*3.2. In Vitro Release of Elements*

Elements released from ALIPS9, ALIG30@10 and ALIG30@20 hydrogels are summarized in Table 2. As expected from the nature and composition of the pristine ingredients of both hydrogels, the release of major elements (Ca, K, S, Mg, Na) was not only confirmed but desirable due to their physiologic activities, which will be discussed later. In particular, Ca showed significant release levels in all hydrogels, which is in agreement with the high levels of this element in pristine materials (Table 1). Moreover, S is the major element present in ALI, which also explains the high release levels of this element from the formulations.

**Table 2.** Mobility of elements after Franz diffusion cell tests. Major elements are expressed in mg/100 g of hydrogel, while the rest of the elements are expressed as μg/100 g of hydrogel. Mean values ± s.e. (*n* = 6). "ND" stands for "Not Detected". Release levels of elements marked with \* were obtained from [36].


Release levels of Mg were very similar for the three hydrogels. The release of Al increased with time in all cases, not being detected in any of the young hydrogels. On the other hand, the amount of B released after 1 month was lower. The most remarkable release regarding trace elements was shown by Zn and Sr, followed by Cu. Cu release significantly decreased after 1 month in the three hydrogels. As previously reported, the amount of Cu detected in G30 was higher than PS9 (Table 2). This was in agreement with the lower release of both elements in ALIPS9 versus ALIG30@10 and ALIG30@20. Mn release increased with time in ALIPS9 and ALIPS9@20, while it was under the detection limit of the technique for ALIG30@10 experiments. Levels of Mn were the same for both PS9 and G30 (and absent in ALI, Table 1) but ALIG30@20 showed a remarkably higher release of this element.

The rest of the elements were not released or released in very low amounts. Except for Au, Cu and Ag, the rest of the trace elements are not included/mentioned in the EC 1223/2009 regulation [33]. This means that their safety has not been thoroughly assessed or their toxicity is considered non-significant. It is worth mentioning that In and Re were not present in the pristine materials and that they were also not detected during the in vitro release tests. This fact confirmed the absence of contamination with these elements during ALIPS9, ALIG30@10 and ALIG30@20 formulation processes and preservation.
