Intrinsically Thermoresponsive Hydrogels from Molecularly Engineered Chitosan

Abstract

Thermoresponsive chitosan hydrogels hold significant promise for advancing biomedical technologies, yet their frequent reliance on petroleum-based polymers raises biosafety and environmental concerns. The present study utilized a molecular functionalization strategy to transform chitosan into thermoresponsive alkylated chitosan (ICS). The ICS was subsequently covalently crosslinked to construct a fully degradable, all-chitosan thermoresponsive hydrogel (TR-ICS_gel), showcasing the effective integration of structural design and functionality. By adjusting the ICS concentration, TR-ICS_gels with varying volume phase transition temperatures (VPTTs) were obtained. Above the VPTT, strengthened alkyl chain hydrophobic interactions triggered hydrogel dehydration and pronounced, reversible shrinkage–swelling. The hydrogel maintained a stable swelling response over 20 consecutive temperature-stimulus cycles. Further investigation was conducted on the effects of ionic strength and small-molecule solvents on the thermoresponsive behavior of TR-ICS_gel. Soil burial and buffer solution tests demonstrated that the hydrogel underwent almost complete degradation within 27 and 15 days, respectively, and the degradation rate could be regulated by the ICS concentration. The TR-ICS_gel’s all-chitosan framework ensured excellent biocompatibility, with cell viability maintained above 95%. This study presents a strategy for developing fully bio-based, degradable smart hydrogels, enhancing biosafety and environmental friendliness. Moreover, these results provide crucial performance data and theoretical support for their practical application.

1. Introduction

Thermoresponsive hydrogels have garnered significant research interest for their ability to undergo reversible physicochemical transitions in response to temperature variations. This makes them promising candidates for drug delivery [1], smart sensing [2], Biomedical Sector [3] and tissue engineering applications [4,5]. To date, research in this field has been largely dominated by petroleum-based thermoresponsive polymer [6] poly (N-isopropylacrylamide) (PNIPAM) and its derivatives [7], which exhibit a lower critical solution temperature (LCST) of around 32 °C. These materials are commonly fabricated into hydrogels through covalent crosslinking, graft copolymerization, or physical blending with natural or synthetic polymers [8,9]. To overcome the limited biocompatibility and poor degradability of PNIPAM, as well as to introduce additional biological functionalities, biomass-derived polymers such as chitosan, cellulose [10], and hyaluronic acid [11] are often incorporated to construct composite hydrogels [12].

Among these biomaterials, chitosan, a natural alkaline polysaccharide, has attracted significant attention due to the numerous free amino groups present along its backbone, which confer excellent biological activities such as antibacterial properties and wound-healing promotion [13,14]. Chitosan stands out due to its extraordinary combination of favorable biological features, including biocompatibility, biodegradability, nontoxicity, and bacteriostatic properties [15]. Currently, the majority of thermoresponsive chitosan-based hydrogels are prepared through copolymerization or blending with synthetic thermoresponsive polymers such as PNIPAM [16,17]. In such systems, chitosan primarily serves as a biocompatible scaffold, while the temperature responsiveness is entirely derived from the introduced synthetic polymers. In certain instances, the incorporation of polysaccharides may even disrupt or weaken the intrinsic thermoresponsive behavior of the hydrogel network.

Therefore, developing a chitosan material with inherent thermoresponsive property that can function as the sole structural framework of a hydrogel provides distinct benefits [18]. This strategy would streamline material composition and fabrication processes, reduce reliance on petroleum-derived monomers, and maximize the preservation and utilization of the inherent bioactivity of chitosan [19]. This approach is of great significance for advancing high-performance, fully bio-based smart materials toward practical applications. However, achieving this goal remains challenging [20]. The solubility of chitosan is greatly influenced by acidic aqueous environments, which severely limits homogeneous chemical modification. In acidic environments, key functional groups, particularly primary amino groups, are predominantly protonated, leading to reduced reactivity and lower modification efficiency [21]. Moreover, conventional chemical modification processes often lead to the loss or deactivation of the functional amino groups [22], thereby compromising the biological activity of chitosan [23]. Therefore, constructing intrinsically thermoresponsive hydrogels solely from chitosan, without relying on any synthetic thermoresponsive polymers, is both theoretically important and practically meaningful.

To address these challenges, the present study proposes a molecular-level functionalization strategy; this strategy endows the chitosan hydrogel with thermoresponsive properties through alkylation, achieving the structural and functional integration. Briefly, chitosan (CS) was first dissolved in an alkaline solution and then reacted with isopropyl glycidyl ether (IPGE) via a ring-opening reaction, which enabled the successful grafting of hydrophobic isopropyl groups onto the chitosan backbone and yielded alkylated chitosan (ICS) with inherent thermoresponsive properties. This strategy not only imparts pronounced temperature sensitivity to chitosan but also significantly enhances its solubility and processability. By using the resulting ICS as the primary gel framework, a fully chitosan-based thermoresponsive hydrogel (TR-ICS_gels) was constructed by covalently crosslinking it with ethylene glycol diglycidyl ether (EDGE). The chemical structure, microstructure, and thermoresponsive behavior of the resulting hydrogel were systematically characterized. Furthermore, the effects of NaCl concentration, as well as the type and concentration of alcohol solvents on thermoresponsive performance of the hydrogel, were comprehensively investigated, providing critical data for the precise regulation of hydrogel behavior in biomedical and related applications. Finally, the degradation behavior of the hydrogel in soil and phosphate-buffered saline (PBS) containing lysozyme, simulating in vivo conditions, was evaluated, along with its cytotoxicity. These assessments are crucial not just for environmental sustainability but also for determining the feasibility of biomedical applications. Overall, this study demonstrates the successful fabrication of a fully bio-based thermoresponsive hydrogel and establishes a strong performance foundation and theoretical framework for its utilization in smart and intelligent biomedical systems and beyond.

2. Results and Discussion

Alkylated chitosan solutions with different concentrations (ICS-1, ICS-2, and ICS-3) were used to fabricate thermoresponsive hydrogels, which are denoted as TR-ICS_gel-1, TR-ICS_gel-2, and TR-ICS_gel-3, respectively.

2.1. Structural Characterization of TR-ICS_gel

FTIR spectra of CS, ICS, and TR-ICS_gel-2 were analyzed to verify the successful isopropyl functionalization and crosslinking reactions (Figure 1a). The broad absorption band centered at 3430 cm⁻¹ is attributed to O–H and N–H stretching vibrations. The peaks observed in the range of 2800–3000 cm⁻¹ correspond to C–H stretching vibrations of CH₂ and CH₃ groups [24]. The absorption peak at 1632 cm⁻¹ primarily arises from the bending vibration of the amide I band associated with residual acetyl groups in chitosan [25]. The distinctive peak at 1376 cm⁻¹ is attributed to the bending vibration of –CH₃ groups, providing additional confirmation for the successful incorporation of isopropyl moieties [26]. The absorption peak at 1075 cm⁻¹, which originates from the C–O–C stretching vibration of the glycosidic linkage in chitosan, is still clearly observable after modification and gel formation. This suggests that the chitosan backbone structure was not disrupted. Notably, TR-ICS_gel retains the key characteristic peaks of both CS and ICS without the emergence of additional absorption bands, suggesting that the crosslinking process did not damage the original chemical structure [27]. Overall, these findings validate the successful synthesis of ICS and the formation of a stable crosslinked network in TR-ICS_gel.

Further, TGA was employed to investigate the thermal stability of TR-ICS_gel-2 (Figure 1b). The thermal decomposition process exhibits a characteristic three-stage behavior [28]. The initial weight loss below 150 °C is due to the evaporation of physically adsorbed and bound water. The major decomposition stage occurs between 250 and 370 °C, which is associated with the degradation of the polysaccharide backbone and the disruption of intermolecular hydrogen bonds. Native chitosan contains abundant hydroxyl and amino groups, which can form dense intermolecular hydrogen-bonding interactions, thereby constructing a more compact polymer framework [29]. This hydrogen-bonded network restricts polymer chain mobility and delays main-chain scission during thermal treatment.

After alkyl functionalization, the intermolecular hydrogen-bonding interactions are moderately regulated, accompanied by a slight increase in chain flexibility, which leads to a marginally earlier thermal decomposition behavior at this stage [30]. Noteworthy, TR-ICS_gel exhibits a slightly higher onset decomposition temperature and a lower mass loss rate during the main degradation stage compared to ICS, indicating enhanced thermal stability. Furthermore, the DTG curve (Figure 1c) reveals an overall shift in the decomposition peak toward higher temperatures, indicating improved structural stability of the hydrogel due to covalently crosslinked network structure.

EDS analysis results (Figure 1d) indicate that TR-ICS_gel-2 is mainly composed of C, N, and O elements. Among these elements, C exhibits the highest atomic fraction (71.21%), while O and N account for 17.79% and 11.00%, respectively. The presence of nitrogen confirms the successful incorporation of amino groups originating from chitosan into the network structure. Figure 1g–i exhibits the elemental mapping images, revealing a homogeneous distribution of C, N, and O elements throughout the TR-ICS_gel, indicating a uniform internal structure.

SEM was employed to characterize the microstructures of TR-ICS_gel-2 at different temperatures. The samples were equilibrated at 30 and 40 °C, and then freeze-dried to preserve the temperature-dependent internal network structures. Figure 1e illustrates that at 30 °C, the hydrogel exhibits a characteristic three-dimensional porous architecture with well-interconnected pores, which is favorable for water retention. At a temperature close to the Volume Phase Transition Temperature (VPTT) (40 °C), the hydrogel undergoes pronounced shrinkage (Figure 1f), leading to a significant reduction in pore size and a more compact structure. This behavior can be attributed to the enhanced hydrophobic interactions and the collapse of hydrophilic chain segments at elevated temperatures. These findings provide direct microscopic evidence of the pronounced thermoresponsive behavior of TR-ICS_gel.

2.2. Thermoresponsive Behavior and Swelling Properties of TR-ICS_gel

Chitosan, which serves as a hydrophilic backbone, can be endowed with pronounced thermoresponsive properties by introducing hydrophobic isopropyl groups through molecular engineering. This alters the hydrophilic–hydrophobic balance in the system. Figure 2a displays the absorbance–temperature curves of aqueous solutions of ICS (MS = 2.35, Figure S1) at different concentrations, along with inset digital photographs illustrating the changes in the macroscopic state of ICS-2. With increasing temperature, the absorbance of the ICS solutions increases significantly, and the absorbance–temperature profiles exhibit a typical sigmoidal shape, which is characteristic of the phase transition behavior of thermoresponsive polymers. Consequently, the solution transitions gradually from transparent to turbid. Importantly, the Lower Critical Solution Temperature (LCST) of the ICS solutions decreases significantly with increasing ICS concentration (Figure S2a), directly confirming that concentration plays a crucial role in governing the phase separation behavior of this system.

Building on these observations, this study delved deeper into the impact of different ICS concentrations on the regulation of VPTT and swelling behavior of TR-ICS_gel derived from these precursors. The equilibrium swelling ratios of the hydrogels were measured at different temperatures, and the corresponding swelling ratio–temperature curves were plotted. Figure 2b demonstrates the temperature-dependent swelling behavior of TR-ICS_gel, showing its distinct volume phase transition characteristics. The inset macroscopic images vividly illustrate the volume changes in the hydrogels below and above the VPTT. These observations, along with the SEM images, offer direct evidence of the pronounced thermoresponsive behavior of TR-ICS_gel. With increasing ICS concentration, the volume change becomes more pronounced. This behavior can be attributed to the gradual weakening of intermolecular hydrogen bonding and the concurrent enhancement of hydrophobic interactions within the hydrogel network upon heating, leading to rapid deswelling and significant volume shrinkage. The variation trends of VPTT for TR-ICS_gel under different ICS concentrations are presented in Figure S2b, further confirming that the volume phase transition behavior of the hydrogels can be effectively regulated by tuning the ICS concentration.

In practical scenarios, thermoresponsive hydrogels are often required to go through multiple stimulus–response cycles, making structural stability a critical factor. The swelling–shrinkage cycling performance of TR-ICS_gel was therefore evaluated. Figure 2c demonstrates that TR-ICS_gel maintains consistent swelling–shrinkage behavior over 20 consecutive cycles, with minimal variations in the swelling ratio between cycles, demonstrating good cyclic stability of the hydrogel.

2.3. Effect of Ionic Concentration on the Swelling Behavior of TR-ICS_gel

Ionic concentration is a key parameter for regulating the phase transition temperature of hydrogels; therefore, clarification of its influence mechanism is significantly important for related biomedical applications. As a result, the effects of different NaCl concentrations (0, 5, 10, 15, and 20 g·L⁻¹) on the equilibrium swelling behavior of three TR-ICS_gel samples were investigated. Figure 3a–c exhibits that the VPTT of all hydrogel samples decreases markedly with increasing NaCl concentration. In particular, with the increase in the NaCl concentration from 0 to 20 g·L⁻¹, the VPTT values of TR-ICS_gel-1, TR-ICS_gel-2, and TR-ICS_gel-3 decrease from 39.7, 38.9, and 38.3 °C to 32.4, 31.5, and 27.6 °C, respectively (Figure 3d).

These phenomena can be attributed to two distinct mechanisms: (i) a dehydration mechanism, where the strong hydration between NaCl and water disrupts the hydrogen bonding between hydrophobic chains and water molecules when NaCl is added [31]; and (ii) a surface tension mechanism, where higher NaCl concentration enhances the surface tension between polymer chains and water molecules, resulting in higher free energy at the hydrophobic group/water interface and consequently inducing dehydration of the hydrogel [32].

2.4. Effect of Alcohol Solvents on the Swelling Behavior of TR-ICS_gel

In this study, methanol–water, ethanol–water, and isopropanol–water binary solvent systems were employed to systematically explore the thermoresponsive swelling behavior of TR-ICS_gel in various solvent environments. Figure 4a–i displays that the three TR-ICS_gel samples demonstrate distinct and representative volume phase transition processes in different alcohol–water systems, suggesting strong thermoresponsive characteristics of the hydrogels. Overall, with increasing alcohol volume fraction, the VPTT of each hydrogel decreases.

In terms of hydrogel composition, hydrogels prepared with different ICS contents exhibit different sensitivities to alcohol solvents. Considering the methanol system as an example (Figure 4a–c and Figure 5), with the increase in the methanol concentration from 0% to 40%, the VPTT of TR-ICS_gel-3 decreases from 38.2 to 29.5 °C (ΔT = 8.7 °C), which is greater than the changes observed in TR-ICS_gel-2 (ΔT = 8.4 °C) and TR-ICS_gel-1 (ΔT = 8.2 °C). This result indicates that hydrogel networks with higher ICS content exhibit greater sensitivity to changes in the solvent environment. The same patterns are also observed in the ethanol and isopropanol systems (Figure 4d–i).

In terms of solvent properties, further analysis combined with the VPTT variations depicted in Figure 5a–c reveals that different alcohols exhibit systematic differences in their ability to regulate the VPTT of TR-ICS_gel. With the increase in the carbon chain length of the alcohol molecules, the VPTT of the hydrogels demonstrates a more pronounced downward shift. For example, in the case of TR-ICS_gel-2, as the concentration of methanol, ethanol, and isopropanol increases from 0% to 40%, the VPTT decreases from 38.5 to 30.1 °C (ΔT = 8.4 °C), from 38.6 to 28.8 °C (ΔT = 9.8 °C), and from 38.6 to 28.1 °C (ΔT = 10.5 °C), respectively. When comparing alcohol volume fractions, the VPTT of TR-ICS_gel in ethanol and isopropanol systems is generally lower than in the methanol system under the identical conditions (Figure 4d–i), demonstrating a clear dependence on carbon chain length.

Therefore, the order of effectiveness of alcohols in lowering the VPTT is as follows: isopropanol > ethanol > methanol. This sequence is consistent with the longer carbon chain and increased hydrophobicity of isopropanol, indicating that the carbon chain length of alcohol molecules plays a crucial role in regulating the thermoresponsive behavior of TR-ICS_gel. These results align well with the results documented in the previous literature [33]. Mechanistically, the introduction of alcohol solvents reduces the overall polarity of the system, weakens hydrogen bonding between water molecules and polymer chains [34], and simultaneously boosts hydrophobic interactions, thereby providing an effective approach for tuning the VPTT of the hydrogels [35].

2.5. Degradability and Cytotoxicity of TR-ICS_gel

In this study, the degradation behavior of ICS hydrogel was systematically investigated through soil burial tests [36] and degradation under simulated physiological conditions. Figure 6a illustrates the changes in TR-ICS_gel during the soil burial process. Initially, at week 0, the samples retained their original morphology and semi-transparent appearance. After 1–2 weeks, noticeable erosion started to occur on the sample surface, causing the hydrogel to partially collapse and combine with the surrounding soil particles. By week 3, significant volume reduction and fragmentation were observed, indicating the deep penetration of soil microorganisms and moisture into the material, leading to gradual breakdown of the network structure. By week 5, only minimal traces or hollow impressions of the samples remained, demonstrating significant hydrogel degradation. This affirms that TR-ICS_gel exhibits good environmental degradation capability in natural soil. Figure 6b shows the morphological changes in TR-ICS_gel in PBS containing lysozyme. Initially, at week 0, the samples remained intact and smooth. After 1 week, there was visible surface softening and slight swelling. By weeks 2–3, the structural integrity of the hydrogel was significantly lost, leading to its gradual fragmentation. By week 4, the samples had almost completely disintegrated, leaving behind only a small amount of fine particles.

Figure 6c illustrates the mass change in the hydrogel over a 30-day period of soil burial. All three concentrations of TR-ICS_gel exhibited a stable degradation trend over time. The degradation rate was relatively slow in the initial 9 days, followed by a significant acceleration after 12 days. Among them, TR-ICS_gel-1 degraded the most rapidly (approximately 85–90% mass loss in 30 days), while TR-ICS_gel-3 degraded the slowest (approximately 65–70%). This difference mainly stems from the varying concentration of ICS in TR-ICS_gel, which impacts microbial penetration and moisture diffusion. This suggests that TR-ICS_gel is biodegradable in soil environments. Figure 6d displays the degradation trend of TR-ICS_gel in a PBS buffer solution with lysozyme. Degradation in the buffer system occurred at a much faster rate compared to soil degradation. The first 6–9 days constituted a rapid degradation phase, gradually stabilizing after 18–21 days, indicating the substantial breakdown of the hydrogel network. The TR-ICS_gels exhibited almost complete degradation within 15 days in buffer solution, whereas a longer degradation period of approximately 27 days was observed under soil burial conditions.

The image characterization and quantitative data analysis evidently indicate that TR-ICS_gel exhibits excellent degradation performance and stability for a specific period under both natural environmental and enzymatic conditions. Soil degradation is relatively slow, influenced by the synergistic effects of the environment and microorganisms; while lysozyme can significantly accelerate network breakdown, causing rapid hydrogel degradation. The degradation order of the three TR-ICS_gel types remains consistent in both systems, further indicating that their degradation rate can be effectively regulated by adjusting the ICS concentration. This characteristic ensures that the material will not cause persistent environmental pollution after its service life, aligning with the principles of green chemistry and sustainable development. These characteristics offer significant application benefits in environmentally relevant fields such as agricultural mulching film and controlled-release fertilizer coating.

Finally, the cytotoxicity of TR-ICS_gel on L929 cells was assessed via the MTT assay. Figure 6e demonstrates that the cell viability rates after 72 h for TR-ICS_gel-1, TR-ICS_gel-2, and TR-ICS_gel-3 hydrogel samples were 96.4 ± 4.8%, 95.0 ± 4.8%, and 94.3 ± 4.7%, respectively. These findings indicate that the hydrogel samples exhibit low cytotoxicity and more excellent cytocompatibility, providing a safety assurance for the application of TR-ICS_gel in the medical field.

3. Conclusions

This study proposes a universal molecular design strategy and successfully constructs a fully bio-based, degradable thermoresponsive chitosan hydrogel, TR-ICS_gel. FT-IR analysis confirmed the successful introduction of alkyl chains and the formation of a stable crosslinked network structure. TGA and DTG results demonstrated that the TR-ICSgel possesses adequate thermal stability within the temperature range relevant to processing and biomedical applications. SEM observations revealed a well-defined porous network architecture, which is favorable for mass transport and biological interactions, while EDS analysis verified the homogeneous elemental distribution throughout the hydrogel matrix. The hydrogel exhibits outstanding thermoresponsive characteristics: under thermal stimulation, the hydrophobic interactions between alkyl chains strengthen, driving gel dehydration and leading to significant, reversible swelling–shrinking behavior. The hydrogel maintains stable performance over 20 consecutive cycles. By adjusting the precursor ICS concentration, the VPTT can be effectively regulated. Further investigation reveals the consistent impact of NaCl concentration, as well as the type and concentration of alcohol solvents with varying carbon chain lengths, on the LCST of the system. This provides important theoretical support for the practical application of TR-ICS_gel. Furthermore, this fully biomass-based material demonstrates favorable biodegradability in different environments, with mass losses of up to 85–90% within 30 days in soil and proceeding more rapidly in a lysozyme system. Tests on cell cytocompatibility reveal that the viability of cells for all three types of hydrogels exceeds 95%, indicating outstanding safety for biological applications. This research not only presents a novel approach for developing high-performance, fully bio-based thermoresponsive materials but also establishes a robust data and theoretical foundation for the application of TR-ICS_gel in fields such as drug delivery systems, tissue engineering scaffolds, and other stimuli-responsive biomaterials.

4. Materials and Methods

4.1. Materials

IPGE, EDGE, and lithium hydroxide (LiOH, analytical grade) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Chitosan (degree of deacetylation ≥ 95%, viscosity 100–200 mPa·s) and potassium hydroxide (KOH, analytical grade) were procured from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All other reagents were of analytical grade and used as received without further purification.

4.2. Synthesis of ICS

Chitosan (3.3 g) was dispersed in 100 mL of a NaOH/urea/LiOH/deionized water mixed solvent, in which the components were present at mass fractions of approximately 3.0 wt% chitosan, 3.0 wt% LiOH, 3.0 wt% alkali hydroxide, and 15.0 wt% urea, with deionized water as the balance. The mixture was subjected to repeated freeze–thaw cycles at −40 °C until it became transparent, following which the temperature was raised to 40 °C. IPGE was then added under continuous stirring, and the reaction was allowed to proceed for 10 h. The resulting reaction solution was dialyzed (molecular weight cutoff: 8000–14,000 Da) for 3 days and subsequently freeze-dried to obtain ICS powder.

4.3. Preparation of TR-ICS_gel

Aqueous solutions of ICS (1.5, 3.0, and 4.5 wt%, corresponding to ICS-1, ICS-2, and ICS-3, respectively) were individually mixed with NaOH (20 wt%) and EDGE. After ultrasonic treatment, the mixtures were subjected to a crosslinking reaction at 50 °C for 3 h. The resulting hydrogels were then put through multiple swelling–shrinkage cycles, thoroughly washed, and freeze-dried for 48 h. The Preparation Details of TR-ICS_gel are listed in Table S1. Three thermoresponsive hydrogels with different ICS concentrations were obtained and denoted as TR-ICS_gel-1, TR-ICS_gel-2, and TR-ICS_gel-3, respectively.

4.4. Characterization

Fourier transform infrared (FTIR) spectra of chitosan CS, ICS, and TR-ICS_gel were recorded using an FTIR spectrometer (IRTracer-100, Shimadzu, Kyoto, Japan) across the wavenumber range of 600–4000 cm⁻¹. ¹H NMR spectra were recorded on a Bruker AVANCE III HD 400 spectrometer (Bruker, Bremen, Germany) at 25 °C using D₂O as the solvent. The surface morphologies of the hydrogels in both swollen and shrunken states were examined by field-emission scanning electron microscopy (FESEM, JSM-7800F, JEOL, Tokyo, Japan) following gold sputtering. The elemental composition was determined using the attached energy-dispersive spectroscopy (EDS) module. Thermogravimetric analysis (TG/DTG) was conducted on a thermogravimetric analyzer (HITACHI STA200, HITACHI, Tokyo, Japan) in a nitrogen atmosphere with a heating rate of 10 °C·min⁻¹. Prior to testing, all samples were freeze-dried.

4.5. Thermoresponsive Behavior (LCST/VPTT) and Swelling Performance

The LCST of the ICS solutions was determined using an ultraviolet–visible (UV–Vis) spectrophotometer equipped with a temperature control unit. The absorbance was recorded at 590 nm as the temperature was increased from 20 to 60 °C at a heating rate of 1 °C·min⁻¹, with each temperature point equilibrated for 2–3 min [37]. The LCST was determined as the temperature at which the first derivative of the absorbance–temperature curve reached its maximum value.

The volume phase transition temperature (VPTT) of TR-ICS_gel was determined by analyzing the changes in the swelling ratio. To achieve swelling equilibrium, dried hydrogel samples were immersed in deionized water at 20 °C for 48 h. Subsequently, the samples were subjected to 20 consecutive swelling–shrinkage cycles between 20 and 40 °C. The swelling ratio (SR) was calculated according to Equation (1), as follows:

$$SR={\displaystyle \frac{(W_t-W_d)}{W_d}}$$

(1)

Next, the thermoresponsive behavior of the three hydrogels prepared with different ICS concentrations was systematically evaluated. Moreover, the equilibrium swelling ratio and corresponding VPTT were measured under different NaCl concentrations (5–20 g·L⁻¹) and in the presence of methanol, ethanol, and isopropanol with various concentrations (10–40 vol.%). All experiments were conducted in triplicate using independently prepared samples, and the reported values represent the average results.

4.6. Degradation Behavior and Cytotoxicity Evaluation

The overall degradation performance of TR-ICS_gel was assessed through soil burial degradation and enzymatic biodegradation tests. In the soil degradation test, TR-ICS_gel samples (2 cm × 2 cm × 0.5 cm) were dried to a constant weight (W₀) and buried 5 cm below the surface of moist soil. The samples were incubated at 25 ± 2 °C, with the soil moisture content maintained at around 60% of its water-holding capacity throughout the experiment. At predetermined time intervals, the samples were retrieved, residual soil particles were removed, and the samples were dried to a constant weight (W_t) before weighing.

For the enzymatic biodegradation test, TR-ICS_gel samples with the same dimensions were immersed in PBS (0.1 M, pH 7.4) containing lysozyme (1.5 mg·mL⁻¹) and incubated at 37 ± 0.5 °C with gentle shaking. At designated time points, the samples were taken out, washed with deionized water, dried until a constant weight (W_t) was achieved, and then weighed. The degradation rate (DR) for both degradation processes was calculated according to Equation (2):

$$\mathrm{D}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\mathrm{\%})={\displaystyle \frac{W_0-W_t}{W_0}}\times 100$$

(2)

All experiments were performed in triplicate, and the results are reported as mean values.

The cytotoxicity of TR-ICS_gel toward mouse fibroblast cells (L929) was evaluated via the MTT assay [38]. Briefly, freeze-dried hydrogel sample (0.01) was extracted in Dulbecco’s Modified Eagle Medium (DMEM) (10 mL) culture medium containing penicillin–streptomycin under sterile conditions for 24 h to obtain the corresponding extracts [39]. L929 cells in the logarithmic growth phase were detached using trypsin (2.5 g·L⁻¹, PBS, pH 7.2–7.4), centrifuged, washed, and adjusted to a cell density of 2 × 10⁵ cells·mL⁻¹. The cells were then seeded into 96-well plates at a density of 100 μL per well and incubated at 37 ± 0.5 °C for 24 h.

After the culture medium was removed, 100 μL of hydrogel extract was added to each well. The control group received cells with fresh culture medium, while the blank group contained only culture medium; each group was tested in triplicate. After incubation at 37 °C for 72 h, the medium was replaced with 100 μL of MTT solution (5 g·L⁻¹), and further incubated for 4 h. The solution was then discarded, and 100 μL of DMSO was added to each well and gently mixed, followed by incubation at 37 °C for 30 min. The absorbance at 490 nm was measured using a microplate reader. The cell viability (CV) was calculated according to Equation (3):

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})={\displaystyle \frac{{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}-{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}{{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-}{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}}\times 100$$

(3)