## 4 Discussion  
\(\gamma\)-PGA-PEG hydrogels were obtained from an esterification reaction between \(\gamma\)-PGA biopolymers and linear PEG cross-linkers with different chain lengths, as well as with a branched PEG cross-linker. The swelling behaviour of the obtained hydrogels was investigated in deionised water, in phthalate buffer at pH = 3.0, and in phosphate-buffered saline at pH = 7.4. The highest swelling ratio was achieved after soaking the hydrogel samples in deionised water. While the SR of each hydrogel soaked in phthalate buffer and in phosphate-buffered saline was significantly lower than the SR achieved after soaking in deionised water, the achieved SR values at both buffers were quite similar. Differences in swelling in deionised water and in the buffers might be associated with the influence of ionic concentration. According to the literature, the swelling behaviour of \(\gamma\)-PGA hydrogels is associated with repulsive forces between -COO\({}^{-}\) groups. The absence of cations in deionised water allows strong electrostatic repulsion between -COO\({}^{-}\) groups, which enables high swelling values. In buffers, the number of additional cations caused weakened electrostatic repulsion between -COO\({}^{-}\) due to the chelation between -COO\({}^{-}\) groups and cations, which presumably lead to a depressed swelling degree [25]. On the basis of the conducted research, we found that the swelling ratio of hydrogels increased with the increase of the cross-linker's chain length. Such a tendency was observed for each tested condition (deionised water, pH = 3.0, and pH = 7.4). Different cross-linker amounts in each hydrogel might have affected the swelling behaviour. Increasing cross-linker concentration could result in a network with more cross-linking points (higher cross-linking density); therefore, there would be less space for water [26]. However, on the basis of conducted \({}^{1}\)H NMR analysis of the hydrolys hydrogels, the cross-linker amount was similar in all hydrogels. Therefore, it could be assumed that the swelling behaviour of the obtained hydrogels mainly depended on cross-linker chain length. Cross-linker chain length influences swelling behaviour; using a cross-linker with longer chains results in an increase of the hydrogel swelling ratio in comparison to a hydrogel with shorter cross-linker chains [27; 28]. Moreover, it was found that the use of a branched cross-linker resulted in obtaining a hydrogel with a lower swelling ratio in comparison to the application of a linear cross-linker with similar molecular weight. The more water that a hydrogel was able to absorb (the higher SR), the larger the pores that were generated during lyophilisation. Thus, in the SEM image of PEG1000, the biggest pores are visible (Figure 2e,f), in the SEM image of the PEG1000-3 arm, the smallest pores can be observed (Figure 2g,h), and a rather similar size of pores was observed in PEG200 (Figure 2a,b) and PEG400 (Figure 2c,d). On the basis of the obtained results, we established the influence of the molecular weight of the PEG cross-linker, as well as the cross-linker structure (linear or branched), on the swelling ratio of the \(\gamma\)-PGA-based hydrogels. In the current study, the lowest SR = 6.0 \(\pm\) 0.4 g/g was achieved after soaking the \(\gamma\)-PGA-based hydrogel with 3-arm PEG1000 cross-linker in phthalate buffer at pH = 3.0. The materials that achieved a low swelling ratio under acidic conditions may be useful for intestine-delivery vehicles. This is because such vehicles are first subjected to highly acidic conditions in the stomach, where contact between the contents of those delivery vehicles and the harmful medium should be limited. Then, the release of the active substance from intestine-delivery vehicles should occur after subjecting them to gradually increasing pH, from pH = 5.5 in the small intestine to about pH = 7.5 in the ileocolonic region [29].