*Article* **Novel Hydrogel Material with Tailored Internal Architecture Modified by "Bio" Amphiphilic Components—Design and Analysis by a Physico-Chemical Approach**

**Richard Heger 1,2,\* , Martin Kadlec 1,2, Monika Trudicova 1,2 , Natalia Zinkovska 1,2, Jan Hajzler <sup>3</sup> , Miloslav Pekar 2,\* and Jiri Smilek 2,\***


**Abstract:** Nowadays, hydrogels are found in many applications ranging from the industrial to the biological (e.g., tissue engineering, drug delivery systems, cosmetics, water treatment, and many more). According to the specific needs of individual applications, it is necessary to be able to modify the properties of hydrogel materials, particularly the transport and mechanical properties related to their structure, which are crucial for the potential use of the hydrogels in modern material engineering. Therefore, the possibility of preparing hydrogel materials with tunable properties is a very real topic and is still being researched. A simple way to modify these properties is to alter the internal structure by adding another component. The addition of natural substances is convenient due to their biocompatibility and the possibility of biodegradation. Therefore, this work focused on hydrogels modified by a substance that is naturally found in the tissues of our body, namely lecithin. Hydrogels were prepared by different types of crosslinking (physical, ionic, and chemical). Their mechanical properties were monitored and these investigations were supplemented by drying and rehydration measurements, and supported by the morphological characterization of xerogels. With the addition of natural lecithin, it is possible to modify crucial properties of hydrogels such as porosity and mechanical properties, which will play a role in the final applications.

**Keywords:** lecithin; hydrogel; rheology; scanning electron microscopy; drying and swelling; extracellular matrix; mesh size

### **1. Introduction**

Hydrogels are hydrophilic polymers with a three-dimensional network structure that have the ability to absorb a large volume of water due to the presence of hydrophilic moieties, which makes them particularly suitable materials for biomedical applications (e.g., scaffolds) [1]. Selecting the pertinent components for the fabrication of the final hydrogel allows for a functional and applicable material with unique properties (e.g., porosity, biocompatibility, biodegradability) to be obtained. This exact customizable functionality makes these materials appropriate and desirable for a wide range of application areas (tissue engineering, pharmacy, water treatment, material engineering, etc.).

An equally important property of hydrogels is their ability to simulate and mimic biological systems such as the extracellular matrix (ECM), which is, in fact, a structural support network composed of diverse proteins, sugars, and other components. ECM regulates cellular processes including survival, growth, proliferation, migration, and differentiation [2]. Engineering a tailored in vitro environment mimicking the organized

**Citation:** Heger, R.; Kadlec, M.; Trudicova, M.; Zinkovska, N.; Hajzler, J.; Pekar, M.; Smilek, J. Novel Hydrogel Material with Tailored Internal Architecture Modified by "Bio" Amphiphilic Components— Design and Analysis by a Physico-Chemical Approach. *Gels* **2022**, *8*, 115. https://doi.org/ 10.3390/gels8020115

Academic Editor: Yang Liu

Received: 12 January 2022 Accepted: 11 February 2022 Published: 13 February 2022

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**Copyright:** © 2022 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/).

structure of ECM is a huge challenge and a desired goal. Since the scaffolds must offer relevant properties sufficient for cellular function, hydrogels have an advantage as potential materials due to their tunable physico-chemical (electrical charge and pore size) and mechanical (stiffness, tensile strength) properties [3]. The majority of hydrogels are also biocompatible, for example, naturally derived polymers such as agarose, alginate, chitosan, collagen, fibrin, gelatin, hyaluronic acid, and dextran as well as biocompatible synthetic gels based on poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and poly(hydroxyethyl methacrylate) (PHEMA) [4].

Since the 3D network structure of hydrogels is mainly responsible for their mechanical properties and porous microstructure, one of the possibilities of how to modify, upgrade, or tailor properties of hydrogels is to incorporate hydrophobic or micellar domains into the gel structure [5].

Pure hydrophobic association (HA) hydrogels refer to physically crosslinked hydrogels formed by hydrophobic interactions, which account for 5–20% of the total amount of polymer. The bulk of hydrophobic association hydrogels are produced by micellar copolymerization [6]. For instance, Tuncaboylu et al. attempted to improve the low mechanical strength of self-healing hydrogels by creating hybrid hydrogels with strong hydrophobic interactions between hydrophilic polymers mediated by the large hydrophobic moiety of a physical crosslinker (stearyl methacrylate) [7]. The addition of NaCl to the reaction solution during the copolymerization of large hydrophobes (stearyl methacrylate (C18)) with the hydrophilic monomer acrylamide (AAm) in an aqueous solution of sodium dodecyl sulfate (SDS) led to micellar growth and the solubilization of the large hydrophobes within the SDS micelles. Rheological measurements showed that the hydrophobic associations surrounded by surfactant micelles acted as reversible breakable crosslinks responsible for the rapid self-healing of the hydrogels [7].

An alternative approach to enhance the toughness of the hydrogel network is to introduce particles as additional crosslinking points (e.g., latex particles, nanoparticles) [6]. Latex particles (LPs) that are usually prepared via emulsion polymerization ensure effective energy dissipation and provide hydrogels with higher mechanical properties. Gu et al. [8] proposed a method that encompassed the adsorption of the hydrophobic alkyl chains of hydrophobic monomers on the surface of the latex microspheres and their subsequent stabilization in the presence of surfactants, thus forming hydrophobic association centers as the first physical crosslinking points. Moreover, anionic sulfate radicals (originating from the dissociation of the persulfate) were attracted toward the cationic chains of latex microspheres (obtained via surfactant-free emulsion copolymerization of styrene with a vinylidene comonomer bearing a cationic side group) and formed secondary physical crosslinking centers. The incorporation of cationic latex microspheres led to an improvement in the tensile and compression strength of the modified hydrogel compared with pure hydrophobic association hydrogel.

Since inorganic nanoparticles have a high specific surface area, their incorporation into the hydrogel network could also improve its mechanical behavior relating to surface structure and charging [6]. At the same time, the introduction of calcium carbonate nanoparticles [9], hydroxyapatite [10], kaolin [11], and laponite particles [12] could also induce hydrogel adhesion.

On the other hand, the embodiment of polymeric nanoparticles provides the ability to encapsulate both hydrophobic and hydrophilic substances [6]. In addition, Arno et al. investigated how particle morphology (e.g., particle shape, size, and surface) affected the adhesion and mechanical properties of the resultant calcium-alginate hydrogels [13]. The authors demonstrated that 2D platelets substantially improved both the adhesion between hydrogel surfaces and the material's mechanical strength when blended into the polymeric network compared to their 0D spherical or 1D cylindrical counterparts.

The properties of hydrogels, as mentioned previously, can be adapted not only through the appropriate choice of materials and crosslinking techniques, but also by modifying the internal structure of the gel by using a structure modifier such as lecithin during the preparation process. It should be remembered that lecithin is a typical amphiphilic phospholipid mixture primarily containing distearoylphosphatidylcholine, which possesses good biocompatibility and capability to enhance the bioavailability of co-administered drugs [14]. Lecithin in water systems can self-assembly into array of liquid-crystalline structures depending on the amount of water and temperature. The most likely structures formed under normal working laboratory conditions are lamellar liquid-crystalline structures [15]. Moreover, varying the ratio of lecithin in the multi-component hydrogel system may further improve the applicability and functionality of designed gels. The transport and mechanical properties of materials are given by their internal structure and can be greatly affected by its rearrangement.

Among the different types of lecithin-based systems, the most common platforms in this area are liposomes and microemulsions [16]. Liposomes are an example of soft phospholipid nanoparticles with typical diameters of around 100 nm [17]. Due to their closed vesicular structure, hydrophilic active compounds could be embedded into their internal water compartments, while hydrophobic compounds could be loaded into the bilayer of the liposome. In most cases, lecithin-based liposomal hydrogels are used as carriers; nevertheless, such systems still have certain disadvantages such as a slow and uncontrolled process of drug release [18]. In contrast, lecithin microemulsion-based gels or organogels have some advantages over liposomal hydrogels such as an easier preparation procedure, an absence of organic solvents, and higher storage stability due to the thermodynamic stability of microemulsions [19]. The matrix of lecithin microemulsion-based gels is composed of lecithin, which acts as a surfactant as well as a gelling agent in the presence of a nonpolar organic solvent (external phase) or a polar agent, which is usually water.

Substantial research is focused on modifying the internal structures of hydrogels, however, to the best of our knowledge, there has previously been no systematic study investigating the preparation and targeted modification of the internal structures of biocompatible hydrogels that focused on the use of natural amphiphilic substances and their crucial (e.g., mechanical) application properties.

Thus, this work focuses on the effect of the structure modifier lecithin (as stated before, the lecithin is able to self-organize into liquid-crystalline structures) and its concentration on the resultant mechanical properties of differently crosslinked hydrogels. The results of this work could provide a deeper understanding of the interactions between lecithin and the hydrogel network, and, alternatively, between lecithin and model drugs. Lecithin aggregates in hydrogels can also be viewed as a model of phospholipid structures (like cell membranes) occurring in real tissues, and thus as a model of their potential impact on the rheological or transport properties of the extracellular matrix.

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

On the basis of the prior experience of our team and in an attempt to investigate the effect of different crosslinking strategies on the final properties of hydrogels, the following materials were selected: agarose as a physically crosslinked hydrogel, alginate crosslinked by polyvalent ions as an ionically crosslinked hydrogel, and PVA-chitosan as a chemically crosslinked hydrogel.

As stated in Section 4, for each type of crosslinking, four different samples were investigated. Three samples with lecithin additions at different concentrations (0.5, 1, and 2 wt.%) were labeled according to their lecithin concentration (i.e., "0.5", "1" and "2"). The fourth sample was a reference sample without lecithin, simply marked as "R". The lecithin concentrations were selected on the basis of preliminary experiments focused mainly on estimating the maximum amount of lecithin that could be incorporated into the hydrogel matrix.

### *2.1. Physical Crosslinking*

Agarose was a representative of the physically crosslinked hydrogel matrix, whose properties were affected by lecithin content. Hydrogel samples after preparation as well as samples after the drying and rehydration procedure were studied (schematic figure of the preparation procedure can be seen in the Supplementary Materials Figure S1).

## 2.1.1. Rheology

Amplitude sweep results for physically crosslinked hydrogels obtained under an applied oscillatory strain of 1 Hz suggest that differences in lecithin concentration have, from a viscoelastic property point of view, a minimal influence on the hydrogel structure after preparation, especially with respect to the width of the linear viscoelastic region (as can be seen in Figure 1a). The storage as well as the loss modulus gradually increased with increasing lecithin concentration, which might be due to the overall higher dry content of the hydrogels. The effect of lecithin concentration on the viscoelastic properties of agarose hydrogels was also minimal in the linear viscoelastic region (LVR), which is the range of the values of storage modulus where the hydrogel is able to resist the applied oscillatory strain and can thus indicate the strength of non-covalent hydrogel nodes. Probably, the strength of the physically crosslinked hydrogel is provided mainly by non-covalent weak interactions (H-bonding) between the chains of agarose. Lecithin only had a small effect on the viscoelastic properties of 1 wt.% aq. agarose. The obtained values marking the end of the LVR were very similar for all samples physically crosslinked (Table 1). The values reported in the tables were either obtained by rheology software (TRIOS TA Instruments) analyses (cross-over point, average moduli values in LVR) or calculated. The end of LVR was obtained by comparing the average value of storage modulus in LVR with each point, where the deviation greater than 5% marked the end of the LVR. The mesh size calculations are described in Section 4.2. The cross-over point (G0 = G00), the point at which the hydrogel was irreversibly damaged, was very similar for all samples.

**Table 1.** Values for physically crosslinked agarose hydrogels after preparation obtained from strain and frequency sweep tests before drying.


The same amplitude sweep tests were performed on samples dried to the xerogel form and again rehydrated. The amount of absorbed water had a significant effect on these samples. As can be seen from Figure 1b as well as from the dry matter content experiments (Section 2.1.2), the samples with the highest lecithin content were able to reabsorb the largest amount of water (twice as much water as the sample without lecithin). This was also reflected in the amplitude sweep results because the moduli values for these hydrogels decreased proportionately. The reference sample had the highest moduli values, whereas the lowest values were observed for the samples with the greatest lecithin concentrations. The moduli values were somewhat larger than those for the samples studied after preparation (Table 2), mainly due to the elevated values of the swelling degrees of the systems after drying and rehydration in comparison with those of the just prepared hydrogels. Lecithin, therefore, favored water absorption. For the physically crosslinked hydrogels, even the cross-over point was affected, and samples with higher lecithin concentrations shifted the cross-over point to higher strain values. This could be the effect of the attractive interactions between lecithin and the polysaccharide chains, leading to the reinforcement of the hydrogels obtained after their drying and rehydration. In the initially prepared hydrogels, lecithin was dispersed to a greater extent in a liquid medium without this (strong) effect. This could be explained by the H-bonding between polysaccharide chains and lecithin,

*Gels* **2022**, *8*, 115 5 of 23

which are more significant for the rehydrated hydrogels because of the absence of water (in xerogel), which could not interfere. The same could be observed for the cross-over point, which again gradually increased with lecithin concentration. chains and lecithin, which are more significant for the rehydrated hydrogels because of the absence of water (in xerogel), which could not interfere. The same could be observed for the cross-over point, which again gradually increased with lecithin concentration.

the attractive interactions between lecithin and the polysaccharide chains, leading to the reinforcement of the hydrogels obtained after their drying and rehydration. In the initially prepared hydrogels, lecithin was dispersed to a greater extent in a liquid medium without this (strong) effect. This could be explained by the H-bonding between polysaccharide

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


**Table 2.** Values obtained from strain and frequency sweep tests for physically crosslinked agarose hydrogels after drying and rehydration.

Frequency sweep test results are presented in Figure 1 and show that the shape of the rheograms for all hydrogel samples was very similar. The storage modulus was dominant, which means that the samples act as a fully crosslinked gel material with a fully crosslinked internal structure. The trend of the moduli values was the same as that observed for the amplitude sweep tests and therefore indicates that the lecithin addition increased the values of the storage moduli as well as of the loss moduli, which was well correlated with the higher dry matter content, as previously stated. With increasing oscillation frequency values, the moduli values increased, which means that the hydrogel samples were not completely relaxed, and the degree of relaxation was influenced by the type of crosslinking. Practically, the average relaxation time of the hydrogel network exceeds the period associated with the progressively increasing frequency of the applied oscillatory deformations. The values of the mesh size of the internal structure of the hydrogels calculated from the frequency sweep tests using Equations (3) and (4) are recorded in Tables 1 and 2. The results for the freshly prepared hydrogels showed the same trend as other rheological data (i.e., that the mesh size does not differ substantially between the concentrations), whereas for the rehydrated xerogels, a slight increase could be observed at higher lecithin concentrations, which can be explained by lecithin fitting itself into the pores and thus increasing its size. This could be explained by lecithin forming lamellar liquidcrystalline structures in absorbed water along with the already mentioned H-bonding between the polysaccharide chains and lecithin. When comparing the absolute values of mesh sizes for freshly prepared and rehydrated hydrogels, we can see that the pores decreased in size after rehydration.

Based on the results of the strain and frequency sweeps performed onto the freshly prepared agarose hydrogels, it can be seen that lecithin, as an amphiphilic natural component, does not lead to a substantially modified viscoelastic behavior of these physically crosslinked hydrogels in the range of lecithin concentrations used (see Figure 1a,c). Agarose, which forms a thermoreversible physical hydrogel in an aqueous medium in the form of a natural linear polysaccharide, was not expected to interact significantly with amphiphilic lecithin. Thus, it was not expected that agarose could significantly interact with amphiphilic lecithin. Lecithin thus serves only as a filler, and does not interfere significantly with the internal structure of the hydrogel. Therefore, lecithin plays an important role in the rehydration of dried samples. Thus, an increasingly higher content of lecithin in the structure of such type of hydrogels causes the viscoelastic moduli storage and loss moduli to gradually decrease. Practically, the presence of lecithin affects the ability of agarose xerogels (hydrogel after drying) to reabsorb water (i.e., to swell) (see Figures 1b,d and 2). The final viscoelastic properties of hydrogels are definitely affected by the amount of dispersion medium (water) after the swelling of xerogels. If the addition of lecithin, as the modifier of the internal architecture of hydrogels, is able to change the swelling properties, it will also definitely change the viscoelastic properties due to the different amount of water. From the applicative point of view, this finding is absolutely essential, given that by choosing a suitable concentration of additive (lecithin), we were able to prepare hydrogels with the required properties (especially viscoelastic) tailored to a specific purpose.

a specific purpose.

**Figure 2.** Drying (**a**) and rehydration (**b**) of the physically crosslinked agarose hydrogels with different contents of lecithin. **Figure 2.** Drying (**a**) and rehydration (**b**) of the physically crosslinked agarose hydrogels with different contents of lecithin.

able to prepare hydrogels with the required properties (especially viscoelastic) tailored to

### 2.1.2. Drying and Rehydration Measurements 2.1.2. Drying and Rehydration Measurements

The amounts of water and dry matter associated with the studied gels are two of the most important parameters for hydrogel characterization and future applicability. Dry matter affects the behavior of the final material. The same is true for the water inside the hydrogel, which significantly affects, for example, the transport properties. As stated in The amounts of water and dry matter associated with the studied gels are two of the most important parameters for hydrogel characterization and future applicability. Dry matter affects the behavior of the final material. The same is true for the water inside the hydrogel, which significantly affects, for example, the transport properties. As stated in Section 1, these parameters predetermine the applicative nature of the final system.

Section 1, these parameters predetermine the applicative nature of the final system. The results of the drying kinetics of physically crosslinked hydrogels can be seen in Figure 2a. At the start of these experiments, all weights of the hydrogels (2 ± 0.2 g) and xerogels were comparably the same. It can be seen that the lecithin addition had no influence on the drying kinetics. The most likely explanation is that water retained by lecithin is not bound as tightly as water hydrating agarose. Conversely, during the swelling process, hydrogel with lecithin easily draws water (more easily than the agarose hydrogel solely) and this resulted in the lecithin-agarose samples showing a higher swelling ability with corresponding lower moduli (Figure 1). The swelling experiments demonstrated the influence of lecithin on the swelling capacity. Therefore, the lecithin structures insert themselves into the hydrogel pores and support the water intake. The kinetics of the swelling process was very similar for all samples, with a peculiarity noted at the onset of the experiment, where the samples richer in lecithin (1 and 2 wt.%) revealed a greater rate of water absorption. Additionally, the same systems (agarose with 1 and 2 wt.% of lecithin) The results of the drying kinetics of physically crosslinked hydrogels can be seen in Figure 2a. At the start of these experiments, all weights of the hydrogels (2 ± 0.2 g) and xerogels were comparably the same. It can be seen that the lecithin addition had no influence on the drying kinetics. The most likely explanation is that water retained by lecithin is not bound as tightly as water hydrating agarose. Conversely, during the swelling process, hydrogel with lecithin easily draws water (more easily than the agarose hydrogel solely) and this resulted in the lecithin-agarose samples showing a higher swelling ability with corresponding lower moduli (Figure 1). The swelling experiments demonstrated the influence of lecithin on the swelling capacity. Therefore, the lecithin structures insert themselves into the hydrogel pores and support the water intake. The kinetics of the swelling process was very similar for all samples, with a peculiarity noted at the onset of the experiment, where the samples richer in lecithin (1 and 2 wt.%) revealed a greater rate of water absorption. Additionally, the same systems (agarose with 1 and 2 wt.% of lecithin) were able to absorb the largest amount of water.
