2.6.3. Payload Release Studies

Payload release studies were carried out on hydrogels incorporating MBG\_Cu2%\_SG\_Ibu, MBG\_Cu2%\_SD\_Ibu and simply dispersed Ibu. Tests were conducted at 37 ◦C up to 14 days using Trizma ® as release medium. Complete gelation of the samples was ensured through incubation at 37 ◦C for 15 min; then, 1 mL of release medium (previously equilibrated at 37 ◦C) was added to each sample and the release test started. Release media were collected and completely refreshed at 1h, 3h, 5h, 1d, 2d, 3d, 4d, 8d, 10d, and 14d incubation time. Released Ibu was quantified through a high performance liquid chromatography (HPLC, Thermo Scientific, Dionex Ultimate 3000, Waltham, MA, USA) instrument equipped with a C18 column (5 μm, 120 Å) according to the protocol described by Alsirawan et al. [22]. A mixture of acetonitrile (ACN, Carlo Erba Reagents, Cornaredo, Italy, HPLC grade) and phosphoric acid solution at 0.03% *w*/*v* concentration (pH 2.25) at 60/40 *v*/*v* was used as mobile phase at 1.7 mL/min. Analysis were conducted with an injection volume of 20 μL, at RT and 214 nm for 5 min. In order to prepare the samples, the collected extracts were mixed with ACN at 40/60 volume ratio and filtered through a 0.45 μm syringe filter (Macherey-Nagel, Düren, Germany, poly(tetrafluoro ethylene) membrane). Ibu content was finally quantified with respect to a calibration curve based on ibuprofen standards with concentration ranging between 0 and 2.5 mg/mL. The collected extracts were also characterized by inductively coupled plasma atomic emission spectrometry technique (ICP-AES) (ICP-MS, Thermo Scientific, Waltham, MA, USA) to measure the concentration of released copper ions. In order to express the results in terms of released percentage, the amount of copper initially incorporated into the MBG framework was measured by dissolving MBG\_Cu2%\_SG\_Ibu and MBG\_Cu2%\_SD\_Ibu in a mixture of nitric and hydrofluoric acids (0.5 mL of HNO3 and 2 mL of HF for 10 mg of powder) and quantifying copper concentration through ICP-AES. To compare the ion/drug release kinetics from MBGs as such and embedded within the hydrogel, release tests from particle alone were also conducted as follows: drug-loaded powders were dispersed in physiological saline solution at 20 mg/mL MBG concentration and Trizma ® was added as release medium at 37 ◦C (1:1 volume ratio), as previously performed with the hydrogel systems. Release tests from free MBG particles were conducted up to 24 h observation time. In order to better characterize copper ion and ibuprofen release mechanism from the hydrogels incorporating MBG\_Cu2%\_SG\_Ibu, MBG\_Cu2%\_SD\_Ibu and Ibu as such, the Korsmeyer-Peppas equation was used in the form recently reported by Bo ffito et al. [23] to estimate the release exponent *n*, whose value classifies the type of release. Specifically, an *n* value of 0.45 or 0.89 is typical of a di ffusion- or swelling/relaxation-controlled release, respectively. An *n* value within the range 0.45–0.89 characterizes an anomalous release, meanwhile when *n* shows a value higher than 0.89 other processes are ongoing in addition to di ffusion and swelling/relaxation.
