3.3.3. Ion/Drug Release Test

Ibuprofen release profile from CHP407-based gels was assessed in physiological-like conditions, that is, Trizma® buffer at 37 ◦C (Figure 7).

**Figure 7.** (**A**) Ibuprofen release (%) profile from CHP407\_Ibu, CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu hydrogels. (**B**) Comparison among ibuprofen release profiles assessed from CHP407\_MBG\_Cu2%\_SG\_Ibu, CHP407\_MBG\_Cu2%\_SD\_Ibu, MBG\_Cu2%\_SD\_Ibu and MBG\_Cu2%\_SD\_Ibu.

Figure 7A compares ibuprofen release profiles from CHP407\_Ibu, CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu up to 14 days observation time. A complete release of the drug was observed from the gels loaded with ibuprofen as such, with a release of 98.6 ± 5.0% at day 10. On the other hand, MBG-loaded gels showed a sustained and prolonged release of the drug, with a percentage release of 70.0 ± 1.0% and 46.4 ± 0.1% from CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu, respectively, after 14 days incubation. Moreover, starting from day 8 the release profile reached a plateau value for both particle-loaded gels, thus suggesting an incomplete ibuprofen release. However, this phenomenon cannot be avoided, being correlated to the progressive pore occlusion due to the dissolution of silica-based MBG framework and its re-precipitation as silica gel at the pores mouth, in accordance with observations reported by Mortera et al. [40], who, in addition, also reported a significant change of the delivery curves as a function of the release medium volume. To further investigate this aspect, the release profile of ibuprofen from MBG\_Cu2%\_SG\_Ibu and MBG\_Cu2%\_SD\_Ibu alone was studied in the same release conditions (in term of powder/medium volume ratio) adopted for hybrid hydrogel testing. As a matter of fact, both MBG\_Cu2%\_SG\_Ibu and MBG\_Cu2%\_SD\_Ibu showed a plateau ibuprofen release of 61.3 ± 3.7% and 40.6 ± 1.0% after 30 and 10 min incubation time, respectively. The significant di fference in the maximum amount released can be attributed to the very high surface reactivity of spray-dried systems when soaked in aqueous media, leading to enhanced dissolution/re-precipitation phenomena and consequent hindered pore accessibility [16]. By comparing ibuprofen release kinetics from free MBGs and hydrogels containing MBG particles within the first 4 h, the role exerted by the polymeric matrix in modulating the release profile of ibuprofen was clearly highlighted (Figure 7B). In fact, a significant reduction (0.0001 < *p* < 0.001) in the initial burst release upon MBG incorporation within CHP407 gels was observed, with approx. 85% burst release reduction for both kinds of particles investigated. The release profile of copper ions from the developed hybrid formulations was also studied, showing a trend similar to that assessed for ibuprofen (Figure 8).

**Figure 8.** Copper ion release (%) profile from CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu gels.

After 14 days of incubation in aqueous medium, the 66.1 ± 1.6% and 56.1 ± 0.8% (corresponding to 291.5 ± 7.1 ppm and 192.3 ± 2.7 ppm, respectively) of the copper initially present in the MBG framework was released from CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu, respectively. These amounts fully agree with our previous results obtained for Cu-substituted MBGs without ibuprofen [16], highlighting that drug loading within MBG porous structure did not suppress or hinder the capability to release therapeutic ions through ion-exchange reactions. The incorporation of Cu-substituted MBGs within the polymeric phase successfully decreased the undesirable initial burst release of Cu2+ species typically observed for particles alone (0.0001 < *p* < 0.001). Indeed, after 1h incubation in similar releasing conditions, CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu released an amount of copper ions approximately 72 % and 61 % lower compared to the corresponding free MBG particles. Both ibuprofen and copper ions were released faster from CHP407\_MBG\_Cu2%\_SG\_Ibu compared to CHP407\_MBG\_Cu2%\_SD\_Ibu, in accordance with the higher surface area of MBG\_Cu2%\_SG\_Ibu compared to MBG\_Cu2%\_SD\_Ibu, which accounts for a faster molecule di ffusion, either ions or drugs, through MBG porous network.

To clarify the ion/drug release mechanism, the equation of Korsmeyer-Peppas was employed to estimate the release exponent *n* within the timeframe 1–5 h (being Korsmeyer-Peppas equation valid up to 60% release). Ibuprofen delivery from CHP407\_Ibu gels turned out to be a purely Fickian di ffusion-driven release, with an *n* exponent of 0.45 ± 0.04. On the other hand, CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu gels released ibuprofen with an anomalous mechanism (*n* value of 0.66 ± 0.03 and 0.68 ± 0.01 for CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu, respectively), being the drug first di ffused from the MBGs within the hydrogel and then through the hydrogel in the surrounding releasing medium. Interestingly, the release exponent clearly highlighted the coexistence of di ffusion and ion exchange reactions when copper species were released, being *n* values higher than 0.89 (*n* value of 0.94 ± 0.08 and 0.90 ± 0.07 for CHP407\_MBG\_Cu2%\_SG\_Ibu and CHP407\_MBG\_Cu2%\_SD\_Ibu, respectively).

Taken together the obtained data proved the ability of the developed platform to co-deliver copper ions and ibuprofen with a sustained release profile. The final released concentration can be finely modulated to target the required therapeutic e ffect with no associated toxicity, by taking advantage of the several degrees of freedom o ffered by the hybrid system, such as the initial particle concentration within the gel solution (before gelation) and the amount of ion/drug loaded inside the MBG nanocarriers.
