were able to absorb the largest amount of water. 2.1.3. Morphological Characterization of Xerogels

2.1.3. Morphological Characterization of Xerogels Morphological characterization was performed on dried samples; therefore, the results may not correspond to the results obtained from methods where hydrogels are studied in native form (specifically, rheology). From the results obtained by scanning electron microscopy (SEM), the effect of lecithin addition could be observed in sectional view. The surfaces of these xerogels were smooth and with no visible pores on the micrometer scale. Morphological characterization was performed on dried samples; therefore, the results may not correspond to the results obtained from methods where hydrogels are studied in native form (specifically, rheology). From the results obtained by scanning electron microscopy (SEM), the effect of lecithin addition could be observed in sectional view. The surfaces of these xerogels were smooth and with no visible pores on the micrometer scale. In sectional view, the lecithin-free xerogel exhibited a layered structure of polymer fibers with no visible interferences (see Figure 3). The same layered morphology was also observed for xerogels of agarose with different contents of lecithin even though there were regions of fusion of adjacent layers. Overall, the general morphology, practically devoid of pores

as revealed by SEM, is most likely due to a compact structure resulting via the air drying procedure applied to hydrogels to finally obtain xerogels. of pores as revealed by SEM, is most likely due to a compact structure resulting via the air drying procedure applied to hydrogels to finally obtain xerogels.

In sectional view, the lecithin-free xerogel exhibited a layered structure of polymer fibers with no visible interferences (see Figure 3). The same layered morphology was also observed for xerogels of agarose with different contents of lecithin even though there were regions of fusion of adjacent layers. Overall, the general morphology, practically devoid

*Gels* **2022**, *8*, 115 8 of 23

**Figure 3.** Physically crosslinked agarose xerogels with different lecithin contents observed in sectional view by SEM. Magnification 5000×. **Figure 3.** Physically crosslinked agarose xerogels with different lecithin contents observed in sectional view by SEM. Magnification 5000×.

For these xerogels, gas sorption measurements were also performed (Table 3). The low values of the specific surface suggest a lack of the pore structure of xerogels, with a slight dependence on the compactness of layered morphology of these systems in dry state. Even if the results of gas sorption are in line with those of SEM investigation, the gas sorption method is not quite a suitable technique for determining the structure of these For these xerogels, gas sorption measurements were also performed (Table 3). The low values of the specific surface suggest a lack of the pore structure of xerogels, with a slight dependence on the compactness of layered morphology of these systems in dry state. Even if the results of gas sorption are in line with those of SEM investigation, the gas sorption method is not quite a suitable technique for determining the structure of these xerogels.

xerogels. **Table 3.** Specific surface area for physically crosslinked agarose xerogels with the addition of leci-**Table 3.** Specific surface area for physically crosslinked agarose xerogels with the addition of lecithin determined by gas sorption.


2 2.1

### *2.2. Ionic Crosslinking*

Sodium alginate crosslinked by the calcium chloride in the two to one weight ratio was a representative of the ionically crosslinked hydrogel matrix, where the negatively charged poly(guluronic) acid units of alginate (-COO−) interact with the polyvalent ions (Ca2+) to form a bond (schematic figure of the preparation procedure can be seen in Supplementary Materials, Figure S2). The final properties were also affected by lecithin addition. Hydrogel samples, both after preparation and dried and rehydrated, were studied by rheology, drying, and rehydration as well as morphological characterization.

## 2.2.1. Rheology

Ionically crosslinked hydrogels also underwent amplitude sweep tests. What is immediately observable is the decreasing trend of moduli for the freshly prepared samples as lecithin content increase (see Figure 4a). One of the reasons for this is the water intake during gelling, which increases for samples with ascending lecithin concentration (Section 2.2.2), the amphiphilic component playing a major role in the preparation of ionically crosslinked hydrogels. Larger lecithin addition also modified some characteristics of the hydrogels (see Table 4). The average moduli values in LVR steadily decreased after lecithin addition, thus making the gel softer. The most likely explanation is that after the crosslinking of alginate by calcium ions, free calcium chloride is still present in the system and is able to interact with the added lecithin micelles due to its dissociated form. Higher lecithin content causes a competitive interaction and as a result, lecithin displaces the calcium ions in the crosslinked alginate. Further lecithin could interact with the alginate via quaternary ammonia or with the calcium ions via negatively charged phosphate residues. For the moduli decrease, we could suggest that newly formed nodes are weaker and, in a lesser amount compared with the original alginate gel. Such competitive interactions were observable even during sample preparation, where the precipitate was visible on the surface of the solution. They were also confirmed by viscosity measurements, where the solution of calcium chloride and lecithin had higher viscosity values than expected, based on the viscosity of lecithin in water and of calcium chloride in water (figure is available in Supplementary Materials Figure S3). Other rheological data were very similar for the samples and, as stated earlier, the biggest differences were in the moduli values, thus in the hydrogel strength.

**Table 4.** Values for ionically crosslinked alginate hydrogels after preparation obtained from strain and frequency sweep tests before drying.


The rehydrated samples followed a similar trend with respect to the moduli values, where these values decreased with increasing lecithin concentration. Average moduli values in LVR reported in the table below (Table 5) were higher than those presented in Table 4 because the rehydrated samples were not able to reabsorb the same amount of water as the freshly prepared hydrogels. Such behavior could be due to a compact arrangement favored by non-covalent interactions (mainly ionic interactions induced by Ca2+ ions onto both alginate and lecithin components) during the drying process.

**Figure 4.** (**a**) Strain sweep of alginate hydrogels with the addition of different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**b**) strain sweep of alginate hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after drying and rehydration of the xerogels (frequency applied 1 Hz); (**c**) frequency sweep of alginate hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**d**) frequency sweep of alginate hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after drying and rehydration of the xerogels. **Figure 4.** (**a**) Strain sweep of alginate hydrogels with the addition of different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**b**) strain sweep of alginate hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after drying and rehydration of the xerogels (frequency applied 1 Hz); (**c**) frequency sweep of alginate hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**d**) frequency sweep of alginate hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after drying and rehydration of the xerogels.

The rheograms obtained during the frequency sweep tests (expressed as viscoelastic moduli on applied frequency) (Figure 4c) obeyed the same order as those that resulted from the amplitude sweep tests (storage and loss moduli as a function of oscillatory applied strain of 1 Hz) (Figure 4a) for all the studied alginate and alginate-lecithin hydrogels.

structural properties of these hydrogels. The higher addition of lecithin causes a higher


**Table 5.** Values for ionically crosslinked alginate hydrogels after drying and rehydration obtained from the strain and frequency sweep tests.

The rheograms obtained during the frequency sweep tests (expressed as viscoelastic moduli on applied frequency) (Figure 4c) obeyed the same order as those that resulted from the amplitude sweep tests (storage and loss moduli as a function of oscillatory applied strain of 1 Hz) (Figure 4a) for all the studied alginate and alginate-lecithin hydrogels. The calculated mesh size from the rheological (frequency sweep) measurement for freshly prepared ionically crosslinked alginate hydrogels indicated the effect of lecithin on the structural properties of these hydrogels. The higher addition of lecithin causes a higher mesh size (more than 50% if the hydrogels without/with 2 wt.% of lecithin is compared). The effect of lecithin concentration was also not observed for dried and rehydrated hydrogels. Although ionically crosslinked hydrogels have the ability to reabsorb the dispersion medium and again create a network internal structure by water intake, the internal structure of these hydrogels is probably damaged by the air-drying process. Moreover, swelled hydrogels differ in mesh size values in comparison with freshly prepared (e.g., hydrogels with 2 wt.% of lecithin had a mesh size of 17.3 nm while the mesh size of the hydrogels with the same concentration of lecithin after swelling was 7.6 nm). Therefore, the effect of lecithin on the mesh size of hydrogels repeatedly prepared by drying and swelling in water medium was negligible.

### 2.2.2. Drying and Rehydration Measurements

The drying curves for the alginate-lecithin systems were very similar almost irrespective of the lecithin content, in contrast to the drying dependence obtained for the freshly prepared hydrogels of alginate solely (Figure 5a). The different kinetics regarding the rate of water loss during the drying step could be due to the way lecithin fills the hydrogel pores and holds water within, and also due to the favorable electrostatic Ca2+-lecithin interactions, which influence the hydrogel structure and thus enable it to better hold water. As for the swelling after drying, it can be observed that the samples with higher lecithin concentrations were able to absorb water more rapidly and to a higher capacity, which is again due to the modified hydrogel network due to the presence of lecithin.

### 2.2.3. Morphological Characterization of Xerogels

SEM images taken for xerogels prepared by ionic crosslinking show the effect of lecithin on the surface morphology of the samples (see Figure 6). Surface morphology of lecithin-free samples and of those with 0.5 wt.% lecithin exhibited a roughness due to the many micrometer-sized crystals of CaCl<sup>2</sup> resulted after air-drying. Instead, the surface of xerogels with 1 and 2 wt.% lecithin is practically devoid of crystalline aggregates, with some degree of roughness, which led to a more compact structure of these mixed systems in their dry state. The morphological characteristics microscopically revealed are in accordance with the decreasing tendency of the specific surface values (from gas sorption measurements, Table 6) as the lecithin content rose. On the other hand, the lack of CaCl<sup>2</sup> crystalline aggregates for the systems with a higher lecithin content (1 and 2 wt.%) could be related to Ca2+ consumption in favorable electrostatic interactions with lecithin anions, which means that the crystalline structures observed in the case of alginate xerogels

**Lecithin** 

*Gels* **2022**, *8*, 115 11 of 23

 **Cross-Over Point Average Moduli Values in LVR End of LVR Mesh Size** 

**Concentration G' Strain G' G'' Strain Mesh (wt.%) (Pa) (%) (Pa) (Pa) (%) (nm)**  0 (R) 479.2 ± 129.7 210.8 ± 119.7 26,342 ± 13,355 3191 ± 1346 1.6 ± 0.4 4.6 ± 1.4 0.5 894.9 ± 612.4 522.6 ± 51.8 68,513 ± 17,434 9861 ± 1533 0.6 ± 0.6 12.3 ± 2.1 1 1179.5 ± 106.7 209.1 ± 37.3 25,386 ± 741 2912 ± 45 1.2 ± 0.2 8.3 ± 1.8 2 553.5 ± 24.3 189.5 ± 17.4 4599 ± 500 1842 ± 1447 2.4 ± 0.0 7.6 ± 0.5

2.2.2. Drying and Rehydration Measurements

swelling in water medium was negligible.

from the strain and frequency sweep tests.

mesh size (more than 50% if the hydrogels without/with 2 wt.% of lecithin is compared). The effect of lecithin concentration was also not observed for dried and rehydrated hydrogels. Although ionically crosslinked hydrogels have the ability to reabsorb the dispersion medium and again create a network internal structure by water intake, the internal structure of these hydrogels is probably damaged by the air-drying process. Moreover, swelled hydrogels differ in mesh size values in comparison with freshly prepared (e.g., hydrogels with 2 wt.% of lecithin had a mesh size of 17.3 nm while the mesh size of the hydrogels with the same concentration of lecithin after swelling was 7.6 nm). Therefore, the effect of lecithin on the mesh size of hydrogels repeatedly prepared by drying and

**Table 5.** Values for ionically crosslinked alginate hydrogels after drying and rehydration obtained

without lecithin and for those with 0.5 wt.% lecithin could be due to the excess of CaCl<sup>2</sup> contained in these explored samples. concentrations were able to absorb water more rapidly and to a higher capacity, which is again due to the modified hydrogel network due to the presence of lecithin.

The drying curves for the alginate-lecithin systems were very similar almost irrespective of the lecithin content, in contrast to the drying dependence obtained for the freshly prepared hydrogels of alginate solely (Figure 5a). The different kinetics regarding the rate of water loss during the drying step could be due to the way lecithin fills the hydrogel pores and holds water within, and also due to the favorable electrostatic Ca2+-lecithin interactions, which influence the hydrogel structure and thus enable it to better hold water. As for the swelling after drying, it can be observed that the samples with higher lecithin

**Figure 5.** Drying (**a**) and rehydration (**b**) of ionically crosslinked alginate hydrogels with different lecithin content. **Figure 5.** Drying (**a**) and rehydration (**b**) of ionically crosslinked alginate hydrogels with different lecithin content. out lecithin and for those with 0.5 wt.% lecithin could be due to the excess of CaCl2 contained in these explored samples.

determined by gas sorption.

**Figure 6.** Surface morphologies of ionically crosslinked alginate xerogels with the addition of lecithin revealed by SEM. Magnification 5000×. **Figure 6.** Surface morphologies of ionically crosslinked alginate xerogels with the addition of lecithin revealed by SEM. Magnification 5000×.

**Table 6.** Specific surface area for ionically crosslinked alginate xerogels with the addition of lecithin

**Concentration of Lecithin (wt.%) Specific Surface Area (m2/g)** 

0.5 6.3 1 5.9 2 4.7


**Table 6.** Specific surface area for ionically crosslinked alginate xerogels with the addition of lecithin determined by gas sorption.
