Hydrogel here investigated was synthesized starting from a high molecular weight branched polyacrylic acid (carbomer 974P) and agarose, a common polysaccharide. The condensation reaction, occurring in PBS solution, was microwave-assisted to obtain a chemically cross-linked hydrogel, as verified with FT-IR analysis and presented in the following section [21]. Generally, chemical interactions would statistically bring polymer chains together and, indeed, the formation of a stable structure occurs through junction zones between chains. The presence of hydroxyl groups and carboxyl ones suggests that main interactions occur via esterification and hydrogen bonding (chemical hydrogel), as confirmed by FT-IR analysis. Moreover, PBS salts freely solvated in water cause salt carboxylates formation. Due to these reactions, AC hydrogels are quite anionic and this electrostatic nature, confirmed by mass equilibrium swelling at different pH, influences the ability and the kinetics involved in drug delivery [20]. The ability to absorb and retain a large amount of water is the key feature for 3D polymeric networks, such as hydrogel systems: swelling is thus the first point to investigate. Figure 2 shows the swelling kinetics of unloaded (Figure 2a) and loaded (Figure 2b) AC1 hydrogel at 37 °C and 5% CO₂, at different pH values. All samples exhibited fast swelling kinetics and they reached swelling equilibrium within the first hour. On one side the dependence on pH is well visible in Figure 2a: the ability to retain water increases together with solvent pH. A possible explanation could be that, because of the presence of carbomer (having a pKa value around 6.0 in a pH 7.2 phosphate buffer), the carboxylic groups of carbomer are highly dissociated. Therefore the carboxylate moiety on the polymer ionizes, resulting in repulsion between the negative charges, which favors the swelling of the polymer [32]. Adding positive charges (acid pH values of swelling solvent), negative charged backbones are partially shielded and attractive electrostatic forces rule the overall swelling process, favoring network shrinking. On the other hand, adding negative charges (basic pH values) prevailing forces are the repulsive ones, with a consequent higher stretching degree of the structure. Furthermore, mean mesh size of the polymeric network can be tuned by controlling swelling solvent pH, according to the specific requirements of the employed cells, and with respect to the specific regenerative medicine application [22]. Moreover it is easily observable that swelling phenomenon is also strictly dependent on the solute loaded within the polymeric network. Indeed the same pH dependence is present in loaded samples, but the respective mass swelling equilibrium values are lower compared with samples in Figure 2a.

This effect is probably due to repulsion forces between negatively charged polymer backbones and the molecule loaded. This compression reduces natural repulsive forces between polymer chains, with consequent reduction of the amount of water that can be absorbed during swelling mechanism.

In fact, as shown in Figure 2b solute concentration causes the swelling ratio to decrease, which agrees with the above-mentioned hypothesis of charged species shielding polymer chain’s charges. The presence of the drug, loaded within the network, represents an encumbrance to the swelling phenomenon and consequently the percentage of solvent absorbed decreases.

FT-IR spectra of AC1 and AC1_SF gel are plotted in Figure 3. Both hydrogels spectra show a broad peak around 3450 cm⁻¹, which is due to the stretching vibration of O-H bonds, while peaks around 2940 cm⁻¹ are due to the C-H stretch. According with our previous studies, the formation of ester bonds is visible in peaks corresponding to symmetric (around 1600 cm⁻¹) and asymmetric (around 1400 cm⁻¹) CO² stretches. Moreover, the presence of triethylamine (TEA) inside the network, related to C-N vibration, is confirmed by the peaks around 1080 cm⁻¹. Spectra also show, in the range 900–1000 cm⁻¹, peaks related to C-O-C stretch vibration that represents the glycosidic bond between monosaccharide units (typical of agarose structure) and ester groups.

In general the building blocks, or subunits, of macromolecules form a stable structure made up mostly of C-C bonds, usually referred as the “carbon skeleton”. C-C and C-H bonds are said to be non polar and, thus, tend to be less reactive and sometimes result inert at our degradation conditions. Building blocks of macromolecules act as discrete subunit because their internal structure consists of C-C bonds. Furthermore, C-O and/or C-N bonds make the links between the subunits representing degradable bonds constituted by oxygen or nitrogen atoms. Here, the obtained FT-IR results allow stating that the most important groups in the studied gels are: -OH, C-H, CO₂ (i.e., carboxylates), vinyl, C-N, and C-O-C. AC1_SF FT-IR spectrum showed the presence of sodium fluorescein loaded within the polymeric network by peaks (in red) in correspondence of 1460 and 1380, related to C-C aromatic skeletal stretch and C-H stretches in the aromatic ring, respectively. Furthermore the peak around 1310 cm⁻¹ is characteristic of fluorescein dianion, showing the presence of the phenoxide-like stretch [33].

Figure 4a shows the Dynamic Frequency Sweep test (DFS) spectra performed at 37 °C for both loaded (AC1_SF) and unloaded (AC1) gel samples. In both cases, storage modulus (G′) is approximately one order of magnitude higher than the loss modulus (G″), showing an elastic behavior rather than a viscous one. Moreover, both G′ and G″ of each gel are essentially independent from frequency over the entire investigated range, thus indicating dominant viscoelastic relaxations of networks at lower frequencies. This means that network relaxation time, τ, is rather long (τ ≈ 2000 s) [21]. Such rheological behavior matches the characteristic signature of a solid-like gel, confirming this nature for both loaded and unloaded gels. The values obtained for G′ (~700 Pa for AC1 and ~580 Pa for AC1_SF) shows the same order of magnitude as the modulus reported in literature for other biopolymers and hydrogels. Previous investigations about thixotropic properties of loaded hydrogel samples suggest that hysteresis loop areas are lower: SF molecules indeed make the gels less stable and cause lower mechanical properties. Sol-gel transition is faster and this effect could be a relevant improvement for gel injectability [34]. Indeed, in Figure 4b, the results of the dynamic strain sweep test were shown: for both hydrogel samples, the elastic modulus (G′) dominates at low values of strain and above the cross-over strain (γ_c), the contribution of tan (δ) overwhelms the elastic one. It is also evident that cross-over strain decreases loading solute within the three-dimensional matrix, ranging from 0.30 for AC1 to 0.1 for AC1_SF. The last comparison between the two samples can be done in terms of yield stress (Figure 4c), which decreases from AC1 to AC1_SF.

In summary, the higher material instability makes our hydrogel less stable and able to flow under lower stress values and this is in complete agreement with previous thixotropic studies performed [20].

The hydrogel samples were first immersed in PBS for about 24 h, then freeze-dried, weighted (W_d) and poured in excess solvent at pH = 0.5, 7.4 and 14 to achieve complete swelling at 37 °C in 5% CO₂ atmosphere; such conditions were applied since they are typical of in vitro experiments [21,36]. The swelling kinetics was measured gravimetrically. The samples were removed from the solvent at regular times. Then, hydrogel surfaces were wiped with moistened filter paper in order to remove the excess of solvent and then weighed (W_t) [36,37]. Swelling ratio is defined as follows:

where W_d(t) is the weight of the wet hydrogel as a function of time and W_d(0) of the dry one.

Hydrogel samples, after being left dipped for 24 h in excess of solvent, were freeze-dried and laminated with potassium bromide. Infrared spectra were then recorded using a TENSOR Series FTIR Spectrometer (Bruker, Bremen, Germany). FT-IR spectra were recorded using a Thermo Nexus 6700 spectrometer coupled to a Thermo Nicolet Continuum microscope equipped with a × 15 Reflachromat Cassegrain objective.

Rheological analysis on gel samples were performed at 37 °C using a Rheometric Scientific ARES (TA Instruments, New Castle, DE, USA) equipped with parallel plates of 30 mm of diameter and a 4 mm gap between [21]. A human-body like temperature of 37 °C was selected because investigated materials contain substantial amounts of water and higher temperatures could significantly affect their properties, especially long-term ones. Oscillatory responses (G′, elastic modulus, and G″, loss/viscous modulus) were determined at low strain values (0.02%) over the frequency range 0.1–500 rad/s. Dynamic Strain Sweep tests were also performed at frequency of 20 rad/s over the strain range from 0.01% to 100%. Thixotropic behavior was also investigated and shear strain and viscosity as a function of shear rate were evaluated, too [20]. Linearity of all viscoelastic properties was assessed for all samples.

Hydrogel samples were first freeze dried for about 24 h, then immersed in excess of PBS to swell and then degrade, always being kept at 37 °C in a 5% CO₂ atmosphere. Samples were removed from PBS (after 1, 2, 3, 7, 14, 21 and 28 days) and compared with dry samples, i.e., those that were freeze-dried but not immersed in PBS. All these samples were analyzed using FT-IR instrument collecting the transmittance signal [21,35].

Degradations were performed in PBS at 37 °C under agitation. The buffer was changed daily, and at 1, 4, 7, 14 and 28 day of incubation the buffer was removed, the polymer samples were lyophilized and degradation was quantified using the following equation [37]:

where W_d(0) is the initially dry hydrogel mass and W_d(t) is the dry hydrogel mass at time t.