2.3.2. Poly(ether urethane) Synthesis

PEU synthesis was carried out according to Bo ffito et al. [21]. P407, HDI and CDM were reacted at 1:2:1 molar ratio and the synthesis was conducted under N2 atmosphere following a prepolymerization method with a first reaction between P407 and HDI to form an N=C=O-terminated prepolymer which was then chain extended through the addition of CDM. In detail, the required amount of HDI was added to a 20% *w*/*v* concentrated P407 solution previously prepared in DCE and equilibrated at 80 ◦C. DBTDL was also added at 0.1% *w*/*w* concentration with respect to P407 and the reagents reacted for 150 min. Then, the reaction mixture was cooled down at 60 ◦C and a 3% *w*/*v* concentrated CDM solution previously prepared in DCE was added to start the chain extension reaction which lasted 90 min and was finally terminated through the addition of MeOH. The synthesized PEU was collected by precipitation in an excess of petroleum ether (petroleum ether: DCE = 4:1 *v*/*v*) and further purified by solubilization in DCE (20% *w*/*v*) and precipitation in a diethyl ether/MeOH mixture (98:2 *<sup>v</sup>*/*<sup>v</sup>*, 5:1 volume ratio with respect to DCE). PEU was collected by centrifugation (MIKRO 220R, Hettich, Tuttlingen, Germany), dried overnight, grinded and stored under vacuum at 4 ◦C until use. Hereafter, the synthesized PEU will be referred to with the acronym CHP407, where C, H and P407 refer to CDM, HDI and Poloxamer®407, respectively.

## *2.4. Chemical Characterization of the As-Synthesized PEU*

The successful synthesis of CHP407 poly(ether urethane) was assessed by attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopy and size exclusion chromatography (SEC). CHP407 and P407 ATR-FTIR spectra were registered using a Perkin Elmer (Waltham, MA, USA) Spectrum 100 instrument equipped with an ATR diamond crystal (UATR KRS5). Spectra were obtained as an average of 16 scans registered at RT within the spectral range 4000–600 cm<sup>−</sup><sup>1</sup> (resolution 4 cm<sup>−</sup>1). CHP407 molecular weight distribution was assessed by SEC using an Agilent Technologies 1200 Series (Agilent Technologies, Inc., Santa Clara, CA, USA) instrument equipped with a refractive index detector (RID) and two columns (Waters Styragel HR1 and HR4, Waters Corporation, Sesto San Giovanni, Italy). Analyses were conducted at 55 ◦C using N,N-dimethylformammide (DMF, CHROMASOLV Plus, inhibitor-free, for HPLC, Carlo Erba Reagents, Cornaredo, Italy), added with 0.1% *w*/*v* LiBr (Sigma Aldrich, Milano, Italy) as eluent at a flow rate of 0.5 mL/min. Samples were prepared at 2 mg/mL concentration in the mobile phase and filtered through a 0.45 μm syringe filter (Macherey-Nagel, Düren, Germany poly(tetrafluoro ethylene) membrane) before analysis. The Agilent ChemStation software was finally used to estimate CHP407 Number Average Molecular Weight (Mn) and polydispersity index (D) based on a calibration curve previously constructed starting from 10 poly(methyl methacrylate) standards with Mn ranging between 940 and 214,600 g/mol.

#### *2.5. Preparation of CHP407-Based Hydrogels Carrying Cu-MBGs Loaded with Ibu*

CHP407 thermosensitive hydrogels were prepared at a final PEU concentration of 15% *w*/*v* [16,21]. Cu-MBGs loaded with Ibu were encapsulated into CHP407-based hydrogels with final particle concentration of 20 mg/mL. In detail, hybrid hydrogels were prepared by adding an aliquot of particle suspension in ddH2O (100 mg/mL), previously sonicated for 3 min at 20% amplitude, to a CHP407-based solution prepared in physiological saline solution (0.9% NaCl). The starting concentration of CHP407 solution was determined in order to reach the desired PEU and particle contents in the final system. Both CHP407 solubilization and particle addition were carried out at 4 ◦C to avoid undesired gelation during sample preparation. Sample mixing was conducted using a Vortex mixer for 30 s to ensure homogeneous particle dispersion within the sol-gel systems. Particles were added to CHP407 aqueous solutions immediately before use to avoid premature payload release before characterization tests. Pure CHP407 sol-gel systems and CHP407 hydrogels loaded with Ibu were also prepared as control samples. Loading of Ibu in CHP407 sol-gel systems was carried out by adding CHP407 aqueous solutions prepared at higher concentration with a predefined volume of an Ibu stock solution (at 40 mg/mL in ethanol) to reach an average content equal to the Ibu amount incorporated into MBG\_Cu2%\_SG\_Ibu and MBG\_Cu2%\_SD\_Ibu, as assessed through TGA analysis. Based on this calculation, CHP407 sol-gel systems were loaded with Ibu at a final concentration of 2.5 mg/mL. Hereafter, the developed sol-gel systems will be referred to with the acronyms reported in Table 1. For all the conducted tests, hydrogels

were prepared in Bijou sample containers (inner diameter 17 mm, Carlo Erba Reagents, Cornaredo, Italy) at a final volume of 1 mL, to avoid geometry and volume influence on the performed characterizations.


**Table 1.** Compositional information and acronyms of the designed sol-gel systems.

## *2.6. Characterization of Hybrid Sol-Gel Systems*

The thermosensitive behavior of pure CHP407 (i.e., without particle) and hybrid hydrogels (i.e., containing MBG\_Cu2%\_SG\_Ibu, MBG\_Cu2%\_SD\_Ibu or Ibu) was studied through tube inverting test in temperature ramp mode and isothermal conditions (i.e., at 37 ◦C) to assess the effects of drug/particle incorporation on hydrogel sol-to-gel transition. In view of their potential clinical application as injectable depots for the prolonged and sustained payload release, the delivery profile of ibuprofen and copper ion was investigated in physiological mimicking conditions. Furthermore, gel swelling potential and residence time in watery environment at physiological temperature were also assessed.

## 2.6.1. Thermosensitive Behavior of CHP407-Based Sol-Gel Systems

Tube inverting test in temperature ramp conditions was carried out to estimate hydrogel Lower Critical Gelation Temperature (LCGT). In detail, samples were subjected to a step-by-step temperature increase within the range 5–40 ◦C at a rate of 1 ◦C/step. In each step, temperature was kept constant for 5 min and the samples were finally inverted for 30 s to visually inspect their sol, biphasic or gel state (in the gel state no flow was observed within the set inversion time). The same test was carried out in isothermal conditions at 37 ◦C to assess hydrogel gelation time. In this test, instead of temperature, incubation time at 37 ◦C was varied from 1 to 10 min with a rate of 1 min/step. Each step consisted in sample equilibration in the sol state for 10 min, incubation at 37 ◦C for the required time, inversion for 30 s and visual inspection.

## 2.6.2. Gel Swelling and Stability in Aqueous Media

Gel swelling and stability in physiological mimicking conditions, that is, in aqueous media and 37 ◦C, were investigated on the designed sol-gel systems up to 14 days incubation time [16]. Samples (1 mL) were first incubated at 37 ◦C (Memmert IF75, Schwabach, Germany) for 15 min to allow a complete gelation and then added with 1 mL of Trizma® (0.1 M, pH 7.4) previously equilibrated at 37 ◦C. Complete medium refresh was performed every two days. On predefined time points of 6h, 1d, 3d, 7d and 14d three samples were collected, weighted upon removal of the residual buffer, lyophilized (Martin Christ ALPHA 2–4 LSC, Osterode am Harz, Germany) and weighted again. The collected data were finally used to estimate sample percentage of swelling (weight change in wet state %) and polymer weight loss (weight loss in dry state %) [16]. Samples collected on day 14 were also analyzed by SEC to assess changes in polymer molecular weight distribution during incubation in aqueous media at 37 ◦C.
