*3.3. Study by Gel Permeation Chromatography*

After the analysis of the reference standards necessary to obtain the reference values, different batches of the sterile DAC® has been used at a fixed concentration of 1.0 mg/mL. The retention values (R.t.) found are reported in Table 2. The study required us to perform a separate experiment to describe correctly the hydrolysis behavior of the sterile DAC® recovered from the titanium disk surface (D samples) or, alternatively, from bulk exposed to biofluids (simplified as PBS buffer) (F samples). Thus, two series of samples have been studied D and F all in triplicate as listed in Table 1.


**Table 2.** Chromatographic retention values of samples of Table 1 examined as reported in Materials and Methods.


**Table 2.** *Cont*.

An important result is that the two D and F series show a similar behavior thus indicating that the proximity to the titanium disk surface it is not able to induce differences in the chemistry of the degradation.

The obtained results indicated that when the ester bond undergoes hydrolysis, the degraded samples lose progressively the amphiphilic character thus decreasing the micellar aggregation and decreasing the retention time significantly.

The chromatographic analysis results can be summarized as follows: the sterilized HA-PLA DAC® shows an apparent MW lower than that measured of the non-sterilized product; the retention time trend at the different study times suggests that hydrolysis occurs at the ester bond between HA and PLA.

In fact, the retention values (Table 2) have a clear decreasing trend upon the time of exposure to hydrolysis thus confirming what was reported in NMR study's section for HA-PLA DAC® with and without sterilization.

In summary, upon time the hydrolysis of the ester bond between HA and PLA increases thus decreasing the marked amphiphilic character of the copolymer and, then, decreasing the tendency to form micellar aggregation.

At the time T15 a single polymeric species is present identified as HA-Na with an apparent MW ≥ 50 kDa.

In Figure 8, the chromatograms of: HA-Na 13 kDa (blue), Ha-Na 50 kDa (red), and T15F1 (green) are reported, displaying a comparable apparent MW for the T15 sample with HA standard at 50 kDa. All the experiments were performed in triplicate as reported in Materials and Methods and the results showed an excellent reproducibility. ≥

**Figure 8.** Overlay of the chromatograms of sample HA-Na 13 kDa (blue), Ha-Na 50 kDa (red), and T15F1 (green). In the horizontal axis the retention times in minutes are reported.
