*Cryogels as Carrier*

The ability of the partially modified PS to act as a delivery system of active molecules was investigated by solution NMR spectroscopy, as this technique is not limited to specific classes of compounds or functional groups but can be extended to the determination of all hydrocarbon compounds and is very useful for the analysis of mixture. Moreover, together with the release profile, it is possible to observe when the cryogel starts to dissolve. Finally, it is a quantitative and non-disruptive technique.

To test the cryogels' ability to act as drug carrier, caffeine was chosen as model molecule because of the affinity of its functional groups with those of modified PS and because it is a well-known antioxidant and pro-oxidant and has reinforcing properties [35]. All samples in Table 1 were loaded with caffeine by immersing each of them into an Eppendorf tube containing caffeine in water, as illustrated in Section 4.3. (route (a)), and finally, samples A and B were discarded because of their fragile texture and easy water solubility; thus, cryogels from sample C conditions were used like carriers due to their compact and sTable Structure. Successively, the uploading capacity was evaluated for both samples. The main difference between the two loading methods is in the wet or dry use of the cryogels. For wet uses, the idea is to enhance the dissolution rate of poorly water-soluble drugs, thus increasing the therapeutic effects linked to drug availability. In the case of dry-loaded cryogels, wound healing applications are considered. This cryogel may be able to generate a wet gel at the wound site when an exudate is present, thus avoiding perilesional skin damages.

Firstly, for sponge cryogels from route (a), the release profiles of adsorbed caffeine were evaluated by collecting a series of proton NMR experiments acquired as detailed in Section 4.5. Direct quantification of released caffeine was done through a proportional comparison between the signal of the internal standard (TMS at δ = 0 ppm) and a selected signal of caffeine (e.g., a methyl signal at δ = 3.32 ppm,) considering that in a proton spectrum, the area of the signals is directly proportional to the number of protons present in the active volume of the sample [36]. Milligrams of released caffeine were determined by applying Equation (1) and then plotted vs. contact time in Figure 6 (considering cumulative milligrams over time). Data analysis for the sponge cryogel evidenced that the maximum of released caffeine was reached in the first 1560 min of elution, probably due to the quick release of caffeine from the surface of the PS material. Successively, for more prolonged contact time, the concentration goes down, and a more constant profile was reached probably because the caffeine entrapped in the core system was slowly released over time. After prolonged contact time, the cryogel starts to dissolve. Two stock solutions were analyzed to replicate data showing the same trend.

In the case of the dry-loaded cryogel from route (b), the procedure for the quantitative analysis of released caffeine was the same as described above. The plot of NMR data vs. contact time in Figure 6 evidenced no significant differences on the release profile for this material with respect to the sponge cryogel. The only valuable difference is attributable to the stability of this material that is more fragile and difficult to handle. In addition, in this case, two stock solutions were analyzed to replicate data showing the same trend.

**Figure 6.** Percentage/mg medium values of the cumulative caffeine release versus time (standard deviation bars are reported) for the sponge cryogel and dry-loaded sample.

These preliminary studies are encouraging and aspire to develop cryogels for specific applications. The sponge cryogels may contribute to the development of specific administration routes (topical, pulmonary, oral), thus shaping on demand, and on the "site" the drug availability. The dry-loaded cryogel will be useful for wound healing applications; it will mimic the natural tissue environment, and when loaded with antioxidant molecules, it can enhance antibiotic resistance during bacterial invasion [37].

### **4. Materials and Methods**
