**1. Introduction**

Hydrogels are three-dimensional, cross-linked networks of polymers and their individual physical properties are of particular interest for use in drug delivery applications, including in pharmaceutical patches or Transdermal Therapeutical Systems (TTS). Some of the most important characteristics of the hydrogels refer to their soft consistency and elasticity, as well as compatibility to body tissues [1,2]. These properties endorse the hydrogels as very attractive structures for biomaterials uses [1,3–6]. Hydrogels based on combinations between natural and synthetic polymers offer significant advantages, e.g., tunable mechanical properties, increased water content, enhanced biocompatibility and appropriateness to body tissue, and possibility of attaching chemical clues for further superior interfacial interactions. The use of polysaccharides as a base for the three–dimensional network structure preparation—recommended due to their properties such as biocompatibility, obtainment from renewable sources, and possibilities of "green" procedures for their modifying—is of major interest [1,7]. Among these compounds, the alginate has an important place. Alginic acid sodium salt (Alg–Na) is a naturally occurring biopolymer, biodegradable, biocompatible, and non-inflammatory, successfully used in medical applications as a carrier for drug delivery [8–10]. In the form of physically and chemically cross-linked systems, the alginate is an attractive starting material for the construction of hydrogels with desired morphology, stiffness, and bioactivity. However, the short residence of alginate time, due to a fast degradation process and poor mechanical characteristics, strongly limit the possibility of broadening its range of biomedical applications. Several chemical transformations of native alginate have been designed to provide mechanically and chemically robust materials and expand its range of application [8,11–13]. Moreover, the combination of alginate with

**Citation:** Sandu, A.E.; Nita, L.E.; Chiriac, A.P.; Tudorachi, N.; Rusu, A.G.; Pamfil, D. New Hydrogel Network Based on Alginate and a Spiroacetal Copolymer. *Gels* **2021**, *7*, 241. https://doi.org/10.3390/ gels7040241

Academic Editor: Wei Ji

Received: 5 November 2021 Accepted: 23 November 2021 Published: 27 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

synthetic polymers for obtaining hydrogels is of interest because in this way, the resistance and reproducibility of the materials increase. There is a series of studies [14–17] concerning the structures with synergistic properties resulting from combining synthetic polymers with polysaccharides [18]. Our group reported the preparation of poly(itaconic anhydrideco-3, 9-divinyl-2,4,8,10-tetraoxaspiro (5.5) undecane) (PITAU), a synthetic copolymer with pendant functional groups. PITAU presents specific properties, such as the possibility to create networks, biodegradability and biocompatibility, binding properties, amphiphilicity, thermal stability, and also sensitivity to pH and temperature [19,20]. The versatility and untapped potential of these polymeric systems make them promising agents for pharmaceutical delivery systems or support for bioactive compounds, among other biomedical applications. Because of these special characteristics of the PITAU copolymers, and taking into account our previous studies [19,20], the possibility of grafting PITAU onto alginate, to obtain biocompatible gels with improved properties it was investigated in the present work. This study presents the preparation of bioconjugated gels based on alginate and poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane). The new prepared structures were characterized from structural, morphological, and thermal behavior points of view. of the materials increase. There is a series of studies [14–17] concerning the structures with synergistic properties resulting from combining synthetic polymers with polysaccharides [18]. Our group reported the preparation of poly(itaconic anhydride-co-3, 9-divinyl-2,4,8,10-tetraoxaspiro (5.5) undecane) (PITAU), a synthetic copolymer with pendant functional groups. PITAU presents specific properties, such as the possibility to create networks, biodegradability and biocompatibility, binding properties, amphiphilicity, thermal stability, and also sensitivity to pH and temperature [19,20]. The versatility and untapped potential of these polymeric systems make them promising agents for pharmaceutical delivery systems or support for bioactive compounds, among other biomedical applications. Because of these special characteristics of the PITAU copolymers, and taking into account our previous studies [19,20], the possibility of grafting PITAU onto alginate, to obtain biocompatible gels with improved properties it was investigated in the present work. This study presents the preparation of bioconjugated gels based on alginate and poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane). The new prepared structures were characterized from structural, morphological, and thermal behavior points of view.

signed to provide mechanically and chemically robust materials and expand its range of application [8,11–13]. Moreover, the combination of alginate with synthetic polymers for obtaining hydrogels is of interest because in this way, the resistance and reproducibility

*Gels* **2021**, *7*, x FOR PEER REVIEW 2 of 13

#### **2. Results and Discussion 2. Results and Discussion**

The proposed illustration structure of the new synthesized gel based on alginate and PITAU is presented in Figure 1. The proposed illustration structure of the new synthesized gel based on alginate and PITAU is presented in Figure 1.

**Figure 1.** Schematized PITAU–alginate network. **Figure 1.** Schematized PITAU–alginate network.

#### *2.1. FTIR Spectra 2.1. FTIR Spectra*

The FTIR spectra of the gel samples are presented in Figure 2. The chemical composition of PITAU–Alg gels was confirmed by FTIR spectroscopy. The characteristic peaks of the PITAU copolymer appeared at (1) 1780 cm−<sup>1</sup> and 1856 cm−<sup>1</sup> corresponding to C=O

symmetric and asymmetric stretching of the five–member anhydride unit; (2) 1660 cm−<sup>1</sup> from C=C stretching; and (3) 1400 cm−<sup>1</sup> for =CH<sup>2</sup> in plane deformation, peaks evidenced as well by other authors [21]. The presence of a strong band around the 1000–1200 cm−<sup>1</sup> region is attributed to ether C–O–C stretching from the spirochetal moieties. The presence of alginate is confirmed by characteristic alginate peaks registered at 2923 cm−<sup>1</sup> and 2850 cm−<sup>1</sup> that correspond to stretching vibrations of aliphatic C−H; at 1610 cm−<sup>1</sup> , corresponding to the carboxylic groups C–O–O as a result of the asymmetric stretch; and the symmetric stretching at 1419 cm−<sup>1</sup> . The band from 1024 cm−<sup>1</sup> was attributed to the C–O stretching vibration, with contributions from C–C–H and C–O–H deformation data. C=O symmetric and asymmetric stretching of the five–member anhydride unit; (2) 1660 cm−1 from C=C stretching; and (3) 1400 cm−1 for =CH<sup>2</sup> in plane deformation, peaks evidenced as well by other authors [21]. The presence of a strong band around the 1000–1200 cm−1 region is attributed to ether C–O–C stretching from the spirochetal moieties. The presence of alginate is confirmed by characteristic alginate peaks registered at 2923 cm−1 and 2850 cm−1 that correspond to stretching vibrations of aliphatic C−H; at 1610 cm−1 corresponding to the carboxylic groups C–O–O as a result of the asymmetric stretch; and the symmetric stretching at 1419 cm−1. The band from 1024 cm−1 was attributed to the C– O stretching vibration, with contributions from C–C–H and C–O–H deformation data.

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The FTIR spectra of the gel samples are presented in Figure 2. The chemical composition of PITAU–Alg gels was confirmed by FTIR spectroscopy. The characteristic peaks of the PITAU copolymer appeared at (1) 1780 cm−1 and 1856 cm−1 corresponding to

,

**Figure 2.** FTIR spectra of the (**a**) S1, S2, and S3 samples; (**b**) PITAU; (**c**) alginate.

**Figure 2.** FTIR spectra of the (**a**) S1, S2, and S3 samples; (**b**) PITAU; (**c**) alginate. The disappearance of the peaks corresponding to the vibrations of the anhydride ring from 1782 cm−1 and 1862 cm−1 (Figure 2) confirm the covalent bonds between alginate The disappearance of the peaks corresponding to the vibrations of the anhydride ring from 1782 cm−<sup>1</sup> and 1862 cm−<sup>1</sup> (Figure 2) confirm the covalent bonds between alginate and PITAU governed by OH groups of the alginate, which open the ITA anhydride ring.

#### and PITAU governed by OH groups of the alginate, which open the ITA anhydride ring. *2.2. SEM Studies*

*2.2. SEM Studies* The samples were analyzed using SEM microscopy to investigate the hydrogel microstructural architecture. The morphological analysis, illustrated in Figure 3, was performed in cross-section of the freeze–dried polymeric networks. According to the cross-sectional SEM pictures (Figures 3 and S1–S3), the hydrogels present a continuous and porous configuration. As it is well known, the structural characteristics such as the porosity and topography governed by the chemical nature are very important in the performance of a gel. Moreover, a gel structure with a 3D network of interconnected channels and appropriate pores and pore size, allows a deeper penetration of liquids loaded with bioactive substances. The following images, representative of the studied gels, demonstrate the porous 3D architecture of the synthesized bioconjugate networks. SEM analyses confirm the important role of the amount of PITAU–Alg in generating the gel networks with superior properties. The pores of the gels have specific shapes and dimensions, in direct correlation with the PITAU–Alg ratio. Thus, the S3 sample, with higher amounts of alginate and smaller quantities of PITAU, leads to the formation of the irregular networks with larger pores. By increasing the PITAU amount (S2, S1), the gel The samples were analyzed using SEM microscopy to investigate the hydrogel microstructural architecture. The morphological analysis, illustrated in Figure 3, was performed in cross-section of the freeze–dried polymeric networks. According to the crosssectional SEM pictures (Figure 3, S1–S3), the hydrogels present a continuous and porous configuration. As it is well known, the structural characteristics such as the porosity and topography governed by the chemical nature are very important in the performance of a gel. Moreover, a gel structure with a 3D network of interconnected channels and appropriate pores and pore size, allows a deeper penetration of liquids loaded with bioactive substances. The following images, representative of the studied gels, demonstrate the porous 3D architecture of the synthesized bioconjugate networks. SEM analyses confirm the important role of the amount of PITAU–Alg in generating the gel networks with superior properties. The pores of the gels have specific shapes and dimensions, in direct correlation with the PITAU–Alg ratio. Thus, the S3 sample, with higher amounts of alginate and smaller quantities of PITAU, leads to the formation of the irregular networks with larger pores. By increasing the PITAU amount (S2, S1), the gel networks are more ordered and present smaller pores. The morphological aspect of the bioconjugate matrices is correlated with the gel's capacity for swelling (Figure 8) and the ability to further incorporate, transport, and release a therapeutic agent.

networks are more ordered and present smaller pores. The morphological aspect of the bioconjugate matrices is correlated with the gel's capacity for swelling (Figure 8) and the

networks are more ordered and present smaller pores. The morphological aspect of the bioconjugate matrices is correlated with the gel's capacity for swelling (Figure 8) and the

ability to further incorporate, transport, and release a therapeutic agent.

ability to further incorporate, transport, and release a therapeutic agent.

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**Figure 3.** SEM microscopy of the freeze-dried gels: (**a**) S1, (**b**) S2, (**c**) S3. **Figure 3.** SEM microscopy of the freeze-dried gels: (**a**) S1, (**b**) S2, (**c**) S3. **Figure 3.** SEM microscopy of the freeze-dried gels: (**a**) S1, (**b**) S2, (**c**) S3.

#### *2.3. Thermal Degradation 2.3. Thermal Degradation 2.3. Thermal Degradation*

Figure 4 illustrates the thermogravimetric (TG) and derivative thermogravimetric (DTG) curves of the studied samples obtained with different gravimetric ratios between PITAU and alginate co-partners, and Table 1 presents the main thermal parameters of the samples. TG and DTG curves of the samples show similar shapes with different mass losses in the five stages of degradation. In the first stage, the mass losses of 4.15–6.52% were determined on the moisture removal, including the water adsorbed on the surface. The mass losses of 29.28–40.79% were recorded in the second stage of degradation with Tonset between 145–147 °C, due to the cleavage of hydroxyl, carboxyl, and carbonyl groups from samples and release of H2O, CO2, aldehydes, alcohols, and ketones [22]. The increase in the temperature over 220 °C generates two consecutive thermal processes (III, IV), which occur with low decomposition rates (3.5–4.0%/min) depending on the gravimetric ratio between PITAU and Alg in gels. The last thermal process with Tonset above 300 °C led to the structural units' decomposition of PITAU and the alginate glycosidic ring, with mass losses of about 16%, along with the release of CO<sup>2</sup> and high–molecular weight aliphatic derivatives, and the obtaining of Na2CO3 due to Na–alginate dehydration and decarbonylation. At 650 °C, Na2CO<sup>3</sup> partially decomposes into CO<sup>2</sup> and Na2O as residue. The increase in the Na–alginate content in the PITAU–Alg samples determined a slight increase in the thermal stability, which was attributed to the additional crosslinking bridges that appear in the new system. This observation is also supported by T20 and T40 values (temperatures with mass losses of 20% and 40%, respectively). These values are 181 °C and 227 °C in the case of S3, 177 °C and 206 °C for S2, and 172 °C and 203 °C for S1. Therefore, the thermal stability series grows in the following order: S3 > S2 > S1. Figure 4 illustrates the thermogravimetric (TG) and derivative thermogravimetric (DTG) curves of the studied samples obtained with different gravimetric ratios between PITAU and alginate co-partners, and Table 1 presents the main thermal parameters of the samples. TG and DTG curves of the samples show similar shapes with different mass losses in the five stages of degradation. In the first stage, the mass losses of 4.15–6.52% were determined on the moisture removal, including the water adsorbed on the surface. The mass losses of 29.28–40.79% were recorded in the second stage of degradation with Tonset between 145–147 ◦C, due to the cleavage of hydroxyl, carboxyl, and carbonyl groups from samples and release of H2O, CO2, aldehydes, alcohols, and ketones [22]. The increase in the temperature over 220 ◦C generates two consecutive thermal processes (III, IV), which occur with low decomposition rates (3.5–4.0%/min) depending on the gravimetric ratio between PITAU and Alg in gels. The last thermal process with Tonset above 300 ◦C led to the structural units' decomposition of PITAU and the alginate glycosidic ring, with mass losses of about 16%, along with the release of CO<sup>2</sup> and high–molecular weight aliphatic derivatives, and the obtaining of Na2CO<sup>3</sup> due to Na–alginate dehydration and decarbonylation. At 650 ◦C, Na2CO<sup>3</sup> partially decomposes into CO<sup>2</sup> and Na2O as residue. The increase in the Na–alginate content in the PITAU–Alg samples determined a slight increase in the thermal stability, which was attributed to the additional crosslinking bridges that appear in the new system. This observation is also supported by T20 and T40 values (temperatures with mass losses of 20% and 40%, respectively). These values are 181 ◦C and 227 ◦C in the case of S3, 177 ◦C and 206 ◦C for S2, and 172 ◦C and 203 ◦C for S1. Therefore, the thermal stability series grows in the following order: S3 > S2 > S1. Figure 4 illustrates the thermogravimetric (TG) and derivative thermogravimetric (DTG) curves of the studied samples obtained with different gravimetric ratios between PITAU and alginate co-partners, and Table 1 presents the main thermal parameters of the samples. TG and DTG curves of the samples show similar shapes with different mass losses in the five stages of degradation. In the first stage, the mass losses of 4.15–6.52% were determined on the moisture removal, including the water adsorbed on the surface. The mass losses of 29.28–40.79% were recorded in the second stage of degradation with Tonset between 145–147 °C, due to the cleavage of hydroxyl, carboxyl, and carbonyl groups from samples and release of H2O, CO2, aldehydes, alcohols, and ketones [22]. The increase in the temperature over 220 °C generates two consecutive thermal processes (III, IV), which occur with low decomposition rates (3.5–4.0%/min) depending on the gravimetric ratio between PITAU and Alg in gels. The last thermal process with Tonset above 300 °C led to the structural units' decomposition of PITAU and the alginate glycosidic ring, with mass losses of about 16%, along with the release of CO<sup>2</sup> and high–molecular weight aliphatic derivatives, and the obtaining of Na2CO3 due to Na–alginate dehydration and decarbonylation. At 650°C, Na2CO<sup>3</sup> partially decomposes into CO<sup>2</sup> and Na2O as residue. The increase in the Na–alginate content in the PITAU–Alg samples determined a slight increase in the thermal which was attributed to the additional crosslinking bridges that appear new This observation is also supported by T20 and T40 values (temperatures with mass losses of 20% and 40%, respectively). These values are 181 °C and 227 °C in the S3, 177 °C and 206 °C for S2, and 172 °C and 203 °C for S1. Therefore, the thermal stability series grows in the following order: S3 > S2 > S1.

**Figure 4.** TG and DTG curves of PITAU–Alg gels. **Figure 4. Figure 4.** TG and DTG curves of PITAU TG and DTG curves of PITAU–Alg gels. –Alg gels.



Heating rate—10 ◦C/min; Tonset—the temperature at which the thermal degradation starts; Tpeak—the temperature at which the degradation rate is maximum; T20, T40,—the temperatures corresponding to 20% and 40% mass losses, respectively; TGS—the temperature at which the maximum amount of gases was released (determined from Gram–Schmidt curves using *Proteus* software); W—mass losses up to 680 ◦C.

The gases released were also examined by FTIR and mass spectrometry techniques concomitantly with the thermal decomposition of the S3 sample on the TG/FTIR/MS system, in the temperature range of 30–650 ◦C. Their 3D FT–IR spectrum is illustrated in Figure 5, and it can be noticed that the release of major gases during thermal decomposition takes place between 150–400 ◦C, according to the Gram–Schmidt and DTG curves. The main gases were identified based on the IR spectra and MS signals available in the literature and spectral libraries of the NIST [23]. From the 3D FT–IR spectrum, we extracted 2D spectra corresponding to the released gases at 195 ◦C and 426 ◦C (Figure 6). The absorption band from 3253 cm−<sup>1</sup> was assigned to the MCT detector (ice band) of the TGA–IR external module cooled with liquid nitrogen [24]. The major gaseous degradation products from the studied sample were water, carbon dioxide, carboxylic derivatives, alcohols, saturated and unsaturated aliphatic hydrocarbons, cycloalkanes, ketones, aldehydes, anhydrides, and ethers. Thus, the main absorption bands located at 3853–3619, 1375–1304, and 1240 cm−<sup>1</sup> can be assigned to water vapors and alcohols that can appear at the thermal degradation of the secondary hydroxyl or ester groups from alginate and PITAU units. The absorption bands from 3062–2847, 1523, 1462–1454, and 977–893 cm−<sup>1</sup> are assigned to the vibration of CH, CH2, and CH<sup>3</sup> groups located in the chemical structure of the saturated and unsaturated aliphatic hydrocarbons, cycloalkanes, and aldehydes. The higher signal present at 2355 cm−<sup>1</sup> and the small signal at 677 cm−<sup>1</sup> are attributed to carbon dioxide. The absorption bands between 1695 and 1648 cm−<sup>1</sup> are assigned to the asymmetric carbonyl groups in acids, aldehydes, and ketones, and those from 1854–1793 and 1170–1086 cm−<sup>1</sup> (νC=O vibrations) in ethers, anhydrides, and unsaturated aldehydes. These data are in agreement with the chemical structure of PITAU–Alg gels and correspond with gases that may result from their thermal degradation.

**Figure 5.** FTIR–3D spectrum of PITAU–Alg gel structure. **Figure 5.** FTIR–3D spectrum of PITAU–Alg gel structure. **Figure 5.** FTIR–3D spectrum of PITAU–Alg gel structure.

**Figure 6.** FTIR spectra of the evolved gases by thermal degradation of PITAU–Alg gel. **Figure 6.** FTIR spectra of the evolved gases by thermal degradation of PITAU–Alg gel. **Figure 6.** FTIR spectra of the evolved gases by thermal degradation of PITAU–Alg gel.

The data obtained from FTIR analysis of the evolved gases were also confirmed by MS spectrometry, and corresponding *m*/*z* signals are presented in Figure 6. With the increase in the temperature over 300 °C, a higher abundance of the gases developed, and the *m*/*z* ratio reached up to 120 for some high–molecular weight products. This confirms that up to 200 °C, there is a tearing of the thermal labile chemical bonds belonging to the functional groups (hydroxyl, carbonyl, carboxyl, and C–O links) from the structural units, and over 300 °C of the hydrocarbon bonds [25]. The main ionic fragments shown in Figure 7 are assigned as follows: HO+ (*m*/*z* = 17), H2O+ (*m*/*z* = 18), CO<sup>2</sup> + (*m*/*z* = 44). The ionic fragments of some saturated and unsaturated aliphatic derivatives are assigned as follows: CH<sup>2</sup> + (*m*/*z* = 14), CH<sup>3</sup> + (*m*/*z* = 15), ethane C2H<sup>6</sup> + (*m*/*z* =30), cyclopropane C3H<sup>6</sup> + (*m*/*z* = 42), propane C3H<sup>8</sup> + (*m*/*z* = 44), cyclobutene C4H<sup>6</sup> <sup>+</sup>(*m*/*z* = 54), cyclobutene, 2–butene C4H<sup>8</sup> + (*m*/*z* = 56), butane C4H10+ (*m*/*z* = 58), cyclohexane C6H12<sup>+</sup> (*m*/*z* = 84), carbonyl derivatives The data obtained from FTIR analysis of the evolved gases were also confirmed by MS spectrometry, and corresponding *m*/*z* signals are presented in Figure 6. With the increase in the temperature over 300 °C, a higher abundance of the gases developed, and the *m*/*z* ratio reached up to 120 for some high–molecular weight products. This confirms that up to 200 °C, there is a tearing of the thermal labile chemical bonds belonging to the functional groups (hydroxyl, carbonyl, carboxyl, and C–O links) from the structural units, and over 300 °C of the hydrocarbon bonds [25]. The main ionic fragments shown in Figure 7 are assigned as follows: HO+ (*m*/*z* = 17), H2O+ (*m*/*z* = 18), CO<sup>2</sup> + (*m*/*z* = 44). The ionic fragments of some saturated and unsaturated aliphatic derivatives are assigned as follows: CH<sup>2</sup> + (*m*/*z* = 14), CH<sup>3</sup> + (*m*/*z* = 15), ethane C2H<sup>6</sup> + (*m*/*z* =30), cyclopropane C3H<sup>6</sup> + (*m*/*z* = 42), propane C3H<sup>8</sup> + (*m*/*z* = 44), cyclobutene C4H<sup>6</sup> <sup>+</sup>(*m*/*z* = 54), cyclobutene, 2–butene C4H<sup>8</sup> + (*m*/*z* = 56), butane C4H10+ (*m*/*z* = 58), cyclohexane C6H12<sup>+</sup> (*m*/*z* = 84), carbonyl derivatives The data obtained from FTIR analysis of the evolved gases were also confirmed by MS spectrometry, and corresponding *m*/*z* signals are presented in Figure 6. With the increase in the temperature over 300 ◦C, a higher abundance of the gases developed, and the *m*/*z* ratio reached up to 120 for some high–molecular weight products. This confirms that up to 200 ◦C, there is a tearing of the thermal labile chemical bonds belonging to the functional groups (hydroxyl, carbonyl, carboxyl, and C–O links) from the structural units, and over 300 ◦C of the hydrocarbon bonds [25]. The main ionic fragments shown in Figure 7 are assigned as follows: HO<sup>+</sup> (*m*/*z* = 17), H2O+ (*m*/*z* = 18), CO<sup>2</sup> + (*m*/*z* = 44). The ionic fragments of some saturated and unsaturated aliphatic derivatives are assigned as follows: CH<sup>2</sup> + (*m*/*z* = 14), CH<sup>3</sup> + (*m*/*z* = 15), ethane C2H<sup>6</sup> + (*m*/*z* =30), cyclopropane C3H<sup>6</sup> + (*m*/*z* = 42), propane C3H<sup>8</sup> + (*m*/*z* = 44), cyclobutene C4H<sup>6</sup> + (*m*/*z* = 54), cyclobutene, 2–butene C4H<sup>8</sup> + (*m*/*z* = 56), butane C4H<sup>10</sup> + (*m*/*z* = 58), cyclohexane C6H<sup>12</sup> + (*m*/*z* = 84), carbonyl deriva-

tives such as formaldehyde CH2O<sup>+</sup> (*m*/*z* = 30), acetaldehyde C2H4O<sup>+</sup> (*m*/*z* = 44), acetone C3H6O<sup>+</sup> (*m*/*z* = 58), 2–cyclopenten–1–one C5H6O<sup>+</sup> (*m*/*z* = 82), itaconic anhydride C5H4O<sup>3</sup> (*m*/*z* = 112), alcohols as methanol CH3OH+ (*m*/*z* = 32), ethanol C2H5OH<sup>+</sup> (*m*/*z* = 46), propanol C3H8O<sup>+</sup> (*m*/*z* = 60), acids as formic acid CH2O<sup>2</sup> + (*m*/*z* = 46), acetic acid C2H4O<sup>2</sup> + (*m*/*z* = 60), propionic acid C3H6O<sup>2</sup> + (*m*/*z* = 74), vinyl ethyl ether C4H8O<sup>+</sup> (*m*/*z* = 72), diethyl ether C4H10O<sup>+</sup> (*m*/*z* = 74). such as formaldehyde CH2O<sup>+</sup> (*m*/*z* = 30), acetaldehyde C2H4O<sup>+</sup> (*m*/*z* = 44), acetone C3H6O<sup>+</sup> (*m*/*z* = 58), 2–cyclopenten–1–one C5H6O<sup>+</sup> (*m*/*z* = 82), itaconic anhydride C5H4O<sup>3</sup> (*m*/*z* = 112), alcohols as methanol CH3OH+ (*m*/*z* = 32), ethanol C2H5OH<sup>+</sup> (*m*/*z* = 46), propanol C3H8O<sup>+</sup> (*m*/*z* = 60), acids as formic acid CH2O<sup>2</sup> + (*m*/*z* = 46), acetic acid C2H4O<sup>2</sup> + (*m*/*z* = 60), propionic acid C3H6O<sup>2</sup> + (*m*/*z* = 74), vinyl ethyl ether C4H8O<sup>+</sup> (*m*/*z* = 72), diethyl ether C4H10O<sup>+</sup> (*m*/*z* = 74).

**Figure 7.** MS spectra of the evolved gases by thermal degradation of PITAU–Alg gel structures. **Figure 7.** MS spectra of the evolved gases by thermal degradation of PITAU–Alg gel structures.

#### *2.4. Swelling Study 2.4. Swelling Study*

One of the most important properties of the gels with pharmaceutical applicability is their capacity to swell when they come in contact with thermodynamically compatible solvents. In this case, the solvent molecules penetrate the polymeric network and determine the expanding of pores, which allow the incorporation of the drug or of other solvent molecules. As is well known, the swelling process is a consequence of the polymer– fluid interactive forces, which increase with the hydrophilic character of the macromolecules. The swelling degree of the studied gel samples as a function of time is illustrated in Figure 8. All the samples show a burst increase in swelling at the early stage, e.g., within the first 10 min. Then, the process is slowly continued up to 300 min. One of the most important properties of the gels with pharmaceutical applicability is their capacity to swell when they come in contact with thermodynamically compatible solvents. In this case, the solvent molecules penetrate the polymeric network and determine the expanding of pores, which allow the incorporation of the drug or of other solvent molecules. As is well known, the swelling process is a consequence of the polymer–fluid interactive forces, which increase with the hydrophilic character of the macromolecules. The swelling degree of the studied gel samples as a function of time is illustrated in Figure 8. All the samples show a burst increase in swelling at the early stage, e.g., within the first 10 min. Then, the process is slowly continued up to 300 min.

(**b**)

**Figure 8.** The swelling behavior of the gels as a function of time (**a**) and equilibrium swelling degree at different pH values (**b**). **Figure 8.** The swelling behavior of the gels as a function of time (**a**) and equilibrium swelling degree at different pH values (**b**).

The maximum degree of swelling corresponds to the network with the average amount of PITAU and alginate (S2), which is justified by a network structure with regulated pores (Figure 3b), due to PITAU copolymer presence, but at the same time owing to the alginate chains with relative mobility, which facilitates swelling. On the other hand, S1 samples have more possibilities for intense intramolecular bonds between alginate and PITAU governed by OH groups of the alginate, which open the ITA anhydride ring. The samples are pH–sensitive (Figure 8b), with the maximum swelling capacity regis-The maximum degree of swelling corresponds to the network with the average amount of PITAU and alginate (S2), which is justified by a network structure with regulated pores (Figure 3b), due to PITAU copolymer presence, but at the same time owing to the alginate chains with relative mobility, which facilitates swelling. On the other hand, S1 samples have more possibilities for intense intramolecular bonds between alginate and PITAU governed by OH groups of the alginate, which open the ITA anhydride ring. The samples are pH–sensitive (Figure 8b), with the maximum swelling capacity registered at pH = 6.5.

tered at pH = 6.5. The minimum swelling capacity is recorded for the sample with the minimum amount of PITAU and maximum amount of alginate (S3). In this case, the compact structure of the system induced a reduced swelling capacity. Subsequently, with the addition of synthetic polymer and the generation of an intermolecular network, the pene-The minimum swelling capacity is recorded for the sample with the minimum amount of PITAU and maximum amount of alginate (S3). In this case, the compact structure of the system induced a reduced swelling capacity. Subsequently, with the addition of synthetic polymer and the generation of an intermolecular network, the penetration of solvent molecules is easier, and consequently, the swelling capacity increased.

#### tration of solvent molecules is easier, and consequently, the swelling capacity increased. *2.5. Release Study*

*2.5. Release Study* The capacity of the PITAU–Alg network structure as a matrix was tested by the encapsulation and release of carvacrol. The carvacrol release profile presented in Figure 9 illustrates a burst effect highlighted in the first minutes of the samples' immersion in medium, while the equilibrium was reached after 4–11 h, depending on the PITAU–Alg The capacity of the PITAU–Alg network structure as a matrix was tested by the encapsulation and release of carvacrol. The carvacrol release profile presented in Figure 9 illustrates a burst effect highlighted in the first minutes of the samples' immersion in medium, while the equilibrium was reached after 4–11 h, depending on the PITAU–Alg hydrogel composition. Thus, the maximum amount of the drug released from the S2 hydrogel was reached after only 250 min, followed by the S1 hydrogel composition, with a

more prolonged release time and a plateau reached after 540 min. The S3 hydrogel variant proceeds a fast release of bioactive compounds at the beginning, but then an extended release up to 720 min was observed. These observations correlate with the swelling study that attests a smaller degree of swelling in the case of the S3 sample and a higher degree for the S2 sample. a more prolonged release time and a plateau reached after 540 min. The S3 hydrogel variant proceeds a fast release of bioactive compounds at the beginning, but then an extended release up to 720 min was observed. These observations correlate with the swelling study that attests a smaller degree of swelling in the case of the S3 sample and a higher degree for the S2 sample.

hydrogel composition. Thus, the maximum amount of the drug released from the S2 hydrogel was reached after only 250 min, followed by the S1 hydrogel composition, with

*Gels* **2021**, *7*, x FOR PEER REVIEW 9 of 13

**Figure 9.** The release profile of carvacrol from PITAU–Alg hydrogel network. **Figure 9.** The release profile of carvacrol from PITAU–Alg hydrogel network.

The drug release kinetic parameters presented in Table 2 were calculated using the semi–empirical equation proposed by Korsmeyer and Peppas [26]. In the table above, the value of *n* = 0.4931 obtained for S1 indicates a Fickian diffusion mechanism of the drug from the sample, while the value of *n* = 0.5581 obtained for S3, which is between 0.5 < *n* < 1, indicates an anomalous non–Fickian release behavior. The highest value of release rate constant k was obtained for S1, indicating the most accelerated drug release behavior, as observed in Figure 9, due to the chain disentanglement. These observations are also correlated with SEM images (Figure 3) which display a more ordered gel network with The drug release kinetic parameters presented in Table 2 were calculated using the semi–empirical equation proposed by Korsmeyer and Peppas [26]. In the table above, the value of *n* = 0.4931 obtained for S1 indicates a Fickian diffusion mechanism of the drug from the sample, while the value of *n* = 0.5581 obtained for S3, which is between 0.5 < *n* < 1, indicates an anomalous non–Fickian release behavior. The highest value of release rate constant k was obtained for S1, indicating the most accelerated drug release behavior, as observed in Figure 9, due to the chain disentanglement. These observations are also correlated with SEM images (Figure 3) which display a more ordered gel network with smaller pores in the S1 sample, in accord with the Fickian mechanism of release.

**Table 2.** Carvacrol release kinetic parameters.


smaller pores in the S1 sample, in accord with the Fickian mechanism of release.

*n—*release exponent; *k—*release rate constant; R*<sup>k</sup>* <sup>2</sup> and R*<sup>n</sup>* <sup>2</sup>—correlation coefficients corresponding to the slope obtained for determination of *n* and *k*, respectively. *n*—release exponent; *k*—release rate constant; R*<sup>k</sup>* <sup>2</sup> and R*<sup>n</sup>* <sup>2</sup>—correlation coefficients corresponding to the slope obtained for determination of *n* and *k*, respectively.

#### **3. Materials and Methods 3. Materials and Methods**

#### *3.1. Materials*

*3.1. Materials* All reagents were of analytical purity and were used without further purification: alginic sodium salt (Alg) from brown algae was supplied by Acros Organics from Belgium, 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane (U) (purity 98%, Sigma-Aldrich, Hamburg, Germany), itaconic anhydride (ITA) (purity 98%, Aldrich), and 2,2′-Azobis All reagents were of analytical purity and were used without further purification: alginic sodium salt (Alg) from brown algae was supplied by Acros Organics from Belgium, 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane (U) (purity 98%, Sigma-Aldrich, Hamburg, Germany), itaconic anhydride (ITA) (purity 98%, Aldrich), and 2,20 -Azobis (2-methylpropionitrile) (AIBN) (purity 98%, Sigma-Aldrich).

(2-methylpropionitrile) (AIBN) (purity 98%, Sigma-Aldrich). The solvents that we used 1,4 dioxane (D) (purity ≥ 99.0%) and diethyl ether (for precipitation)—were purchased from Sigma–Aldrich. The water used in the experiments was purified using an Ultra Clear TWF UV System.

#### *3.2. Copolymer Synthesis*

The PITAU preparation was described in detail before [20]. In brief, PITAU copolymers were synthesized through a radical process of polymerization with a total monomer concentration of about 20% (ratio between itaconic acid (ITA)/3, 9-divinyl-2,4,8,10-tetraoxaspiro (5.5) undecane (U) = 1.5/1), using AIBN as an initiator (0.9%) and 1,4-dioxane as a solvent. The reaction was conducted under a nitrogen atmosphere, in a constant temperature bath at 75 ◦C, with a stirring rate of 250 rpm, for about 17 h. The reaction mixture was further added dropwise into diethyl ether when the copolymer precipitated. The copolymer was further washed several times with diethyl ether and dried in a vacuum oven at room temperature with a 600 mm HG vacuum for 24 h.

#### *3.3. Bioconjugate Samples Preparation*

Three variants of bioconjugate structures based on PITAU and Alg were prepared as presented in Table 3. Precise amounts of copolymer (in dioxane solution) were mixed with specific amounts of alginate aqueous solution in order to have the following gravimetric ratios: PITAU:Alg = 3:1; 2:1, and 1.5:1. The gels formed rapidly within 20 min after mixing, and were left to mature for 24 h. Then, the gels were freeze–dried to remove the solvents and use the systems for further investigations.

**Table 3.** Codification and chemical composition of the studied samples.

