*2.3. Chemical Crosslinking*

Poly(vinyl alcohol) and chitosan crosslinked by the epichlorohydrin was a representative of the chemically crosslinked hydrogel matrix. Epichlorohydrin reacts with either the hydroxyl group of PVA or amino group of chitosan to form a highly reactive intermediate. This intermediate product reacts with another hydroxyl (PVA) or amino group (chitosan) to form the crosslinked structure. Study of these hydrogels, both in their freshly prepared state, after air-drying at 40 ◦C and their subsequent rehydration and as xerogels, showed some physico-mechanical properties altered by the lecithin content (schematic figure of the preparation procedure can be seen in the Supplementary Materials Figure S4).

### 2.3.1. Rheology

For chemically crosslinked hydrogels, the amplitude sweep results showed that the addition of lecithin modified the rheological properties of hydrogels (see Figure 7a). However, the highest lecithin concentration did not lead to further changes in the mechanical properties. The same can be said after comparing the data points (see Table 7). At the same time, a higher content of lecithin decreased the values marking the end of the LVR as well as the strength of the hydrogels and the cross- over point values. The results are acceptable after taking into account the preparation and final state of the hydrogel. An important step of the preparation procedure is drying of the liquid mixture, which leads to crosslinking of the nodes and its subsequent rehydration. If lecithin is present, the rehydration is improved.

**Table 7.** Values for chemically crosslinked PVA-chitosan hydrogels obtained from strain and frequency sweep tests before drying.


The same experiments were performed for hydrogel samples dried and rehydrated. The dried and rehydrated hydrogels with lecithin assembled into the pores ended up with modified properties (see Figure 7b), specifically, an increase in moduli values and a decrease in the values marking the cross-over point, in contrast to the reference sample. As can be seen in Figure 7b and Table 8, the presence of lecithin makes the hydrogels obtained after the drying–rehydration step much more deformation resistant, characterized by much higher values of strain at the cross-over point. At the same time, for these mixed rehydrated hydrogels, lecithin, irrespective of its content, exerted a larger influence in the enhancement of the hydrogels' strength (average moduli values in LVR) when compared to the rehydrated systems physically and ionically crosslinked.

of decreasing mesh sizes after rehydration could be observed.

of the applied frequencies. The same trend was also observed for the dried and rehydrated samples. Again, for all samples, the storage modulus prevailed in comparison to the loss modulus. The mesh sizes of these samples (Tables 7 and 8) were not affected by the content of lecithin, a result that can be explained by the character of covalent crosslinking, which is stronger than physical and ionic crosslinking. On the other hand, the same trend

**Figure 7.** (**a**) Strain sweep of PVA-chitosan hydrogels with the different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**b**) strain sweep of PVA-chitosan 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 PVA-chitosan hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**d**) frequency sweep of PVA-chitosan hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after drying and rehydration of the xerogels. **Figure 7.** (**a**) Strain sweep of PVA-chitosan hydrogels with the different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**b**) strain sweep of PVA-chitosan 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 PVA-chitosan hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after preparation; (**d**) frequency sweep of PVA-chitosan hydrogels with different lecithin concentrations (0, 0.5, 1, and 2 wt.%) after drying and rehydration of the xerogels.


**Table 8.** Values for chemically crosslinked PVA-chitosan hydrogels after drying and rehydration obtained from strain and frequency sweep tests.

The frequency and amplitude sweep results indicated the same tendency discussed above (see comparatively Figure 7). Thus, a critical lecithin concentration is necessary to modify the properties of this type of chemically crosslinked hydrogels (according to the results lying between 0.5 and 1 wt.%); also, there is a maximum concentration above which further modifications do not occur (differences between 1 and 2 wt.% are negligible). The significant difference in the chemically crosslinked hydrogels (comparing to the physically and ionically crosslinked) is the relaxation phenomenon characterized by much longer relaxation times in contrast to covalently crosslinked systems. Covalently crosslinked hydrogels exhibit almost constant values of storage moduli over the whole range of the applied frequencies. The same trend was also observed for the dried and rehydrated samples. Again, for all samples, the storage modulus prevailed in comparison to the loss modulus. The mesh sizes of these samples (Tables 7 and 8) were not affected by the content of lecithin, a result that can be explained by the character of covalent crosslinking, which is stronger than physical and ionic crosslinking. On the other hand, the same trend of decreasing mesh sizes after rehydration could be observed.

### 2.3.2. Drying and Rehydration Measurements

As can be seen from Figure 8, the drying and swelling kinetics were not significantly altered by the addition of lecithin. Only a marginal influence was observed for samples with the highest lecithin concentrations, which were able to absorb the most water. This generally smaller influence of lecithin can be explained by the structure of chemically crosslinked hydrogels, which are characterized by a high enough crosslinking density and, consequently, by a smaller pore size morphology. The structure is more organized due to the stronger covalent bonds. The water absorption for this kind of hydrogel possessing stronger covalent cross linkages was very fast and occurred almost immediately during the first minutes of the swelling experiments.

### 2.3.3. Morphological Characterization of Xerogels

Results on the structural characterization of chemically crosslinked xerogels were similar to those for physically crosslinked hydrogels. The surface morphology of these xerogels looked smooth with no visible pores. In sectional view, SEM images revealed clear layered structures, with an interlayer roughness increasing with lecithin content (Figure 9), which in turn led to a gradual ascension of the value of specific surface (Table 9). Despite this fact, an apparently less corrugated surface observed for lecithin-free hydrogels had a higher specific surface area (Table 9), which might be explained by a greater compactness associated with the layered structure of the mixed xerogels.

**Lecithin** 

during the first minutes of the swelling experiments.

**Figure 8.** Drying (**a**) and rehydration (**b**) of chemically crosslinked PVA-chitosan hydrogels with different lecithin content. **Figure 8.** Drying (**a**) and rehydration (**b**) of chemically crosslinked PVA-chitosan hydrogels with different lecithin content. hydrogels had a higher specific surface area (Table 9), which might be explained by a greater compactness associated with the layered structure of the mixed xerogels.

**Table 8.** Values for chemically crosslinked PVA-chitosan hydrogels after drying and rehydration

As can be seen from Figure 8, the drying and swelling kinetics were not significantly altered by the addition of lecithin. Only a marginal influence was observed for samples with the highest lecithin concentrations, which were able to absorb the most water. This generally smaller influence of lecithin can be explained by the structure of chemically crosslinked hydrogels, which are characterized by a high enough crosslinking density and, consequently, by a smaller pore size morphology. The structure is more organized due to the stronger covalent bonds. The water absorption for this kind of hydrogel possessing stronger covalent cross linkages was very fast and occurred almost immediately

obtained from strain and frequency sweep tests.

2.3.2. Drying and Rehydration Measurements

 **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) 2470.0 ± 494.7 138.5 ± 13.5 14514 ± 1413 532 ± 33 3.2 ± 0.0 11.6 ± 0.3 0.5 7122.4 ± 633.3 379.1 ± 233.0 62099 ± 6505 1928 ± 65 5.0 ± 1.0 7.1 ± 0.1 1 4964.6 ± 275.8 502.2 ± 277.5 52833 ± 10153 2089 ± 246 3.0 ± 1.7 6.2 ± 1.3 2 4074.2 ± 182.3 900.1 ± 97.5 43685 ± 3177 1761 ± 211 5.9 ± 2.3 8.1 ± 0.0

**3. Conclusions** 

**Figure 9.** SEM images of chemically crosslinked PVA-chitosan xerogels with the addition of lecithin taken from the sectional view. Magnification 1000×. **Figure 9.** SEM images of chemically crosslinked PVA-chitosan xerogels with the addition of lecithin taken from the sectional view. Magnification 1000×.

**Table 9.** Specific surface area for chemically crosslinked PVA-chitosan xerogels with different leci-

**Concentration of Lecithin (wt.%) Specific Surface Area (m2/g)**  0 (R) 2.9

> 1 1.2 2 1.6

This work studied the influence of lecithin (L-α-phosphatidylcholine) on three differently crosslinked hydrogels (physically crosslinked agarose, alginate ionically crosslinked by calcium ions, and a mixture of PVA and chitosan chemically crosslinked by epichlorohydrin). The bulk of this work was to study differences between the gels investigated immediately after preparation and the corresponding rehydrated xerogels (prepared by swelling). By choosing the lecithin content, we were able to modify some of the mechanical properties of the hydrogels with a modified internal structure, especially in the case of the rehydrated ones. In this regard, the addition of lecithin had the strongest influence in enhancing the strength of chemically crosslinked PVA-chitosan gels, which is partially consistent with the mesh size and by the amount of water absorbed into their structure after being previous air-dried. Apart from the rheological data and those obtained from the kinetics of water loss during hydrogel dehydration, these conclusions

thin content determined by gas sorption.


**Table 9.** Specific surface area for chemically crosslinked PVA-chitosan xerogels with different lecithin content determined by gas sorption.

### **3. Conclusions**

This work studied the influence of lecithin (L-α-phosphatidylcholine) on three differently crosslinked hydrogels (physically crosslinked agarose, alginate ionically crosslinked by calcium ions, and a mixture of PVA and chitosan chemically crosslinked by epichlorohydrin). The bulk of this work was to study differences between the gels investigated immediately after preparation and the corresponding rehydrated xerogels (prepared by swelling). By choosing the lecithin content, we were able to modify some of the mechanical properties of the hydrogels with a modified internal structure, especially in the case of the rehydrated ones. In this regard, the addition of lecithin had the strongest influence in enhancing the strength of chemically crosslinked PVA-chitosan gels, which is partially consistent with the mesh size and by the amount of water absorbed into their structure after being previous air-dried. Apart from the rheological data and those obtained from the kinetics of water loss during hydrogel dehydration, these conclusions were supported by the scanning electron microscopy and gas sorption experiments performed on the xerogels. For this type of material, even though gas sorption appears to be inappropriate, however, it serves to confirm the non-porous structure of the xerogels.

In this work, we determined that the addition of phospholipid lecithin into the hydrogel matrix can alter their mechanical properties, which might be highly beneficial knowledge for the use of such hydrogels in particular applications. However, the transport properties also need to be investigated. Therefore, further transport experiments are required, which are absolutely crucial for a better understanding of such hydrogel materials and how they can be used in final applications.

### **4. Materials and Methods**

Hydrogels with distinct gelation mechanisms (physical, ionic, chemical crosslinking) [20] were studied. As an example of a physically crosslinked matrix, the linear thermoreversible polysaccharide agarose (Agarose E, Condalab, Madrid, Spain) at 1 wt.%, was used [21]. As an example of an ionically crosslinked matrix, sodium alginate (Sigma-Aldrich, Prague, Czech Republic) at 2 wt.% crosslinked by calcium chloride (Lach-Ner, Neratovice, Czech Republic) at a two to one weight ratio was chosen [22]. For chemically crosslinked hydrogels, poly(vinyl alcohol) (Sigma-Aldrich, Prague, Czech Republic) mixed with chitosan (low molecular weight, Sigma-Aldrich, Prague, Czech Republic) and crosslinked by epichlorohydrin (Sigma-Aldrich, Prague, Czech Republic) was employed [23]. L-α-Phosphatidylcholine (lecithin) was incorporated into all hydrogel samples before gelation at three different weight percentage concentrations (Sigma-Aldrich, Czech Republic, Prague).

The materials and their concentrations and ratios were selected on the basis of data previously reported [20–24] and can be seen in the table below (Table 10).


**Table 10.** Concentrations of each individual component in the final hydrogel form (agarose, sodium alginate, calcium chloride, PVA, chitosan, and lecithin).

### *4.1. Water Loss during Drying and Rehydration Measurements*

The ability to hold, release, and absorb water was tested by different approaches. Water loss was monitored by means of simple drying tests. All samples were dried either in the laboratory dryer at 40 ◦C and regularly weighed, or in a semi-automatic moisture analyzer (IR-35, Denver Instrument, Denver, CO, USA), where the weight was recorded automatically. The relative weight of the hydrogel (*x*) during drying was calculated using the following formula:

$$\mathbf{x} = \frac{m\_{\mathbf{t}}}{m\_0} \cdot 100 \tag{1}$$

where *m*<sup>t</sup> is the weight of the gel at time t, and *m*<sup>0</sup> is the weight of the hydrogel in the swollen state.

Often very small weight losses of water from the hydrogel samples made using drying scales more difficult. For this reason, drying kinetics were mostly studied using the combination of laboratory driers and analytical scales, upon which samples were weighed every twenty minutes. After the samples were dried to the xerogel form, they were inserted into a water bath, where they were kept until they reached their maximum water absorption capacity. The degree of water absorption (*m*a) was calculated by:

$$m\_{\rm a} = \frac{m\_{\rm t}}{m\_{\rm x}} \cdot 100\tag{2}$$

where *m*t is the weight of the hydrogel at time t, and *m*<sup>x</sup> is the weight of the xerogel. The hydrogel samples were regularly weighed on analytical scales to study their swelling kinetics.

### *4.2. Rheology*

Hydrogels are semi-solid materials that exhibit distinctive mechanical characteristics lying between those of solids and liquids. Therefore, rheology is indeed an appropriate technique for studying their behavior [25–29]. The mechanical properties of the prepared hydrogels were determined by rheological characterization using a rotational rheometer (Discovery HR-2, TA Instruments) employing cross-hatched 20 mm plate–plate geometry

to avoid potential sensor wall-slippage during measurement. The complex rheological procedure consisted of strain sweep and frequency sweep tests. The strain sweep test is a useful tool for obtaining information about samples if fluid-like or gel-like behavior under different values of applied strain prevails. In addition, it is possible to determine the region where the deformation is non-destructive (the linear viscoelastic region-LVR) as well as the behavior of the sample when the LVR strain limit is exceeded. The other mentioned test, the frequency sweep test, serves the purpose of describing hydrogel behavior in the non-deformation range (LVR) and provides information about different crosslinking sites (if applicable) in the internal structure of the hydrogel. Both tests were carried out on freshly prepared samples and rehydrated ones. The rehydrated samples were first dried to constant mass in the laboratory dryer for two days at a constant temperature of 40 ◦C and further rehydrated for three days in distilled water. Freshly prepared agarose and alginate samples were measured within a gap of 1000 µm. The gap for rehydrated samples varied according to the thickness of the gel, which depended on its swelling capacity, 500 µm for agarose gels and 1000 µm for alginate gels. PVA-chitosan hydrogels (both fresh and rehydrated) were measured within a gap of 200 µm due to the limited thickness of the prepared hydrogel foils. Prior to each applied test, samples were allowed to temper and rest for 180 s after loading into the measuring gap.

To obtain a suitable value of constant amplitude strain for the linear viscoelastic region (LVR), which was an essential parameter for ongoing frequency sweep tests, strain sweep tests were conducted first within the amplitude strain range of 0.01–1000% under a constant frequency of oscillation of 1 Hz in at least two repetitions, using a freshly loaded sample for each test. From these measurements, a strain of 0.1% was chosen as a suitable value of deformation for ongoing frequency tests, because this strain value lays within the LVR for all fresh and rehydrated samples. The range of oscillating frequencies for the frequency sweep tests was set to 0.01–100 Hz. Like the former strain sweep tests, the frequency sweep tests were also conducted in at least two repetitions. A summary of settings for both rheology tests is presented in Table 11.


**Table 11.** Summary of settings for rheology measurements (conditioning step, amplitude sweep, and frequency sweep).

Routine techniques that are usable for the characterization of the internal structures of many materials (e.g., scanning electron microscopy) have some limitations in the study of hydrogels. One of the most limiting factors is that the structures of hydrogels are mostly studied in a dried state. The internal structures of a hydrogel in the presence of water and in the absence of water must certainly differ. Moreover, the preparation of the hydrogel in its dried state is also critical because the dispersion medium (water) must be removed (mostly by evaporation or by sublimation if lyophilization is used). Unfortunately, both of these processes (evaporation as well as sublimation) have a significant impact on the final xerogel morphology. Simply, the fragile internal structure of the hydrogel may be critically damaged by the removal of the dispersion medium. Thus, such a resulting structure (specifically, the porous structure) revealed by scanning electron microscopy often has low informative value with respect to the internal structure of the hydrogel in its swollen state. Therefore, an alternative way to determine the pore size (and then obtain information about the internal structure of the hydrogel) must be found. An interesting solution to this problem is offered by the rheological characterization of the hydrogel, which involves the calculation of the mesh size.

Mesh size, as one of the most critical parameters in hydrogel characterization, was calculated by means of relaxation spectra (relaxation moduli *G* and relaxation time *λ*) from the frequency sweep oscillation measurements in accordance with the Maxwell model [30]. The frequency sweep (viscoelastic moduli as a function of oscillation frequency) was interpolated by continuous relaxation spectra in TRIOS software (TA Instruments, New Castle, DE, USA).

Typical relaxation spectra can be found in the Supplementary Materials (Figure S5). On the basis of previous rheological investigation [25], it was concluded that the optimal number of Maxwell elements was 4, in order to fit the frequency sweep measurements of the hydrogels. Four relaxation moduli were obtained from continuous relaxation spectra analyses. The sum of relaxation moduli was calculated in order to determine the crosslinking density [31] (see Equation (3), where *<sup>ρ</sup>*<sup>x</sup> represents the crosslinking density (mol·m−<sup>3</sup> )) and provides information on the density of the junction in the swollen hydrogel form. *<sup>G</sup>* (Pa) is the sum of 4 relaxation moduli, *<sup>R</sup>* (J·mol−<sup>1</sup> ·K−<sup>1</sup> ) represents the universal gas constant, and *T* is the thermodynamic temperature in Kelvins.

$$
\rho\_\chi = \frac{G}{RT} \tag{3}
$$

If all criteria are met (in particular, frequency sweep measurements are realized in the linear viscoelastic region and the mechanical properties of hydrogels with different crosslinking are consistent with rubber elasticity theory [32]), finally the mesh size can be calculated using Equation (4), where *ξ* is the mesh size (unit: m) and N<sup>A</sup> represents Avogadro's number.

$$\mathfrak{F} = \sqrt[3]{\frac{6}{\pi \rho\_{\text{x}} \mathbf{N}\_{\text{A}}}} \tag{4}$$

### *4.3. Morphological Characterization of Xerogels*

Since the structure affects properties that are crucial for hydrogel applications, determining the hydrogel morphology is one of the most important characterizations. There are many direct (microscopy) and indirect (scattering-based) methods to characterize hydrogel morphology [33]. Several direct visualization techniques (light microscopy, laser scanning confocal microscopy, and micro-computed tomography) that can handle swollen hydrogels have considerable disadvantages (e.g., limited resolution) [34]. On the other hand, commonly used scanning electron microscopy includes a critical step (i.e., the inevitable solidification of the sample using drying or freezing, during which the collapse of the structure or the creation of artifacts can occur) [35,36]. Kaberova et al. [37] tested the usability of scanning electron microscopy and concluded that the results from this method should always be confirmed by microscopy techniques applicable for gels in their swollen state.

For the characterization of dry samples, the specific surface area (the Brunauer– Emmett–Teller (BET) approach) is typically determined. The specific surface area is not suitable for characterizing hydrogels because of the already mentioned artifacts that appear during the preparation of dried samples. However, it can be used, for example, for the characterization of materials used in a dried state and that can form hydrogels (adsorbent) [38], or for the confirmation of reversible porosity [39].

The structure of the xerogels was studied in this work. Specifically, scanning electron microscopy and gas sorption were chosen as suitable techniques for determining the internal architecture of xerogels. Since the mechanical properties were studied for hydrogels right after preparation and also for swollen hydrogels after dehydration, it seemed convenient to investigate the structural properties of the hydrogels in these forms. Since this form is a dry form, it was possible to avoid deformation of the structure caused by the preparation of hydrogels for scanning electron microscopy.

### 4.3.1. Scanning Electron Microscopy

To determine changes in hydrogel structure, xerogels of all prepared samples were subjected to direct visualization using scanning electron microscopy. The samples were dried in a laboratory dryer at 40 ◦C. A few small specimens were taken from each studied sample to maintain objective observation. These specimens were subsequently gold-coated in a sputtering device (POLARON) and investigated using a ZEISS EVO LS 10 scanning electron microscope.

Both the surface morphologies and sectional images of samples were recorded. Observations were realized in secondary electron (SE) mode and the accelerating voltage was set to 5 kV to avoid charging of the samples.

### 4.3.2. Gas Sorption

A NOVA 2200e high-speed gas sorption analyzer (Quantachrome Instruments) was used to determine the specific surface area. The samples were weighed into a measuring cell (0.05–0.1 g). The measuring cell was placed in a degassing station, where the degassing process was carried out at 75 ◦C for 20 h. After cooling, the degassed sample was weighed to four decimal places. The samples were placed in a measuring station. The adsorption and desorption isotherms were measured under liquid nitrogen (77 K) from 0.05–0.95 of the relative pressure P/P0. The obtained data were processed by NovaWin software and specific surface area was calculated by the multi-point BET method.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/gels8020115/s1, Figure S1: Preparation procedure of physically crosslinked agarose hydrogels; Figure S2: Preparation procedure of ionically crosslinked alginate hydrogels; Figure S3: Dynamic viscosity measurements for combinations of solutions of lecithin, CaCl<sup>2</sup> and alginate; Figure S4: Preparation procedure of chemically crosslinked PVA-chitosan hydrogels; Figure S5: Typical relaxation spectra for mesh size calculations, TRIOS software (TA Instruments).

**Author Contributions:** Conceptualization, R.H. and J.S.; Methodology, R.H., M.K., M.T. and J.H.; Validation, R.H., M.K. and M.T.; Formal analysis, R.H. and J.S.; Investigation, R.H., M.K., M.T., J.H. and N.Z.; Data curation, R.H., M.T. and M.K.; Writing—original draft preparation, R.H., M.K., M.T. and N.Z.; Writing—review and editing, J.S. and M.P.; Visualization, R.H. and N.Z.; Project administration, R.H.; Funding acquisition, R.H.; Supervision, J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project was supported by the project Quality Internal Grants of BUT (KInG BUT), Reg. No. CZ.02.2.69/0.0/0.0/19\_073/0016948, which is financed from the Operational Program: Research, Development, and Education.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data used in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

