**4. Conclusions**

From the results of chromatographic analysis, HPLC-GPC-UV and NMR spectroscopy included DOSY NMR of sterilized HA-PLA DAC® it is possible to conclude that the sterilization process obtained with ionizing radiation induces a reduction of MW of HA-PLA DAC® as already reported by several authors.

Moreover, HA-PLA DAC® sterilized in physiological conditions (PBS), as in the proprietary Novagenit protocols for checking stability, shows the expected behavior obtaining a complete hydrolysis after about 15 days and the degradation into monomeric, dimeric, and trimeric species within the sensitivity of the techniques used.

The copolymer HA-PLA DAC® sterilized after 15 days in the PBS stability test shows an apparent MW of almost 50 kDa in both the techniques applied. The sterilized HA-PLA DAC® shows an amphiphilic character with micellar aggregation properties in the polar media as indicated by the results in agreement for all the applied techniques.

Finally, in our case, the diffusion studies by DOSY NMR spectroscopy confirm to be a very useful tool in investigating the behavior of chemically conjugated and bioconjugated compounds in pharmaceutical industry research.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/10/11/1478/s1, Figure S1: Behaviour of HA-PLA conjugate DAC® after sterilization in different solvent mixture indicated, Figure S2: DOSY experiments conducted as reported in Materials and Methods on the mixture of two fragments of HA with different MWs, Figure S3: DOSY experiments conducted as reported in Materials and Methods on the mixture of two fragments of HA with different MWs.

**Author Contributions:** Conceptualization, A.T., T.G., D.B., M.P., C.M.; Methodology, T.G., R.N., W.M., F.B.; Validation, A.T., R.N., W.M.; Investigation, T.G., C.M., D.B., R.N., W.M., F.B., A.T., M.P.; Resources, A.T., D.B., T.G., M.P.; Data Curation, T.G., R.N., W.M., F.B.; Writing—Original Draft Preparation, C.M., A.T., T.G., M.P.; Writing—Review & Editing, A.T., T.G., D.B., M.P.; Visualization, T.G., C.M.; Supervision, A.T.; Project Administration, A.T., D.B., M.P.; Funding Acquisition, A.T., D.B.,M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
