannot suppress inflammatory pain mediators [31,32]. Thus, the development of a hydrogel network that could control the kinetics of curcumin release based on the crosslinking density, polymer composition, and matrix structure is essential to the desired successful and maintained pain management [13,20,26].

There are multiple prior studies that cover the different hydrogel systems to deliver curcumin and to heal wounds, but each system has different shortcomings, as will be covered in our present work. As an example, hydrogels made of chitosan and curcumin showed excellent biocompatibility but were poorly degraded, and burst release was uncontrolled, which shortened the duration of therapeutic action [33,34]. The use of alginate–curcumin hydrogels for wound healing showed potential, but lacked mechanical stability in physiological environments, which causes an early collapse of the structure [35]. The biocompatible single-crosslinked gellan gum systems did not have the mechanical strength needed to wear continuously and release drugs precisely [5]. Dual-network hydrogel systems based on physical and chemical crosslinking have been more recently discussed as a technique to improve mechanical strength; although these systems were based on synthetic polymers that might be cytotoxic or could not efficiently balance mechanical strength and drug release kinetics [36,37]. More importantly, none of the past studies have employed response surface methodology to systematically optimize the dual-crosslinking parameters to obtain the concurrent control of mechanical properties and continuous curcumin release, nor have they systematically assessed the anti-inflammatory processes that may be of particular importance to pain control (ROS/NO suppression and macrophage migration regulation).

This study optimally uses a two-crosslinked PVOH/CMC/gellan hydrogel of gum with oil of curcumin to achieve continuous anti-inflammatory and analgesic pain relief. The interactive effects of major formulation variables such as low-acyl gellan gum concentration, high-acyl gellan gum concentration, DCMC concentration, and citric acid concentration were investigated using response surface methodology (RSM), having a central composite design (CCD), in order to study their effects on firmness, rupture strength, and compressive strength [3]. Through the ideal formulation parameters and mathematical models that predict, this study will develop a hydrogel system that balances mechanical strength and long-term delivery of the anti-inflammatory drug to treat pain. The structural characteristics and molecular interactions of the hydrogel matrix were characterized using SEM, FTIR, and TGA. The analgesic efficacy of the optimized hydrogel was quantified in cellular in vitro, release kinetics, and ROS and NO inactivation of lipopolysaccharide (LPS)-induced macrophages, which is directly associated with pain caused by inflammation [16,38]. Macrophage migratory modulation was also examined since the prolonged macrophage infiltration leads to excessive inflammatory pain and hyperalgesia [4,16]. The paper offers a rational understanding of the next-generation pain-relief hydrogel dressing that integrates structural integrity and control of anti-inflammatory delivery to improve patient clinical pain management and wound care, in which inflammatory pain is the most significant risk factor to patient outcomes and quality of life [2,4,6].

2. Results and Discussion

2.1. CCD for Optimization of Process Parameters

A central composite design (CCD) was employed to optimize the mechanical properties by systematically varying the ratio of low-acyl and high-acyl gellan gum (1–3 g), DCMC concentration (0.0125–0.0375% w/w), and citric acid concentration (5–15% w/w). The experimental design was conducted as detailed in

Table 1. Experimental design along with predicted (model-generated) and actual (experimental) mechanical properties of hydrogel formulations.

Run	Independent Variables				Responses
	Low-acyl Gellan Gum (g)	High-acyl Gellan Gum (g)	DCMC Concentration (%w/w)	Citric Concentration (%w/w)	Firmness (N)		Rupture Strength (N)		Compressive Strength (kPa)
					P †	A ‡	P †	A ‡	P †	A ‡
1	2.00	2.00	0.025	10.00	2.63	1.87	12.06	36.30	25.77	61.64
2	2.00	0.28	0.025	10.00	1.60	0.92	21.89	20.39	58.72	22.47
3	2.00	2.00	0.025	10.00	0.61	1.68	17.33	37.13	34.09	73.28
4	1.00	1.00	0.0125	5.00	0.08	0.09	41.04	6.73	77.53	8.28
5	2.00	2.00	0.025	1.41	0.93	0.37	4.74	16.04	9.44	31.91
6	2.00	2.00	0.025	10.00	1.58	1.61	31.80	27.21	59.90	62.81
7	3.00	1.00	0.0125	5.00	0.17	0.10	4.82	7.69	5.87	15.17
8	3.00	3.00	0.0375	5.00	0.47	0.77	5.81	16.68	14.90	32.93
9	3.00	1.00	0.0375	5.00	0.74	0.11	16.32	46.91	45.79	82.05
10	3.00	1.00	0.0125	15.00	0.55	2.63	10.04	5.69	20.14	16.88
11	1.00	3.00	0.0375	15.00	1.27	1.19	6.31	7.02	14.24	15.37
12	1.00	3.00	0.0125	5.00	1.96	0.55	15.35	6.79	35.57	13.86
13	2.00	2.00	0.0464	10.00	1.58	0.54	31.80	21.07	59.90	42.46
14	2.00	2.00	0.025	10.00	0.86	1.35	10.75	29.11	23.03	57.44
15	2.00	2.00	0.025	10.00	1.58	1.62	31.80	29.91	59.90	59.03
16	3.00	3.00	0.0375	15.00	1.58	2.66	31.80	11.38	59.90	22.45
17	3.00	3.00	0.0125	15.00	1.04	2.45	8.64	21.50	21.05	42.43
18	1.00	3.00	0.0125	15.00	0.36	0.97	7.69	21.51	17.40	46.66
19	2.00	2.00	0.0035	10.00	0.05	0.49	8.39	6.95	14.56	15.71
20	1.00	3.00	0.0375	5.00	2.40	0.96	19.40	9.56	42.75	19.87
21	1.00	1.00	0.0375	5.00	0.98	0.29	23.88	28.56	32.41	60.37
22	3.00	3.00	0.0125	5.00	2.39	0.40	9.22	10.14	20.60	20.30
23	2.00	2.00	0.025	18.59	1.58	2.48	31.80	9.62	59.90	20.02
24	0.28	2.00	0.025	10.00	0.71	0.96	23.49	16.63	46.17	44.41
25	3.72	2.00	0.025	10.00	2.35	1.15	2.53	16.06	11.44	51.97
26	1.00	1.00	0.0375	15.00	0.37	1.02	30.66	12.36	58.30	24.40
27	2.00	2.00	0.025	10.00	1.60	1.59	19.47	36.90	27.30	53.70
28	1.00	1.00	0.0125	15.00	1.58	0.75	31.80	5.38	59.90	12.34
29	3.00	1.00	0.0375	15.00	0.96	1.86	23.31	14.37	46.92	38.36
30	2.00	3.72	0.025	10.00	0.45	1.42	19.11	17.44	34.30	29.10

^† P: Predicted value, ^‡ A: Actual value.

, featuring a matrix of 30 formulations that facilitated a thorough statistical analysis of variable interactions and quadratic effects on key mechanical properties: firmness, rupture strength, and compressive strength [39,40].

The firmness of gel pads was measured to be between 0.09 and 2.66 N (Table 1), and the results were substantially influenced by the factors, as evidenced by regression and ANOVA analyses (Table S1). The most important factor was found to be citric acid concentration (p < 0.0001). The supports hydrocolloid science results indicating that changing acidulants is necessary to improve gellan networks by facilitating more ionic cross-linking [7,24]. The mechanism is linked to the protonation of carboxylate groups on gellan gum chains, which promotes direct ionic bonding and the formation of multivalent ion bridges, resulting in more cohesive and stiffer networks [41]. Both low-acyl and high-acyl gellan gums significantly influenced firmness (p = 0.0001 and p = 0.0025), which confirmed that the gelling mechanism of gellan gum is facilitated by the formation of double-helical structures and cation-mediated aggregation [8]. Increasing total gellan content proportionally increases the number and density of these network junctions, thus enhancing mechanical integrity and resistance to deformation [9]. Interestingly, DCMC, although not a significant linear factor (p = 0.3687), exhibited a substantial influence in its quadratic term (C², p < 0.0001), indicating nonlinear and threshold-dependent effects. This behavior illustrates the complicated structure of multi-hydrocolloid systems, wherein secondary polysaccharides can transition from serving as supportive cross-linkers to functioning as network disruptors upon exceeding saturation levels, as evidenced by research findings on phase stability and structure breakdown at higher additive concentrations [10,12]. The resulting quadratic model for firmness was statistically significant, as indicated by the p-value (p < 0.0001), with an R² value of 0.95, an adjusted R² of 0.91, and a predicted R² of 0.77. An adequate precision of 16.46 measures the signal-to-noise ratio, for which a ratio greater than 4 is desirable. Notably, the interaction between low gellan gum and citric acid (AD, p < 0.0001) demonstrated a strong interaction effect, suggesting that optimal acidification enhances the cross-linking efficiency of the gellan matrix, thereby facilitating the formation of a tighter and stronger network. This finding aligns with models in the biopolymer gel literature, which illustrate the combinatorial enhancement of stiffness through cooperative interactions between gelling agents and acidic substances [8,24]. The quadratic equation generated from the models (Figure 1) is the following Equation (1):

C_Firmness = −1.84 + 0.19A + 0.51B + 114.44C + 0.04D + 0.005AB − 6.46AC + 0.08AD + 7.54BC − 0.013BD − 1.04CD − 0.14A2 − 0.099B2 − 2049.76C2 − 0.0006D2

(1)

The rupture strength of the gel pads exhibited significant variability (5.38–46.91 N), indicating the complex compositional dependencies and mechanical strength present in various formulations (Table 1). A quadratic response surface model was employed to confirm the adequacy and widespread utility of the model for material optimization. The model exhibited a statistically significant F-value and p-value (F = 13.47, p < 0.0001), accounting for 92.6% of the variance (R² = 0.9263; adjusted R² = 0.8875; Table S2), and exhibited a non-significant lack of fit (p = 0.6348). DCMC and citric acid were significant main effects (p = 0.0006 and p = 0.0375, respectively), demonstrating recognized ideas in hydrocolloid gel systems concerning the thickening action of DCMC and the cross-linking role of acids [2]. The concentrations of low-acyl and high-acyl gellan gum exhibited statistical non-significance (p = 0.0919 and p = 0.1735), indicating that these polysaccharides alone have no impact on rupture resistance. However, critical two-way interactions—DCMC × citric acid (CD, p = 0.0003) and high-acyl gellan gum × DCMC (BC, p < 0.0001)—were important indicators of mechanical strength, highlighting the collaborative behavior of the hydrocolloid networks, indicating more intermolecular bonding between the biopolymer matrix. This relationship corresponds with recent theories that characterize food gel integrity as dependent on the distribution of network linkages and interactions with secondary components [8,24]. The significant quadratic effects of DCMC concentration, citric acid concentration, low-acyl, and high-acyl gellan gum concentrations (C² and D², p < 0.0001; A², p = 0.0007; and B², p = 0.0039) illustrate that there are specific optimal ranges for these factors that maximize rupture strength. When these optimal levels are exceeded, the rupture strength decreases—probably because the gel network becomes too structured or separates into different phases. These problems are well-documented in recent studies on hydrocolloids and represent significant challenges when designing advanced gel materials [9]. These findings align with previous research on hydrocolloid gels, demonstrating the criticality of both linear and nonlinear polysaccharide-acidulant effects in achieving targeted textural properties [2]. The quadratic equation generated from the models (Figure 2) is the following Equation (2):

Rupture strength = −71.72 + 19.14A + 15.99B + 3381.93C + 4.78D − 0.43AB + 136.21AC − 0.29AD − 460.12BC + 0.88BD − 79.31CD − 4.30A2 − 32560.72C2 − 0.22D2 − 3.43B2

(2)

The compressive strength response surface model was highly significant (F = 22.36, p < 0.0001, Table S3), accounting for 91.16% of the total variation (R² = 0.9116, adjusted R² = 0.9116, predicted R² = 0.834) with a non-significant lack-of-fit (p = 0.683), confirming the model reliability. Low-acyl gellan gum (p = 0.011) and DCMC concentration (p = 0.001) showed significant linear effects, indicating their role in enhancing the density and uniformity of the cross-linked network. This mechanism has been previously emphasized in foundational studies on biopolymers and physical gels [3,10]. Although citric acid (p = 0.075) and high-acyl gellan gum (p = 0.266) did not achieve statistical significance when analyzed independently, but high-acyl gellan gum interaction with DCMC or citric, and citric-DCMC interaction (p < 0.001) demonstrated a significant relationship. Non-linear modeling and verified response surface methodology (RSM) are essential tools for gel engineering, as evidenced by the presence of significant quadratic terms across key factors (p < 0.001), except for low-acryl gellan gum (p = 0.095). The quadratic equation is generated as Equation (3) and illustrated in models (Figure 3):

Compressive strength = −141.49 + 15.23A + 51.19B + 6485.95C + 9.30D − 1.54AB + 210.75AC − 0.33AD − 925.71BC + 1.42BD − 155.34CD − 2.59A² − 10.17B² − 57938.75C² − 0.40D²

(3)

To verify the RSM models further and more clearly visualize the impact of individual factors, experimental values were compared against the projected trends (Figure S1). The plots show that there was a great level of agreement between the distribution of the experimental data and how the models were expected to curve. The consistency of these relationships is justified by the statistics of the global model that demonstrated Global R² of 0.95, 0.91, and 0.88 of firmness, rupture strength, and compressive strength, respectively. Moreover, the adequate precision of 16.46 of the firmness model means that there is a strong signal-to-noise ratio. Figure S1 demonstrates that, with a rise in citric acid concentration (5–15 w/w), the firmness (0.3–2.5 N), and compressive strength (15–45 kPa) were progressively enhanced, and the necessity of chemical crosslinking in the formation of the network. Likewise, there was a non-linear behavior of DCMC concentration; the optimum mechanical properties were found to be at intermediary levels (0.02–0.025% w/w), and excessive ionic crosslinking at higher concentration resulted in a decreased network integrity.

The response surface methodology was employed to validate the model by determining the optimal concentrations of low-acyl and high-acyl gellan gum, DCMC, and citric acid. The optimized gel pad contains low-acyl gellan gum (2.23 g), DCMC (0.02% w/w), citric acid (10.21% w/w), and high-acyl gellan gum (1.00 g). The mechanical properties revealed that the gel pads exhibited a firmness of 1.27 ± 0.06 N, a rupture strength of 24.24 ± 0.52 N, and a compressive strength of 41.91 ± 0.62 kPa. The mechanical properties indicate a moderately firm gel structure, consistent with the dual-crosslinking approach that combines physical and chemical mechanisms [10]. The combination of low and high-acyl gellan gum results in a balanced network structure that enhances both flexibility and stability. The low concentration of DCMC (dialdehyde CMC) plays a key role in stabilizing the network through aldehyde-hydroxyl crosslinking, forming covalent acetal linkages that enhance mechanical strength while maintaining network flexibility [23]. In addition, the higher concentration of citric acid acts as a significant crosslinking agent through esterification, enhancing network density and thermal stability, which is associated with increased compressive strength suitable for soft tissue applications [28,29]. The rupture strength indicates the hydrogel’s ability to resist mechanical damage, possibly due to the beneficial interaction between gellan gum and chemical crosslinkers that balances toughness and elasticity [10,11].

2.2. Gel Pads with Curcumin Oil Characterization

2.2.1. Mechanical Properties of Gel Pad with Curcumin Oil

The incorporation of curcumin oil significantly influenced the mechanical properties of the gel pads (p < 0.001, Table S4). Gel pads without curcumin oil exhibited a firmness of 1.27 ± 0.06 N, a rupture strength of 24.24 ± 0.52 N, and a compressive strength of 41.91 ± 0.62 kPa. Upon addition of 20% curcumin oil, these values decreased to 0.89 ± 0.01 N for firmness, 17.47 ± 0.31 N for rupture strength, and 30.87 ± 0.53 kPa for compressive strength. The reduction in mechanical performance is due to the disruption of the gel matrix by hydrophobic curcumin oil, which acts as a plasticizer and interferes with the polymer network, resulting in a softer, less rigid gel structure [13,14]. These trends align with prior research indicating that incorporating hydrophobic compounds into hydrogel systems reduces mechanical strength by reducing crosslinking density [13,14]. Although mechanical properties are reduced, curcumin-loaded hydrogels offer enhanced bioactive functionality, including increased antioxidant and anti-inflammatory effects, as well as superior drug-release profiles [13,20]. Therefore, while the incorporation of curcumin oil provides valuable therapeutic benefits, the mechanical properties must be optimized to ensure suitability for the intended biomedical applications [13,14].

2.2.2. Morphology of Gel Pad with Curcumin Oil

As shown in Figure 4A,B, the scanning electron micrograph of the freeze-dried gel pad revealed a porous, irregular structure and a loosely packed surface, indicating an open polymer network with weak physical cohesion between polysaccharide chains, which is consistent with the study by Khorshidi et al. (2023) [25]. Such a structure is typical of ungelled or minimally crosslinked gellan gum matrices and has been reported in similar hydrocolloid systems. The available holes are related to the storage of ice crystals within the frozen gel structure, which is then sublimed in the freeze dryer. Moreover, dialdehyde carboxymethyl cellulose (DCMC) and citric acid can serve as crosslinking agents, improving surface smoothness and compactness. Due to DCMC and citric acid forming additional ester and ionic crosslinks, reinforcing the structural integrity of the matrix [5,25]. In addition, digital microscopy provided evidence of successful encapsulation. Compared to the uniform matrix of the gel pad without oil (Figure 4C), the essential oil-loaded hydrogel exhibited numerous distinct light reflections dispersed throughout the polymer structure (Figure 4D). These reflections correspond to essential oil microdroplets entrapped within the interstitial spaces of the 3D hydrogel network. This result is consistent with the scanning electron microscopy (SEM) analysis, which confirmed the encapsulation of the essential oils within the pores and interstitial spaces of the 3D hydrogel structure. The evidence between our microscopic observations and the other reports confirms the role of the hydrogel matrix as a reservoir matrix, considering the intrinsic volatility and instability of essential oils. Similar sponge-like structures have been reported in composite systems where essential oils are stabilized within a physically or covalently cross-linked wall architecture to achieve a sustained and regulated release profile [30]. This entrapment in a 3D network not only proved the encapsulation success but also showed that the material could keep the bioactivity of essential oil by keeping them safe from the effects of the environment.

2.2.3. FTIR of Gel Pad

The FTIR spectrum of the gel pad displayed typical absorption bands of polysaccharides. A broad O–H stretching vibration at 3150 cm⁻¹ indicated hydroxyl groups and intermolecular hydrogen bonding. A C–H stretching band near 2900 cm⁻¹ originated from the glucose backbone in gellan gum. Strong symmetric and asymmetric stretching vibrations at 1325 and 1211 cm⁻¹, respectively, are linked to carboxylate (–COO⁻) groups. Additionally, peaks between 1150 and 1000 cm⁻¹ corresponded to C–O and C–O–C stretching in glycosidic linkages, as shown in Figure 5, panel A. These assignments correspond with previously reported gellan gum spectra [14,15,16,17,18]. In the gel pad with curcumin oil (Figure 5, panel B), the O–H stretching band shifted slightly, indicating hydrogen bonds between hydroxyl groups of gellan gum and phenolic groups of curcumin oil. Changes were also seen in the 1510 cm⁻¹ region, related to aromatic C=C stretching, and at 1720 cm⁻¹, possibly linked to curcumin’s conjugated diketone C=O vibrations. The glycosidic region from 1100 to 1000 cm⁻¹ showed subtle modifications, indicating structural adjustments in the polysaccharide backbone caused by curcumin oil addition. Spectral changes have been widely reported in biopolymer matrices containing curcumin or other hydrophobic bioactive compounds, indicating molecular interactions and the even dispersion of the active compound within the polymer network [18,19,29].

2.2.4. Thermal Property of Gel Pad

The TGA/DTG curves for the gel pad without or with curcumin oil are illustrated in Figure 6. The gel pad exhibited a single-step degradation, with weight loss occurring at 250.83 °C, indicating thermal depolymerization of the polysaccharide backbone, including degradation of the DCMC structure and citric acid’s role as a cross-linker (Figure 6A). The gel pad with curcumin oil reached a temperature of 260.83 °C (Figure 6B). The overall mass loss in the primary degradation step was approximately 24.52%, with a loss of about 7.47% for the gel pad with curcumin oil. This degradation pattern is consistent with previous reports on gellan gum and similar polysaccharides, in which thermal breakdown is dominated by dehydration, depolymerization, and cleavage of glycosidic linkages [26,42,43]. For the gel pad with curcumin oil, the initial degradation onset shifted to a higher temperature, from 260.83 to 414.13 °C, suggesting that thermal stability was enhanced due to the formation of a complex cross-linking biopolymer network. These findings align with previous studies that indicate the maximum degradation rate of curcumin occurs at 394 °C [9,15]. Because of the hydrophobic nature of curcumin oil, it acts as a physical barrier, reducing heat transfer and restricting the mobility of polymer chains. Also, the curcumin aromatic structure contributes to thermostability, thereby increasing resistance to thermal decomposition. This effect has been observed in other biopolymer systems containing hydrophobic bioactive oils, in which the dispersed phase reinforces the polymer matrix and improves thermal stability [15,44].

2.3. Curcumin Oil Released from Gel Pad

The release of curcumin oil from the gel pad exhibits a biphasic kinetic pattern (Figure 7), characterized by a rapid initial release phase during the first 120 min, followed by a slower, constant release phase lasting up to 360 min, resulting in approximately 82% cumulative release. The initial rise is attributed to the diffusion of loosely bound curcumin molecules from the hydrogel surface into the release media. This phase ensures rapid therapeutic concentrations, which are crucial for applications that require immediate access to the drug. The following slower release phase involves a combination of non-Fickian diffusion through the dense hydrogel matrix and the gradual breakdown of the polymer network, hence prolonging curcumin delivery and maintaining drug levels over time [4].

Kinetic modeling explained the complex mechanisms governing the release of curcumin from the dual-crosslinked PVOH/CMC/gellan gum hydrogel system (

Table 2. Fitting parameters and correlation coefficients (R²) of curcumin oil from PVOH/CMC/gellan gum hydrogel based in PBS buffer on various mathematical models. The parameters (k and n) and correlation coefficients (R²) were determined for five mathematical models.

Kinetic Model	Equation	Parameter	Value
Zero-order	${\displaystyle \frac{M_t}{M_{\infty }}}=k_{0}t$ + F0	k0	0.0001
		R2	0.9601
First-order	${\displaystyle \frac{M_t}{M_{\infty }}}=1-e^{-k_{1}t}$	k1	0.0086
		R2	0.9802
Higuchi	${\displaystyle \frac{M_t}{M_{\infty }}}=k_H{t}^{1/2}$	kH	0.0676
		R2	0.9742
Hixson–Crowell	${\displaystyle \frac{M_t}{M_{\infty }}}=100\left[1-{\left(1-k_{HC}t\right)}^3\right]$	kHC	0.0155
		R2	0.9730
Korsmeyer–Peppas	${\displaystyle \frac{M_t}{M_{\infty }}}=k_{KP}{t}^n$	kKP	0.0083
		n	0.8773
		R2	0.9887

and Figure 8). The Korsmeyer–Peppas model had the highest correlation (R² = 0.9887; n = 0.8773), signifying anomalous, non-Fickian transport influenced by the relationship of diffusion and polymer chain relaxation [32]. A significant correlation with the first-order model (R² = 0.9802) indicated concentration-dependent release behavior, whereas the Higuchi (R² = 0.9742) and Hixson–Crowell (R² = 0.9730) models revealed the primary diffusion and concurrent matrix erosion processes, respectively [31]. These findings collectively illustrate a biphasic release profile, characterized by a brief initial spike followed by sustained drug release, which effectively enhances curcumin bioavailability while addressing challenges associated with its hydrophobicity and rapid metabolism [4,13]. The controlled-release characteristics illustrate the potential of this hydrogel substrate for biomedical applications, particularly in wound healing and extended therapeutic treatments.

2.4. Cytotoxicity and Antioxidant Activities of Gel Pad with Curcumin Oil

The cell compatibility of the curcumin-released medium was confirmed via MTT cell viability assays, which showed a high safety profile for the hydrogel matrix (Figure 9a). The cell compatibility was conducted without reference to the anti-inflammatory assays. Notably, the cytotoxicity test did not involve the use of LPS; cells were incubated with curcumin-releasing medium (collected at 15, 60, 120, and 360 min of the drug release test) only, and incubated for 24 h. The medium of curcumin released kept the cells alive, at least up to 120 min of curcumin release, and the levels remained at or below 70% despite exposure to the 360-min release medium. The viability values are all higher than the ISO 10993-5-biocompatibility acceptance level of 70%, and this confirms the safety profile of the hydrogel system in the biomedical applications [ISO 10993-5:2009] [45]. This brief loss of viability at 360 min is explained by the cumulative exposure to curcumin and natural metabolism drop over the long culture times, which is in line with the published literature on curcumin-loaded biomaterials [21,27,43]. PVOH (polyvinyl alcohol), CMC (carboxymethyl cellulose), and gellan gum are a polymer blend that is thought to be highly biocompatible and non-cytotoxic [46]. This confirms that the hydrogel and crosslinkers, as well as their release products, are non-toxic and suitable for extended biomedical applications [21,27]. Studies consistently demonstrate that these materials promote cell viability and are safe for use in biomedical applications such as tissue engineering, drug delivery, and wound dressings. The antioxidant potential was evaluated under LPS-induced stress conditions. LPS induction significantly elevated intracellular ROS levels to approximately 1.8-fold relative to the control (p < 0.001; Figure 9b). However, treatment with the curcumin-releasing medium induced a strong, time-releasing-dependent reduction in ROS generation, with the most significant inhibition occurring at 60 and 120 min of releasing medium (p < 0.001). This bioactive performance results from a sustained-release profile, governed by a transport mechanism, that consistently delivers phenolic groups of curcumin to scavenge free radicals and protect cells from oxidative damage [16].

2.5. Inhibition of Nitric Oxide Production and Macrophage Migration in LPS-Stimulated Raw 264 Cells by Gel Pad with Curcumin Oil

The hydrogel effectively modulated cell migration in a scratch wound healing test (Figure 10a,b). The stimulation of LPS caused an inflammatory hyper-migration, with a wound covered more than 80% in 48 h, a disease process of the excessive infiltration of macrophages, which results in chronic inflammation and chronic pain. Notably, therapeutic inhibition of this excessive migration was affected by the curcumin-releasing medium to a level of 40–58 reduction in closure (p < 0.001). This regulated migration is also clinically desirable because it avoids over-activation of macrophages, release of pro-inflammatory cytokines (IL-1β, TNF-α), and inflammatory hyperalgesia and chronic pain [4,16]. Additionally, the anti-inflammatory effects of the medium were assessed after establishing good cell compatibility and antioxidant activity, as shown in Figure 9. LPS markedly increased pro-inflammatory signaling, doubling nitric oxide (NO) production compared with control (Figure 10c)—a hallmark of inflammatory activation that directly contributes to pain sensitization. Treatment with the curcumin medium caused a significant, timepoint-dependent decrease in NO levels at all release time points (15, 60, and 120 min), with the most significant reduction relative to the releasing medium at 120 min (p < 0.001). This NO suppression is of critical therapeutic importance, as NO serves as a key mediator of inflammatory pain through nociceptor sensitization and transient receptor potential (TRP) channel activation [6,17]. By reducing NO production, the curcumin-loaded hydrogel directly attenuates pain signaling pathways, supporting its application as a localized analgesic platform. Overall, these results show that the 3D hydrogel network effectively delivers bioactive curcumin, creating a regulated cellular environment ideal for tissue remodeling and relief from inflammation.

In summary, the relationships between the structural components of the hydrogel and the bioactivity of its functions are conceptualized in Figure 11. The dual-crosslinking method ensures the mechanical stability, as well as provides a controlled reservoir of curcumin oil. The architecture enables a biphasic release profile, which effectively suppresses oxidative stress and pro-inflammatory signaling (NO/ROS), focusing on the inflammatory microenvironment. This combined platform is opportune information-based research toward local pain relief dressing development.

3. Conclusions

This study was able to develop and optimize a dual-crosslinked system of PVOH/CMC/gellan gum hydrogel in which curcumin oil was incorporated that has a significant potential as a localized pain-relief system that is applicable in the management of wounds and tissue damage-related inflammatory pain. We optimally balanced the formulation parameters (citric acid 10.21% w/w, DCMC 0.02% w/w, low-acyl gellan gum 2.23 g, high-acyl gellan gum 1.00 g) using response surface methodology and central composite design to achieve an optimal compromise of mechanical integrity (firmness 1.27 ± 0.06 N, rupture strength 24.24 ± 0.52 N, compressive strength 41.91 ± 0.62 kPa).

The most important innovation of this piece is the dual-crosslinking construction based on ionic gelation through the use of DCMC and chemical esterification (through the use of citric acid), which forms a mechanically strong three-dimensional polymer network that can release curcumin continuously and preserve its structural integrity at physiological conditions. The hydrogel was found to have great biocompatibility (cell viability >70% over the release period) and an anti-inflammatory effect, which reduced several important pain-causing mediators of inflammation, such as reactive oxygen species (ROS) and nitric oxide (NO), in LPS-stimulated macrophages (p < 0.001). Significantly, the system regulated the migration of macrophages, avoiding excess inflammatory cell infiltration linked to the manifestation of chronic pain.

Predictive mathematical modeling and detailed anti-inflammatory evaluation are proposed to offer a rational and data-driven approach to the design of hydrogels to be used in pain management. This dual-crosslinked hydrogel platform overcomes key flaws of current systems-low mechanical stability, uncontrolled burst release, and absence of localized anti-inflammatory action placing it as a promising candidate for clinical translation in the treatment of wound-related inflammatory pain, burns, post-surgical pain, and other localized inflammatory diseases.

Even with these promising findings, more rigorous, in vivo pain assessment studies are required to confirm these findings to be used in clinical practice. Future research might be interested in the hydrogel analgesic efficacy in validated models of animal pain behavior (thermal hyperalgesia, mechanical allodynia, spontaneous pain behavior), measurement of the reduction of pain-related inflammatory mediators in wound exudates, long-term safety, and tissue integration as well as its use in a diversity of pain-associated wound models. Pain outcomes (visual analog scale, numerical rating scale, quality-of-life indicators) should be tested to assess the efficacy of the clinical and the best dose schedules. This platform may be enhanced with nerve growth factor inhibitors, local anesthetic or synergistic analgesic agents. Finally, the research will provide a foundation for next-generation pain-relief hydrogel dressings, which will combine structural integrity with anti-inflammatory, regulated, and analgesic release, thereby enhancing the transfer of functional biomaterials into clinical wounds and tissue-related inflammatory pains and improving outcomes.

4. Materials and Methods

4.1. Chemicals and Reagents

DMEM (12800017), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and trypsin-EDTA solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Additionally, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), dimethyl sulfoxide (DMSO), and lipopolysaccharides (LPS) (L7770) were purchased from Sigma Chemical, Inc. (St. Louis, MO, USA). Curcumin oil was purchased from Specialty Natural Products Public Company Limited (Chonburi, Thailand). The amount of curcumin in turmeric extract was found to be about 5% w/w.

4.2. Cell Culture

The murine macrophage cell line (RAW 264.7) was provided by Prof. Dr. Ratana Banjerdpongchai (Department of Biochemistry, Faculty of Medicine, Chiang Mai University). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplement with 10% heat-inactivated fetal bovine serum. Cells were culture at 37 °C in an incubator supplemented with 5% CO_2.

4.3. Preparation of Gel Mixture

Carboxymethyl cellulose (CMC) and polyvinyl alcohol (PVOH) were prepared and dispersed in distilled water at 0.5% w/v using a stirrer (IKA, Baden-Württemberg, Germany). Low-acyl and high-acyl gellan gum with different weight ratios were dissolved in distilled water at a concentration of 0.75% w/v. CMC and PVOH solutions were blended in a 4:1 ratio for 15 min. The gellan gum solution was poured into the CMC/PVOH solution at a 1:1 ratio. The DCMC and citric acid with varying concentrations were then added to the CMC/PVOH/GG solution. The mixture was stirred at 1000 rpm for 20 min. The CMC/PVOH/GG 60 mL was poured into the mold and left at room temperature for 15 min to allow the gels to set.

The preparation of gel pads utilized the optimal ratio of low-acyl and high-acyl gellan gum at a concentration of 0.75% w/v, mixed with the CMC/PVOH solution for 20 min. Then, 0.5% v/v of liquid paraffin and 25% v/v of curcumin oil were added to the CMC/PVOH/GG solution, and the mixture was stirred well for 30 min using a stirrer. Following this, the optimal concentrations of DCMC and citric acid, as determined by the CCD, were added to the mixed solution. The curcumin oil gel mixture was poured into the mold and left at room temperature for 15 min to allow the gel to set.

4.4. Experimental Design and Model Development

A central composite design (CCD) within the framework of response surface methodology (RSM) was implemented using the Design Expert Software (version 11, Stat-Ease Inc., Minneapolis, MN, USA). This design was selected to efficiently evaluate the linear, quadratic, and interaction effects of the chosen variables on the mechanical performance of the gel pads. Four independent variables were designated as factors: the ratio of low-acyl gellan gum (A), the ratio of high-acyl gellan gum (B), the concentration of the crosslinking agent DCMC (C), and the concentration of citric acid (D). These variables were selected due to their critical role in modulating the structural, crosslinking, and mechanical characteristics of gellan gum-based gels.

The CCD was structured with three coded levels (−1, 0, and +1) for each factor, as shown in

Table 3. Independent variables and their levels.

Variables	Symbol	Unit	Code Level
			−1	0	1
Low gellan gum	A	g	1	2	3
High gellan gum	B	g	1	2	3
DCMC concentration	C	%w/w	0.0125	0.025	0.0375
Citric concentration	D	%w/w	5	10	15

. The response variables targeted for optimization were firmness, rupture strength, and compressive strength. The regression analysis of the experimental data was carried out using the following polynomial model, as shown in Equation (4).

$$Y={\beta }_0+\sum _{i=1}^k{\beta }_i{X}_i+\sum _{i=1}^k{\beta }_{ii}{X}_i^2+\sum _{i<j}^k\sum {\beta }_{ij}{X}_i{X}_j+\epsilon$$

(4)

4.5. Mechanical Properties

A universal testing machine (LS1, Lloyd Instruments, Bognor Regis, UK) with a maximum capacity of 1 kN was employed to evaluate the mechanical properties of the hydrogels, including firmness, rupture strength, and compressive strength. Hydrogel samples were prepared with a diameter of 5 cm and a height of 5 cm. Measurements were performed using a cylindrical probe (10 mm diameter). The firmness tests were conducted at a pre-test speed of 8.0 mm/s, followed by both test and post-test speeds of 10.0 mm/s, with a trigger force of 10.0 g. During testing, the probe was immersed to a depth of 3.2 cm into the hydrogel sample and then returned to its initial position. All measurements were carried out in triplicate.

Compression tests were performed on cylindrical composite gel samples (diameter: 5 mm, height: 5 mm) using a universal testing machine (LS1, Lloyd Instruments, Bognor Regis, UK) equipped with a 1 kN force sensor. The samples were compressed at a constant rate of 0.5 mm/min until a maximum compressive strain of 90% was reached. All measurements were carried out in triplicate.

4.6. Fourier-Transform Infrared (FT-IR) Spectroscopy

The FTIR spectra of freeze-dried samples were acquired using an attenuated total reflectance (ATR) system. An FTIR spectral analysis was conducted on the JASCO FT/IR-4700 (JUSCO, Hachioji, Tokyo, Japan). For each sample, FTIR spectra were recorded in the absorbance mode from 3400 to 600 cm⁻¹ with a resolution of 4 cm⁻¹. Three replicates were collected for each sample.

4.7. Thermogravimetric Analysis (TGA)

The thermal stability of the dried samples was evaluated with a thermogravimetric analyzer (Thermal Analysis System TGA/DSC 3+; Mettler-Toledo International, Columbus, OH, USA). The samples were weighed (6 ± 1.0 mg, OD weight) and used to analyze the thermal stability of the sample under a nitrogen atmosphere (flow of 50 mL/min) from 25 to 600 °C at a heating rate of 10 °C/minute with a gas flow of 10 mL/min. The average of three experiments was used to determine the thermal stability of the films.

4.8. Scanning Electron Microscopy (SEM)

The surface morphology of freeze-dried gel was imaged using a scanning electron microscope (JSM-5910LV; JEOL Ltd., Akishima, Tokyo, Japan) operating at an accelerating voltage of 15 kV. The carbon adhesive was employed to mount the dried samples on aluminum stubs. A vacuum sputter coater (GSL-1100X-SPC12-LD, MTI Corporation, Richmond, CA, USA) was used to coat the samples with gold for 60 s, resulting in a thickness of 20 nm. Each image was acquired at a magnification of 100–500X.

4.9. Curcumin Oil Releasing

The in vitro release kinetics of curcumin oil from the gel pads were evaluated under controlled conditions. Gel pads (2 cm in diameter and 1 cm in height) were individually immersed in 17 mL of phosphate-buffered saline (PBS, pH 7.4) and incubated at 37 °C with continuous agitation at 90 rpm. Samples of the release medium, curcumin-released medium, were collected at predetermined time intervals over a 360-min period for analysis of curcumin oil release. The concentration of curcumin oil was estimated using a spectrofluorimetric 96-well plate. The relative fluorescence intensity (FI) was measured in comparison to PBS alone with an emission and excitation wavelength at 528 nm and at 485 nm, respectively. The standard curve of curcumin oil was conducted at concentrations ranging from 1.39 to 22.28 mg/mL. The fluorescence intensity (FI) of the sample was subtracted from the blank reading to obtain corrected FI.

4.10. Cytotoxicity Assay

RAW264.7 cells were seeded at 3000 cells/well in a 96-well culture plate. After 16 h, cells were treated with curcumin-released medium (15, 30, 60, 120, 240, and 360 min time points) and incubated for 24 h at 37 °C under 5% CO₂. After removing the culture medium, MTT stock dye solution (5 mg/mL in PBS), 15 µL was added to each well and incubated for 2 h. The medium from each well was taken out as much as possible, leaving purple formazan crystals. DMSO (200 µL) was added to each well to dissolve the crystals, and the plates were shaken for 10 min. The absorbance was detected at 540 nm with a reference wavelength of 630 nm by an ELISA plate reader.

4.11. Intracellular Antioxidant Activity

RAW264.7 cells were seeded at 5000 cells/well into 96-well plates and incubated overnight. Cells were induced with 10 ng/mL LPS for 1 h, then counteracted with a curcumin-released medium from different time points for 2 h at 37 °C. The culture was removed and washed twice with Ca²⁺-free PBS. Cells were incubated with 10 μM DCFH-DA (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. After washing twice with Ca²⁺-free PBS, cells were replaced with 200 μL Ca²⁺-free PBS. The fluorescent intensity was read with excitation and emission wavelengths at 485 and 535 nm, respectively, on a fluorescence plate reader (BioTek, Santa Clara, CA, USA).

4.12. Cell Migration Assay

RAW264.7 cells were seeded in 6-well culture plates at a density of 1 × 10⁵ cells to create a monolayer. All scratches were created by a 200 μL pipette tip and washed with PBS. LPS was used at 10 ng/mL for 1 h of pretreatment, and the curcumin-released medium from different time points was added before initial scratch areas were captured by an inverted microscope (Motis, AE2000) equipped with a digital camera (Canon EOS700D; Canon Inc., Ohta-ku, Tokyo). After 48 h, scratch areas were re-captured, and the width of the scratch was determined using ImageJ software (Version release 1.48q) with the Wound Healing Size Tool plugin [38].

4.13. Nitric Monoxide Production

RAW264.7 cell line was plated at 10,000 cells/mL into 24-well plates and incubated for 12 h. The cells were introduced by 10 ng/mL LPS for 1 h, after that, cultures were challenged by curcumin-released medium at different time points for 3 h at 37 °C under 5% CO₂. Culture supernatants were collected and mixed with the Griess Reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) in a 96-well plate. After incubating at room temperature for 30 min, the absorbance was determined on a microplate reader at 548 nm.

4.14. Statistical Analysis

All results were independently repeated at least three times and are given as mean ± standard deviation (S.D.). Statistical analyses were performed using IBM SPSS Statistics version 30 for macOS. The Student’s t-test was used to test for differences between two groups. Differences among more than two groups were compared using a one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test.