Synthesis of NVCL-NIPAM Hydrogels Using PEGDMA as a Chemical Crosslinker for Controlled Swelling Behaviours in Potential Shapeshifting Applications

Stimuli-responsive hydrogels have recently gained interest within shapeshifting applications due to their capabilities to expand in water and their altering swelling properties when triggered by stimuli, such as pH and heat. While conventional hydrogels lose their mechanical strength during swelling, most shapeshifting applications require materials to have mechanical strength within a satisfactory range to perform specified tasks. Thus, stronger hydrogels are needed for shapeshifting applications. Poly (N-isopropylacrylamide) (PNIPAm) and poly (N-vinyl caprolactam) (PNVCL) are the most popular thermosensitive hydrogels studied. Their close-to-physiological lower critical solution temperature (LCST) makes them superior candidates in biomedicine. In this study, copolymers made of NVCL and NIPAm and chemically crosslinked using poly (ethylene glycol) dimethacrylate (PEGDMA) were fabricated. Successful polymerisation was proven via Fourier transform infrared spectroscopy (FTIR). The effects of incorporating comonomer and crosslinker on the LCST were found minimal using cloud-point measurements, ultraviolet (UV) spectroscopy, and differential scanning calorimetry (DSC). Formulations that completed three cycles of thermo-reversing pulsatile swelling are demonstrated. Lastly, rheological analysis validated the mechanical strength of PNVCL, which was improved due to the incorporation of NIPAm and PEGDMA. This study showcases potential smart thermosensitive NVCL-based copolymers that can be applied in the biomedical shapeshifting area.


Introduction
Hydrogels are three-dimensional hydrophilic polymeric networks that can absorb large amounts of water into their polymeric matrix [1]. Depending on the types of crosslinking, chemically crosslinked hydrogels are stable, whereas physically crosslinked hydrogels disintegrate and dissolve sooner or later [2]. Chemically crosslinked hydrogels are also known as permanent or chemical gels. Their molecular chains are connected by covalent bonds; while physically crosslinked hydrogels are known as reversible or physical gels, and their polymeric network is formed by molecular entanglements and often with secondary forces, such as ionic, hydrogen bonding, and hydrophobic forces [3].
In fact, hydrogels were the first biomaterials created for applying in the human body, they were used as filling after enucleation of the eye [4,5]. There are many benefits for utilising hydrogels as biomaterials because of their biodegradability, biocompatibility, hydrophilicity, superabsorbancy, viscoelasticity, softness, and fluffiness [6]. To date, hydrogels Gels 2023, 9,248 3 of 17 drug delivery applications [39]; however, there are no FDA-approved PNVCL commercial products yet [40].
Implementation of PNVCL in clinical applications might seem limited presently; however, the tunability of the LCST of PNVCL has been proven practical by recent work [41]. Our team has also published the effect of applying 3D-printing and UV-curing techniques on fabricating an NVCL-based polymer using two different photoinitiators [42]. Continuous research on PNVCL will one day all add up to its successful application in numerous areas, including biomedical and shapeshifting areas.
Therefore, in this research, our aim is to study the effects of incorporating NIPAm as comonomer along with poly (ethylene glycol) dimethacrylate (PEGDMA) as a chemical crosslinker based on different concentrations with the main monomer, NVCL, on UV-cured copolymer properties, such as swelling behaviours and LCST, for their future use in shapeshifting applications. The results show that the NVCL-based polymers are mechanically improved through the incorporation of NIPAM and PEGDMA, and the effect of such incorporation has minimal impact on the alteration of the copolymers LCST.

Photopolymerisation
The photopolymerisation method consumed less time and a much smaller volume of materials than most other polymerisation techniques [38]. Furthermore, the inclusion of Irgacure 2959 as a photoinitiator was essential in the photopolymerisation process, as it absorbed UV light, which used the light energy to form free radicals that subsequently initiated the polymerisation process [43]. Irgacure 2959 was chosen as it has lower cytotoxicity when compared to other photoinitiators [44].
PEGDMA was used as a chemical crosslinker to obtain chemical gels so that swelling studies could be performed without dissolving the gels. Unlike previous studies that applied low percentage of PEGDMA [41,42], higher percentages of PEGDMA were used to strengthen the gels and limit their maximum swelling ratio. In the shapeshifting applications, the materials would not keep up if they are too soft, and vice versa, their shape would not return to the pre-programmed shape if the shapeshift-able smart materials are too stiff [45].
In addition, the incorporation of NIPAm in the formulations influenced the mechanical properties of cured samples due to its molecular weight and size being different from NVCL. The samples were UV-cured for 10 min on each face to ensure consistent irradiation. Silicone moulds were used to contain the prepolymerised mixtures due to their flexibility and indestructibility. Eventually, the samples cured were transparent and glass-like (Figure 1), showing amorphous features as light penetrated through the polymers instead of being absorbed and reflected.

Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy
ATR-FTIR is a common technique conducted to detect molecular fingerprints and to identify the presence of any functional group of both monomers and polymers. In this study, ATR-FTIR was conducted to determine if the chemically crosslinked gels were successfully polymerized through the UV irradiation. All tested samples were oven-dried at 50 °C for 24 h prior to analysis. Figure 2 illustrates the spectra of two monomers: NVCL and NIPAm, and a few chemically crosslinked gels (FC14, FC32, and FC34). Furthermore,

Cloud-Point Measurements
Cloud-point measurements were performed on the aqueous solution of physically crosslinked gels. The cloud point was the temperature where the solution started to show the first sign of turbidity. The aqueous solutions prepared were transparent at room temperature ( Figure 3a); by heating, they became turbid, and polymers precipitation occurred (Figure 3b) due to stronger hydrophobic chain reactions. This is the smart feature of thermosensitive hydrogels, such as PNVCL and PNIPAm, that allows them to have different properties below and above their LCST.
The disappearance of the peak at around 1655 cm −1 is due to the breakage of C=C bonding indicating the successful polymerisation of FC14, FC32, FC33, FC34, FC35, FC36, and FC37. The successful polymerisation was also proven by the disappearance of the peak assigned to 988 cm −1 in the fingerprint region that corresponded to =CH 2 bonding for both NVCL and NIPAm, and the fading of 3108 and 3072 cm −1 peaks from NVCL and NIPAm that associated with the =CH bonding. Nonetheless, due to the polymerized samples absorbing moisture from the air, the peaks detected at around 3500 cm −1 for the chemically crosslinked gels indicated O-H stretching, which corresponded to the hydrophilic nature of the gels. The disappearance of peaks in copolymers is represented by the arrows indicated in Figure 2.   The aqueous solutions of physically crosslinked NVCL-based polymers' LCST was also analysed using UV spectroscopy. At the set wavelength of 500 nm, the solutions absorbed light differently from a temperature range of 23-50 °C. As shown in Figure 4, the absorbance levels increased drastically starting at 30 °C. High absorbance indicated an opaque appearance of the tested samples was being detected. It is observed that with 30 wt% of NIPAm comonomer, the LCST was detected to be 1 °C higher than formulations with less NIPAm concentration ( Table 2).
In addition, the aqueous solution of formulations with higher NIPAm content showed higher viscosity than the others. A sharp decrease in absorbance was observed in Based on the literature [47], generally 10 wt% concentration of aqueous solutions were prepared, yet in this study, 5 wt% concentration of aqueous solutions were made as the gels only fully dissolved at 5 wt%. We noticed that, by increasing the PNIPAm percentage from 10 to 30 wt%, the LCST increased from 31 to 32 • C. However, due to the similar LCST range between PNVCL [38] and PNIPAm [22], the incorporation of NIPAm proved insignificant effects on the NVCL-based copolymers' LCST ( Table 2).

UV Spectroscopy
The aqueous solutions of physically crosslinked NVCL-based polymers' LCST was also analysed using UV spectroscopy. At the set wavelength of 500 nm, the solutions absorbed light differently from a temperature range of 23-50 • C. As shown in Figure 4, the absorbance levels increased drastically starting at 30 • C. High absorbance indicated an opaque appearance of the tested samples was being detected. It is observed that with 30 wt% of NIPAm comonomer, the LCST was detected to be 1 • C higher than formulations with less NIPAm concentration ( Table 2).

Differential Scanning Calorimetry
DSC is a technique that is used for thermal characterisation of polymeric materials. During the heating or cooling stage, the heat flow linked with material transitions as a function of temperature and time is plotted [48]. DSC analysis was conducted to determine the following: the glass transition temperature (Tg) and the LCST of the chemically crosslinked gels.
Chemically crosslinked hydrogels were not tested for LCST using cloud-point measurements and UV spectroscopy due to the existence of covalent bonding that restricted their dissolution in water. For the determination of LCST, fully swollen chemically crosslinked hydrogels were used. A fully swollen state would improve the chance of noticing the breakage of hydrophilic bond using the DSC method. Furthermore, the heating rate was set at 1 °C/min to ensure the accuracy of the results. The LCST was found as the onset temperature of an endothermic peak ( Figure 5). The LCST values ranging from 30-32 °C were observed for all chemically crosslinked gels using the DSC method. Hence, it was concluded that the incorporation of PEGDMA did not significantly affect the LCST properties of the hydrogels prepared.

31.71°C
Onset Temperature  In addition, the aqueous solution of formulations with higher NIPAm content showed higher viscosity than the others. A sharp decrease in absorbance was observed in samples FP34 and FP37 at a temperature of 33 • C, which could be attributed to the refraction of light caused by the presence of air bubbles formed during pipetting of aqueous samples into wells after the temperature exceeded their LCST. However, UV spectroscopy performed was to determine the LCST of the samples under the same concentration; therefore, the spectroscopy results confirmed again that the effect on the LCST through incorporating NIPAm together with NVCL was negligible.

Differential Scanning Calorimetry
DSC is a technique that is used for thermal characterisation of polymeric materials. During the heating or cooling stage, the heat flow linked with material transitions as a function of temperature and time is plotted [48]. DSC analysis was conducted to determine the following: the glass transition temperature (Tg) and the LCST of the chemically crosslinked gels.
Chemically crosslinked hydrogels were not tested for LCST using cloud-point measurements and UV spectroscopy due to the existence of covalent bonding that restricted their dissolution in water. For the determination of LCST, fully swollen chemically crosslinked hydrogels were used. A fully swollen state would improve the chance of noticing the breakage of hydrophilic bond using the DSC method. Furthermore, the heating rate was set at 1 • C/min to ensure the accuracy of the results. The LCST was found as the onset temperature of an endothermic peak ( Figure 5). The LCST values ranging from 30-32 • C were observed for all chemically crosslinked gels using the DSC method. Hence, it was concluded that the incorporation of PEGDMA did not significantly affect the LCST properties of the hydrogels prepared. linked hydrogels were used. A fully swollen state would improve the chance of noticing the breakage of hydrophilic bond using the DSC method. Furthermore, the heating rate was set at 1 °C/min to ensure the accuracy of the results. The LCST was found as the onset temperature of an endothermic peak ( Figure 5). The LCST values ranging from 30-32 °C were observed for all chemically crosslinked gels using the DSC method. Hence, it was concluded that the incorporation of PEGDMA did not significantly affect the LCST properties of the hydrogels prepared.  Based on the literature, PNVCL has a Tg of 147 • C [49], and PNIPAm has a Tg of 133 • C [50]. The discovered peak Tg values ( Figure 6) of all chemically crosslinked gels were within the range of 115-145 • C. The incorporation of three monomers: NVCL, NIPAM, and PEGDMA had a slight effect on the glass transition properties. In fact, the obtained Tg values would not matter as most hydrogels are applied in biomedicine. Therefore, the Tg results in this case are not significant for utilizing the developed formulations in the shapeshifting applications. Based on the literature, PNVCL has a Tg of 147 °C [49], and PNIPAm has a Tg of 133 °C [50]. The discovered peak Tg values ( Figure 6) of all chemically crosslinked gels were within the range of 115-145 °C. The incorporation of three monomers: NVCL, NIPAM, and PEGDMA had a slight effect on the glass transition properties. In fact, the obtained Tg values would not matter as most hydrogels are applied in biomedicine. Therefore, the Tg results in this case are not significant for utilizing the developed formulations in the shapeshifting applications.

Swelling Studies
Preliminary swelling analysis was performed for all chemically crosslinked gels at room temperature. Hydrogels swell in water due to the expansion of the polymeric chains network caused by the interaction between the polymeric chains network and water molecules [51].
However, the mechanical properties of hydrogels degrade as the degree of swelling increases [29,52], yet the mechanical properties are essential in the shapeshifting applications. Therefore, it is crucial to develop formulations that offer satisfactory mechanical properties for making shapeshifting assemblies. First, FC14, which contained 90 wt% of NVCL and 10 wt% of PEGDMA, showed very poor surface quality after 72 h of swelling  Figure 6. One of the dried chemically crosslinked gels analyzed for Tg using the DSC method.

Swelling Studies
Preliminary swelling analysis was performed for all chemically crosslinked gels at room temperature. Hydrogels swell in water due to the expansion of the polymeric chains network caused by the interaction between the polymeric chains network and water molecules [51]. However, the mechanical properties of hydrogels degrade as the degree of swelling increases [29,52], yet the mechanical properties are essential in the shapeshifting applications. Therefore, it is crucial to develop formulations that offer satisfactory mechanical properties for making shapeshifting assemblies. First, FC14, which contained 90 wt% of NVCL and 10 wt% of PEGDMA, showed very poor surface quality after 72 h of swelling with flakes forming on the surface as shown in Figure 7. While FC33 contained 20 wt% of NIPAm like FC36, the crack may be due to higher crosslinker percentage that caused more restriction to the polymer chain movement. Moreover, the swelling ratios of formulations FC32-FC37 are plotted in Figure 9. It is worth mentioning that FC32-FC34 that contained 10 wt% of crosslinker swelled about 25% less than FC35-FC37 that contained 7.5 wt% of crosslinker. The crosslinking density affecting swelling capacity of hydrogels inversely matches with the literature [53]. Eventually, FC34, FC36, and FC37 were selected for pulsatile swelling studies.  After being fully swollen at 20 • C for 72 h, FC14 reached a swelling ratio of 144%. However, the discs became very brittle, and the flakes formed started to peel off from the surface. This was due to the high percentage of chemical crosslinker that caused a great level of restriction to the polymer chain movement during swelling and resulted the breakage of polymer chains. Then, a great improvement in the hydrogel's integrity was achieved for all the other chemically crosslinked hydrogels.
As can be seen in Figure 8, flakes were no longer formed on the disc surface although minor cracks occurred on some of the discs. The incorporation of NIPAm as a comonomer has a positive impact on the structural integrity of NVCL-based hydrogel when a high percentage of crosslinker was used. Accordingly, it was observed that higher concentrations of NIPAm yielded better hydrogel's integrity. For instance, FC34 (30 wt% of NIPAm), FC36 (20 wt% of NIPAm), and FC37 (30 wt% of NIPAm) were the three formulations that showed perfect appearance after swelling to equilibrium at room temperature. While FC33 contained 20 wt% of NIPAm like FC36, the crack may be due to higher crosslinker percentage that caused more restriction to the polymer chain movement. Moreover, the swelling ratios of formulations FC32-FC37 are plotted in Figure 9. It is worth mentioning that FC32-FC34 that contained 10 wt% of crosslinker swelled about 25% less than FC35-FC37 that contained 7.5 wt% of crosslinker. The crosslinking density affecting swelling capacity of hydrogels inversely matches with the literature [53]. Eventually, FC34, FC36, and FC37 were selected for pulsatile swelling studies.  While FC33 contained 20 wt% of NIPAm like FC36, the crack may be due to higher crosslinker percentage that caused more restriction to the polymer chain movement. Moreover, the swelling ratios of formulations FC32-FC37 are plotted in Figure 9. It is worth mentioning that FC32-FC34 that contained 10 wt% of crosslinker swelled about 25% less than FC35-FC37 that contained 7.5 wt% of crosslinker. The crosslinking density affecting Gels 2023, 9, 248 9 of 17 swelling capacity of hydrogels inversely matches with the literature [53]. Eventually, FC34, FC36, and FC37 were selected for pulsatile swelling studies.

Pulsatile Swelling Studies
To unearth more about the swelling ability of the selected formulations, pulsatile swelling studies were performed. At two different temperatures (20 and 50 • C), swelling of FC34, FC36, and FC37 samples in distilled water was performed until equilibria weight was achieved. The swelling begun at 20 • C until equilibrium then continued at 50 • C in an oven, and the entire process was repeated twice more. It was observed that either formulation reached their maximum swelling ratio at 72 h, and it took less than 24 h for all discs to reach equilibria weight after being placed in the oven. In addition, in the second cycle of swelling the discs spent merely 24 h (instead of 72 h in the first cycle) to reach to their maximum swelling ratio.
The pulsatile swelling continued until all three cycles were completed, and the result is plotted in Figure 10. Both FC34 and FC37 completed three cycles of pulsatile swelling, yet FC36 broke into halves in second cycle (Figure 11b). This could be due to the lower percentage of NIPAm (20 wt%) when compared to both FC34 and FC37, which comprised 30 wt% of NIPAm. Furthermore, after three complete cycles, the tested FC34 and FC37 discs were fully dried in the oven at 50 • C. FC37 discs were partially broken (Figure 11c) while FC34 discs still maintained their structural integrity (Figure 11a) after drying, although, cracks were observed throughout the discs ( Figure 12). To unearth more about the swelling ability of the selected formulations, pulsatile swelling studies were performed. At two different temperatures (20 and 50 °C), swelling of FC34, FC36, and FC37 samples in distilled water was performed until equilibria weight was achieved. The swelling begun at 20 °C until equilibrium then continued at 50 °C in an oven, and the entire process was repeated twice more. It was observed that either formulation reached their maximum swelling ratio at 72 h, and it took less than 24 h for all discs to reach equilibria weight after being placed in the oven. In addition, in the second cycle of swelling the discs spent merely 24 h (instead of 72 h in the first cycle) to reach to their maximum swelling ratio.
The pulsatile swelling continued until all three cycles were completed, and the result is plotted in Figure 10. Both FC34 and FC37 completed three cycles of pulsatile swelling, yet FC36 broke into halves in second cycle (Figure 11b). This could be due to the lower percentage of NIPAm (20 wt%) when compared to both FC34 and FC37, which comprised 30 wt% of NIPAm. Furthermore, after three complete cycles, the tested FC34 and FC37 discs were fully dried in the oven at 50 °C. FC37 discs were partially broken (Figure 11c) while FC34 discs still maintained their structural integrity (Figure 11a) after drying, although, cracks were observed throughout the discs (Figure 12).
The phenomena of crack development and breakage of discs were due to the sudden formation of internal stresses, while the temperature rose to above the LCST that caused the polymer chains shrink and expel water molecules through hydrophobic segments [54]. The FC34 discs preserving their structural integrity after three cycles of pulsatile swelling may be due to less internal stresses generated within their polymer network when compared to FC36 and FC37.
Such outcome also indicates that NVCL formulation with high percentage of PEGDMA requires another comonomer like NIPAm to improve their structural integrity. FC34, a chemically crosslinked NVCL/NIPAm hydrogel with the best swelling integrity was successfully developed. Future improvement is required to prevent the formation of cracks within the gel during pulsatile swelling for prolonging their application lifetime in the thermosensitive area.

Rheological Analysis
It is challenging when it comes to examining the mechanical properties of hydrogels. Hydrogels generally become much weaker after swelling and the conventional instruments used for analysing thermoplastics mechanical properties are not all applicable for analysing the same for hydrogels. For instance, most conventional mechanical testing instruments expect samples to be clamped on the fixture while carrying out the analysis, while this requirement would have possibly destroyed the hydrogels. Therefore, to compare the mechanical strength of each formulation by causing minimal destruction to the hydrogels, a rheometer equipped with parallel plate was used. In general, storage modulus (G′) is the indication of the energy stored in the elastic structure of the sample. In this case, only G′ tested for each sample were compared as it brings the most interest about the effect of including the crosslinker and comonomer on the hydrogel's stiffness.
Distinct from the usual testing techniques for hydrogels in liquid form [55], a constant normal force of 3 +/− 0.3 N was applied to the chemically crosslinked hydrogels due to different swelling ratios that resulted different thicknesses of the samples. As shown in Figure 13, the incorporation of NIPAm as a comonomer has increased the G′ of FC14 (90 wt% of NVCL with 10 wt% of PEGDMA) from about 85 to above 150 kPa. Another point worth mentioning is that copolymer formulations having a higher percentage of PEGDMA (10 wt%) showed comparably higher G′ at above 200 kPa in the linear viscoelastic region, while copolymer formulations having lower percentage of PEGDMA (7.5 wt%) showed lower G′ at below 200 kPa in their linear viscoelastic region.

Rheological Analysis
It is challenging when it comes to examining the mechanical properties of hydrogels. Hydrogels generally become much weaker after swelling and the conventional instruments used for analysing thermoplastics mechanical properties are not all applicable for analysing the same for hydrogels. For instance, most conventional mechanical testing instruments expect samples to be clamped on the fixture while carrying out the analysis, while this requirement would have possibly destroyed the hydrogels. Therefore, to compare the mechanical strength of each formulation by causing minimal destruction to the hydrogels, a rheometer equipped with parallel plate was used. In general, storage modulus (G′) is the indication of the energy stored in the elastic structure of the sample. In this case, only G′ tested for each sample were compared as it brings the most interest about the effect of including the crosslinker and comonomer on the hydrogel's stiffness.
Distinct from the usual testing techniques for hydrogels in liquid form [55], a constant normal force of 3 +/− 0.3 N was applied to the chemically crosslinked hydrogels due to different swelling ratios that resulted different thicknesses of the samples. As shown in Figure 13, the incorporation of NIPAm as a comonomer has increased the G′ of FC14 (90 wt% of NVCL with 10 wt% of PEGDMA) from about 85 to above 150 kPa. Another point worth mentioning is that copolymer formulations having a higher percentage of PEGDMA (10 wt%) showed comparably higher G′ at above 200 kPa in the linear viscoelastic region, while copolymer formulations having lower percentage of PEGDMA (7.5 wt%) showed lower G′ at below 200 kPa in their linear viscoelastic region. The phenomena of crack development and breakage of discs were due to the sudden formation of internal stresses, while the temperature rose to above the LCST that caused the polymer chains shrink and expel water molecules through hydrophobic segments [54]. The FC34 discs preserving their structural integrity after three cycles of pulsatile swelling may be due to less internal stresses generated within their polymer network when compared to FC36 and FC37.
Such outcome also indicates that NVCL formulation with high percentage of PEGDMA requires another comonomer like NIPAm to improve their structural integrity. FC34, a chemically crosslinked NVCL/NIPAm hydrogel with the best swelling integrity was successfully developed. Future improvement is required to prevent the formation of cracks within the gel during pulsatile swelling for prolonging their application lifetime in the thermosensitive area.

Rheological Analysis
It is challenging when it comes to examining the mechanical properties of hydrogels. Hydrogels generally become much weaker after swelling and the conventional instruments used for analysing thermoplastics mechanical properties are not all applicable for analysing the same for hydrogels. For instance, most conventional mechanical testing instruments expect samples to be clamped on the fixture while carrying out the analysis, while this requirement would have possibly destroyed the hydrogels. Therefore, to compare the mechanical strength of each formulation by causing minimal destruction to the hydrogels, a rheometer equipped with parallel plate was used. In general, storage modulus (G ) is the indication of the energy stored in the elastic structure of the sample. In this case, only G tested for each sample were compared as it brings the most interest about the effect of including the crosslinker and comonomer on the hydrogel's stiffness.
Distinct from the usual testing techniques for hydrogels in liquid form [55], a constant normal force of 3 +/− 0.3 N was applied to the chemically crosslinked hydrogels due to different swelling ratios that resulted different thicknesses of the samples. As shown in Figure 13, the incorporation of NIPAm as a comonomer has increased the G of FC14 (90 wt% of NVCL with 10 wt% of PEGDMA) from about 85 to above 150 kPa. Another point worth mentioning is that copolymer formulations having a higher percentage of PEGDMA (10 wt%) showed comparably higher G at above 200 kPa in the linear viscoelastic region, while copolymer formulations having lower percentage of PEGDMA (7.5 wt%) showed lower G at below 200 kPa in their linear viscoelastic region. The phenomenon is due to more covalent bonds formed between the polymer chains when higher PEGDMA concentration is incorporated, which, in turn, enhances the stiffness of the swollen samples. The outcome of rheological analysis proved that the mechanical performance of NVCL-based hydrogels was improved overall by including NIPAm as a comonomer and PEGDMA as a crosslinker.

Conclusions
By using the photopolymerisation technique, the fabrication of NVCL-NIPAm copolymers chemically crosslinked with PEGDMA was successful, which was proven by FTIR. Cloud-point analysis and UV spectroscopy were performed on an aqueous solution of the physically crosslinked copolymer, and the authors confirmed that NIPAm has a negligible effect in tilting the copolymer's LCST due to its similar LCST range with NVCL. Differential scanning calorimetry was used to further conclude that PEGDMA did not significantly affect the LCST.
The glass transition temperature detected for the chemically crosslinked copolymers lay within the irrelevant range for their applications in biomedicine. FC34, FC36, and FC37 showed excellent structural integrity after being fully swollen at room temperature. However, only FC34 and FC37 completed three cycles of pulsatile swelling at both 20 and 50 °C. The pulsatile swelling revealed that FC34 had the best structural integrity post-process, although the formation of cracks was noticed. Finally, the rheological analysis discovered that the incorporation of NIPAm and PEGDMA significantly improved the hydrogels mechanical performance in their viscoelastic region.
While further improvements to the best formulation are to be made, FC34 demonstrated its ability to swell at temperatures below and above its LCST repeatedly for three cycles. This creates great potential for FC34 to be used as a thermosensitive bio-actuator. Future work that includes the incorporation of nanoparticles with F34 will be pursued for the prolonged application lifetime in thermosensitive areas. Since the applications of hydrogels in biomedicine usually involve environmental factors, such as pH, ions, and The phenomenon is due to more covalent bonds formed between the polymer chains when higher PEGDMA concentration is incorporated, which, in turn, enhances the stiffness of the swollen samples. The outcome of rheological analysis proved that the mechanical performance of NVCL-based hydrogels was improved overall by including NIPAm as a comonomer and PEGDMA as a crosslinker.

Conclusions
By using the photopolymerisation technique, the fabrication of NVCL-NIPAm copolymers chemically crosslinked with PEGDMA was successful, which was proven by FTIR. Cloud-point analysis and UV spectroscopy were performed on an aqueous solution of the physically crosslinked copolymer, and the authors confirmed that NIPAm has a negligible effect in tilting the copolymer's LCST due to its similar LCST range with NVCL. Differential scanning calorimetry was used to further conclude that PEGDMA did not significantly affect the LCST.
The glass transition temperature detected for the chemically crosslinked copolymers lay within the irrelevant range for their applications in biomedicine. FC34, FC36, and FC37 showed excellent structural integrity after being fully swollen at room temperature. However, only FC34 and FC37 completed three cycles of pulsatile swelling at both 20 and Gels 2023, 9, 248 12 of 17 50 • C. The pulsatile swelling revealed that FC34 had the best structural integrity postprocess, although the formation of cracks was noticed. Finally, the rheological analysis discovered that the incorporation of NIPAm and PEGDMA significantly improved the hydrogels mechanical performance in their viscoelastic region.
While further improvements to the best formulation are to be made, FC34 demonstrated its ability to swell at temperatures below and above its LCST repeatedly for three cycles. This creates great potential for FC34 to be used as a thermosensitive bio-actuator. Future work that includes the incorporation of nanoparticles with F34 will be pursued for the prolonged application lifetime in thermosensitive areas. Since the applications of hydrogels in biomedicine usually involve environmental factors, such as pH, ions, and blood, the performances and characterizations of the developed formulation must be verified under the applying conditions when a specific prototype is fabricated.

Synthesis of NVCL-Based Copolymers
The fabrication of the NVCL-based copolymers was performed through free-radical polymerisation using UV irradiation. A UV-irradiation chamber (Dr. Gröbel UV-El-

Materials
Chemical

Synthesis of NVCL-Based Copolymers
The fabrication of the NVCL-based copolymers was performed through free-radical polymerisation using UV irradiation. A UV-irradiation chamber (Dr. Gröbel UV-Elektronik GmbH, Ettlingen, Germany) that contained 20 UV tubes was utilised. This UVirradiation chamber provides a spectral range of UVA between 315-400 nm at an average

Synthesis of NVCL-Based Copolymers
The fabrication of the NVCL-based copolymers was performed through free-radical polymerisation using UV irradiation. A UV-irradiation chamber (Dr. Gröbel UV-Elektronik GmbH, Ettlingen, Germany) that contained 20 UV tubes was utilised. This UVirradiation chamber provides a spectral range of UVA between 315-400 nm at an average

Synthesis of NVCL-Based Copolymers
The fabrication of the NVCL-based copolymers was performed through free-radical polymerisation using UV irradiation. A UV-irradiation chamber (Dr. Gröbel UV-Elektronik GmbH, Ettlingen, Germany) that contained 20 UV tubes was utilised. This UV-irradiation chamber provides a spectral range of UVA between 315-400 nm at an average intensity of 10-13.5 mW/cm 2 . Seven different formulations that contained NVCL, NIPAm, PEGDMA, and Iragacure 2959, as shown in Table 4, were pre-mixed based on the table-listed percentages in individual beakers. Subsequently, the mixtures were stirred using a magnetic stirrer Gels 2023, 9,248 13 of 17 at 150 rpm and 50 • C for 20 min until homogeneous. The pre-polymerized solutions were then pipetted into several disc impression-containing silicone moulds. The circular disc impressions had a thickness of 4 mm and a diameter of 30 mm. Photopolymerisation was then conducted for 10 min on each face of the circular discs. Cured samples were dried in a vacuum oven at 50 • C for 24 h. To complete the characterisations of all formulations, physically crosslinked versions of each formulation were fabricated following the same method according to Table 5.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed on a Perkin Elmer Spectrum One FT-IR Spectrometer (Perkin Elmer, Waltham, MA, USA). equipped with a universal ATR sampling accessory. All examinations were conducted at room temperature in the spectral range of 4000-650 cm −1 , utilizing a fourscan-per-sample cycle under a constant universal compression force of 75 N. The collected data was analysed using the Spectrum software (2022.1.0, Tokyo, Japan).

Preparation of Aqueous Solutions
The fabricated physically crosslinked gels (Table 5) were dissolved in distilled water with a ratio of 0.5:9.5 (gel: distilled water) to form aqueous solutions of 5 wt% hydrogel concentration. The mixtures were kept in capped mechanical flasks at room temperature until the hydrogels were completely dissolved.

Cloud-Point Analysis
Cloud-point measurement was used to discover the phase transition temperature of the dissolved hydrogels. The aqueous solutions that were previously prepared were pipetted into separate test tubes, and the test tubes were immersed in a Memmert water bath system. A thermometer with accuracy of +/− 0.2 • C was placed in the water bath during the analysis for temperature monitoring. Starting from 20 • C, the temperature was slowly increased for 1 • C per 2 min to ensure consistency of temperature change. The temperature was recorded when the solution started to show turbidity until the degree of turbidity became constant.

UV Spectroscopy
UV spectroscopy was another method used to discover the phase transition temperature of the fabricated aqueous physically crosslinked formulations. A Synergy HT BioTek plate reader fitted with a heating system was operated. The spectroscopy was set at 500 nm for optical absorbance from 23-50 • C. For every 1 • C, the 96-well plate was allowed to equilibrate for 20 min before the measurement of absorbance.

Differential Scanning Calorimetry
The chemically crosslinked xerogels were analysed using a Pyris 6 DSC (Perkin Elmer, Waltham, MA, USA). An empty aluminium sample pan sealed with a lid was used as a reference sample for the test. The gel samples were weighed between 8-10 mg using a Sartorius scale with 0.01 mg resolution. Both swollen and dry gels for each chemically crosslinked formulation (Table 2) were tested based on different parameters. The swollen gels were tested from 10-60 • C at 1 • C/min. Moreover, the dried gels were tested from 20-200 • C at 10 • C/min. A constant nitrogen gas flow at 20 mL/min was applied while performing all tests. The obtained curves were plotted as a function of heat flow (W/g) against temperature ( • C) and further processed using the TA Universal Analysis software (2000 Manual, New Castle, DE, USA).

Swelling Studies
The swelling studies were performed in triplicate by placing the chemically crosslinked gels (Table 2) into the Thermo Scientific™ Sterilin™ (Waltham, MA, USA) petri dishes (diameter = 90 mm, height = 15.9 mm), which were later fully filled with distilled water. All the analyzed samples had dry weight measured at 2.5 +/− 0.25 g. The swelling studies were performed at room temperature. Intermittently, the gel samples were removed from the petri dishes and blotted free of surface water using filter papers, and the swollen weights were measured using a Sartorius scale at room temperature.
The gel samples were then resubmerged in distilled water until the next measurement was taken, and the cycle was repeated until the constant weight of the gel samples was achieved. The swelling percentage of the samples was calculated using Equation (1), where W t represents the weight of the swollen gel at a predetermined time and W d represents the dry weight of the gel.

Pulsatile Swelling Studies
Based on the result from swelling studies, candidates showing the best integrity at a fully swollen state were chosen for further swelling back and forth repeatedly at both 20 • C (room temperature) and 50 • C. A cycle consisted of an equilibria weight achieved at both temperatures, and three cycles were performed for each selected formulation. The swelling percentages were calculated using Equation (1), and the results are plotted in a graph ( Figure 10).

Rheological Analysis
The amplitude sweep was conducted on fully swollen chemically crosslinked hydrogels as a comparative test for their mechanical strength. A Discovery HR30 rheometer (TA Instruments, New Castle, DE, USA) installed with a stainless-steel Peltier plate, including temperature control was utilised for the analysis. A 20 mm diameter stainless-steel parallel plate was used for testing all samples. The amplitude sweep was conducted from 0.0001 to 10% of strain at 1 Hz frequency. A constant normal force of 3 +/− 0.3 N was applied to all tests at a set temperature of 20 • C. The storage moduli (G ) were compared using a graph.