*3.2. Stability Studies of HA-PLA DAC*® *Sterilized*

The stability studies have been performed by NMR and with DOSY NMR upon the time on the samples reported in Table 1. The NMR and DOSY of sample T15F1 are reported in Figure 6a–d and the NMR spectrum of T7F1 in D2O are displayed in Figure 7.

In the spectrum in Figure 6a, the decrease of the resonance at 5.2 ppm and the rising of the resonances characteristic of the degradation of PLA into dimers and trimers of lactic acid are evident (Monomer: (CH3) δ 1.23; (CH) δ 4.03; Dimer: (CH3) δ 1.27–1.38; (CH) δ 4.19–4.92; Trimer: (CH3) δ 1.27–1.40–1.44; (CH) δ 4.20–4.98–5.10) [16]. Moreover, DOSY experiments showed evidently that different diffusive fronts due to different hydrodynamic radius are present (a) HA resonances without the resonances characteristic of PLA, (b) the resonances due to PLA moiety and the resonances due to oligomers of lactic acid.

In Figure 6b the left projection circled in red displays species with higher diffusivity than the (a) trace and, then with lower MW than the HA residual. These are also visible in the left projections of the DOSY spectrum, circled in red.

In Figure 6c,d the slices (a) and (b) of the DOSY in Figure 6b are reported and correspond to the spectral profiles of HA and PLA fragments. Furthermore, the diffusion coefficient of the HA polymer for this last study time appears close to that of the HA standard at 50 kDa.

δ

The spectrum of the sample T7F1 in Figure 7 indicates that the ester bond is nearly completely hydrolyzed, and the PLA is completely transformed in lactic monomer, dimer, and trimer, for this study time.

(**b**)

**Figure 6.** *Cont*.

− − − − − − **Figure 6.** (**a**) NMR spectrum of the sample of T15F1 of HA-PLA DAC® sterilized and degraded as in Table 1 as reported in Materials and Methods. (**b**) DOSY spectrum of the sample T15F1. The DOSY indicates the presence of two diffusive fronts due to chemical species "**a**" and "**b**". Front "**a**" represents the residual HA polymer with a MW slightly decreased with respect to the result in Figure 3, showing D = 1.30 × 10−<sup>10</sup> m<sup>2</sup> s −1 corresponding to a MW of 37 kDa. Front "**b**" represents the fragments of PLA released by hydrolysis of the conjugated bridge. The region of the vertical profile circled in red shows the presence of the fragments with a higher diffusivity (3.9–2.50 × 10−<sup>9</sup> m<sup>2</sup> s −1 ). The region circled in blue is attributed to the diffusive front of the residual deuterium hydrogen oxide HDO at 4.76 ppm. For D2O residual protons, the value of D found was 3.79 <sup>×</sup> <sup>10</sup>−<sup>9</sup> <sup>m</sup><sup>2</sup> s −1 . (**c**) Slice of front "**a**" of the DOSY spectrum. (**d**) Slice of front "**b**" of the DOSY spectrum.

**Figure 7.** NMR spectrum of sample T7F1 in D2O.
