*4.1. Release of Elements and Potentially Useful Therapeutic Activities*

According to the ICH Q3D(R1) guideline, no PDE limits have been established for class 4 elements [32]. The presence of Al in cosmetics is allowed according to EC 1223/2009 since it specifies that "natural hydrated aluminum silicates (Al2O3·2SiO2·2H2O) containing calcium, magnesium or iron carbonates, ferric hydroxides, quartz-sand, mica, etc. as impurities" are allowed. Aluminum has shown to be released from 1-month-old hydrogels (Table 2). The WHO has established a tolerable weekly intake of 7 mg/kg of body weight for aluminum [37]. In view of the low bioavailability of aluminum from cosmetic products (≤0.07%) [38–40], applications with more than 213 kg of hydrogel would be necessary to subject patients to potentially dangerous Al doses. Therefore, it is possible to guarantee that ALIPS9, ALIG30@10 and ALIG30@20 are totally safe regarding aluminum release. Additionally, some Al3<sup>+</sup> "misfolds cell membrane proteins", which gives it antibacterial activity [41].

Ca, Fe, Mn, Zn and S are not listed in this regulation [33], which means that, legally speaking, the presence of these elements does not limit the use of the present hydrogels as cosmetics from a legal point of view. Major elements such Mg, Ca, Na and K are considered as "essential" for both animals and human beings, and their presence in the pristine materials is considered totally safe and, sometimes, even favorable in certain cases. The usefulness of metals during wound healing has also been pointed out by some studies. For instance, it has been demonstrated that wound supplementation of Zn, Cu and Mg would be advisable during the healing process [42].

The amount of K in solids was higher than Na and Ca, though its release from hydrogels was remarkably lower than that of Ca and Na. This result is in agreement with the cation exchange capacity (CEC) of PS9 and G30 reported in previous studies [17], which showed calcium as one of the main exchangeable cations. Additionally, Ca is the second most abundant element in ALI. It has been reported that low concentrations of extracellular potassium may accelerate and favor fibroblast differentiation, thus forming scar tissue [43]. Low intracellular K<sup>+</sup> concentrations favor

interleukin-8 expression, which plays an important role in stimulating re-epithelialization, migration and proliferation of dermal cells during wound healing [26]. Therefore, a limited potassium release from both hydrogels would be beneficial during wound healing treatments.

Sodium is the second/third element with higher in vitro release levels (Table 2) and the third/forth element in terms of abundance in the pristine materials (Table 1). Moreover, Na was one of the minor exchanged cations of PS9 and G30. This apparently contradictory result has previously been observed for other clay-based hydrogels subjected to the very same in vitro release methodology [44]. This result could be related to the hydrophilicity of the exchangeable cations of the clay, that follow the order Ca2<sup>+</sup> > Na<sup>+</sup> > K<sup>+</sup> [45]. The higher the hydrophilicity of the element, the higher the ability of water to enter the interlayer space and the higher the exchange capacity. The very same trend has been found for Ca, Na and K release (Table 2) and CEC [17], despite this not being the same exact order of abundance in the pristine materials (Table 1).

Mg release increased with time in ALIPS9 and ALIG30@10, whereas it reduced in ALIG30@20 (Table 2). This element has been shown to easily permeate the skin [46] and possess anti-inflammatory activity, and is thus able to treat skin disorders such as psoriasis and atopic dermatitis [47,48]. The combination of Mg and Ca has been reported to accelerate skin barrier repair, as well as skin hydration by synergic effects [49]. Moreover, apart from the beneficial effects of Mg in the skin, this element, along with Ca, is also essential for good bone and muscle health. Therefore, if any of these elements are able to reach the bloodstream during the hydrogel treatment, they could also help treat other systemic musculoskeletal disorders, such as fibromyalgia [50].

Boron compounds have been demonstrated to be beneficial for wound healing of burned skin and in diabetic wound healing processes, both in vitro and in vivo [51,52]. B has proved useful in several metabolic pathways as well as in the increase of the wound healing rate [53,54]. Release of B decreased with time in the three hydrogels until it reached undetectable levels. Consequently, if any benefit should be obtained from B, those benefits would be at its maximum in young hydrogels.

ALI composition also played an important role in the levels of elements released during the in vitro tests. In fact, the release of S can be totally ascribed to the natural spring water composition (ALI) (Table 1). The release of sulphur reduced with time in all cases (Table 2). Higher S release was reported for ALIG30@10 48 h. For ALIPS9 and ALIG30@20, the release amounts of S were very similar. Differences in ALIG30@10 and ALIG30@20 can be ascribed to the clay mineral concentration. Balneotherapy with sulphurous waters and peloids has been proven to help with several disorders and diseases [55,56]. Specifically, keratolytic, anti-inflammatory, keratoplastic and antipruritic effects have been related to S [57]. Sulphurous mineral waters may be absorbed through the skin causing vasodilation, analgesia, immune response inhibition, and keratolytic effects that reduce skin desquamation [58]. Moreover, S could potentiate angiogenesis (endothelial cell proliferation) and regulate skin immunity. Consequently, the mobility of this element would be positive, since it can ameliorate several skin disorders. In this particular case, to obtain the maximum beneficial effects from sulphur, young hydrogels should be used, when the mobility of this element is maximum.

Mn works as a coenzyme in several biological processes, such as the transition between quiescent and proliferative phases of fibroblasts [59]. Nonetheless, Mn levels contained in healthcare formulations should be controlled due to possible toxic brain accumulation [60–62]. Levels of Mn were the same for pristine PS9 and G30 (while absent in ALI, Table 1). Consequently, it is possible to state that the release of this element is solely due to the clay mineral. Mn release increased with time in ALIG30@20, while it was not measurable in ALIG30@10, probably due to the lower concentration of G30 in this formulation. A study on the bioavailability of manganese from soils revealed that in acid soils, Mn bioavailability grows [63]. Previously it has been shown that G30 and PS9 hydrogels prepared with ALI water suffer from a reduction in pH values during the first 6 months [64]. This modification of the pH could be the explanation for a higher release of Mn after 1 month in ALIPS9 and ALIG30@20. In terms of safety, ALIG30@10 would be the safest formulation, since Mn release was not detectable during Franz cells study.

Zinc is a class 4 element, but it is not listed in EC 1223/2009. The ALIPS9 hydrogel showed an increase in Zn release with time, while ALIG30@20 showed stable levels (Table 2). The increase in Zn release in ALIPS9 and ALIG30@10 could also be related to pH changes in the formulation with time, although the literature results are contradictory [63]. Regarding safety and regulations, Zn did possess a defined PDE level in the Q3D(R1) [32] (13,000 μg/day for both oral and parenteral routes). Moreover, the WHO defined a provisional maximum tolerable daily intake amount of 18–60 mg/day for an adult of 60 kg. As previously mentioned, it has been reported that this element could compromise renal and hepatic functions when high doses reach the bloodstream. Nonetheless, Zn has also been demonstrated to be essential for keratinocyte and fibroblast proliferation, differentiation and survival. Its deficiency has been related to different disorders such as acquired acrodermatitis enteropathica, biotic deficiency, alopecia and delayed wound healing. Moreover, Zn concentration is usually higher in the epidermis than in the dermis [65,66]. Consequently, the mobility of Zn from the studied hydrogels is seen as a positive and potentially useful feature for wound healing. Moreover, the released amount of Zn in Franz cells can be considered safe, since it was below the WHO and PDE limits previously mentioned and they are intended to be topically administered.

Together with Zn, Cu is a useful element in terms of wound healing [67] and its presence is allowed in cosmetics by EC 1223/2009. This element has been demonstrated to increase the expression of TGF-β1 in ex vivo skin models, thus leading to higher pro-collagen 1 and elastin production by fibroblasts [67]. Moreover, Cu has been demonstrated to enhance skin cell migration (keratinocytes and fibroblasts), which is crucial for wound healing [68,69]. ALIPS9 and ALIG30@10 were shown to favor fibroblast migration in a previous study [17], which could be related to copper release. Additionally, copper possesses an antimicrobial effect and has been proposed as an ingredient for wound dressings [70]. In fact, some clay minerals with Cu were demonstrated to be the most effective against *Escherichia coli* and *Staphylococcus aureus*. Release levels of Cu revealed that, to obtain the aforementioned effects, extemporaneous hydrogels should be used (Table 2).

Ga showed minimum mobility in both hydrogels (Table 2) and significantly reduced mobility in ALIG30@20 after 1 month. Higher release levels in ALIG30@20 versus ALIPS9 can be ascribed to a higher concentration of this element in G30 pristine material (Table 1). This element is not addressed in any of the aforementioned regulations [32,33,71,72] since it is currently considered a relatively non-toxic element for humans. Antimicrobial activity of Ga has been reported [73,74], which could be of use for the treatment of infected wounds. A biocompatible, gallium-loaded, antimicrobial, artificial dermal scaffold has been recently proposed [75]. Other biomedical uses of Ga have also been previously reported due to its low toxicity [76–81]. In view of the existing bibliography and the present results, extemporaneous ALIG30@20 hydrogels would be a proper choice to obtain antimicrobial activity.

Strontium mobility was one of the most remarkable among the trace elements, mainly because of its presence in ALI. The presence of this element in cosmetics is not considered determinant in terms of safety, maybe because symptoms of Sr overdose are not yet clear in humans. What is more, despite the in vivo studies performed in animals, no Sr limits have been established for humans (since dietary intake variations did not induced acute toxicity symptoms) [82,83]. Wound healing effects of strontium chloride hexahydrate has been evaluated in vivo. This strontium salt was shown to reduce TNF-α expression in the wound site and, therefore, reduce inflammation [84], which is of special use in chronic inflammatory disorders. The antioxidant effect is also related to Sr, according to previous studies [85] that used strontium-substituted bioglass for tissue engineering purposes. Strontium has also been included in wound dressings as a wound healing promoter [86] and has been demonstrated to exert useful systemic effects when it reaches the bloodstream [87–90]. In conclusion, the release of Sr release is desirable, ALIPS9 being the formulation providing the highest levels of this element.
