**4. Conclusions**

Six new nanoconjugates were synthesized using polyhedral oligosilsesquioxanes (POSS) as nano-carriers for anthracyclines (DOX and DAU) and PEGs (water solubilizing agent). The new conjugates contain an ester bond capable of hydrolysis under the conditions of lowered pH (5.5), characteristic for cancer cells. The structure of the new prodrugs were confirmed by NMR (1H-NMR, 13C-NMR, 1H-13C HSQC and DOSY), FTIR and DLS. The analytical methods applied in this work can serve as the important tools and model approach in studies of other nanocarrier-anthracycline conjugates. This is evidenced by the disappearance of the signal at ~2.5 ppm (CH2OH) associated with the formation of an ester bond (in the NMR analysis) and the appearance of the 1730 cm<sup>−</sup><sup>1</sup> peak in the FTIR analysis. Using simple and efficient NMR techniques, it was possible to detect formation

of the prodrug conjugates. The conjugates are larger in size, compared to anthracycline antibiotics themselves, which is favorable due to the presumably limited normal cell penetration during chemotherapy. In addition, they show longer release time, which makes them potential candidates for biomedical applications in anticancer therapy. POSS type nanocarriers were proven again [10,11] to be useful systems in formation of nanoconjugates and nanocomplexes with anthracycline drugs. The present work shall be expanded by in vitro studies as soon as the test laboratory reopens after COVID-19 closure.

**Supplementary Materials:** The following are available online, Figure S1: Scheme for the synthesis of SAMDOX and SAMDAU; Figure S2: Scheme for the synthesis of POSSDAU-MR.; Figure S3: Structure of POSSDAU-MR; Table S1: NMR results for POSSDAU-MR.; Table S2: Concentrations of the reagents used in conjugation reaction **4**–**9**; Table S3: Concentrations of the conjugates **4**–**9** in drugs release study; Figure S4: Calibration curves of: (A) DOX in H2O/DMF (5:1) (B) DAU in H2O/DMF; Figure S5: Dependence of anthracycline concentration (A. DOX, B. DAU) on the absorbance intensity in the UV-Vis spectrum; Figure S6: A. Study of DOX/DAU release from nanoconjugates at pH 5.5 at 310 K quantified by UV-Vis method after 21 h (A) and after 42 h (B); 1H NMR spectra of **4**–**9** (500 MHz, 295 K, DMSO-d6).

**Author Contributions:** K.P. has written the research proposal, carried out part of the synthetic work and interpreted NMR, UV-Vis and FTIR results, J.K. interpreted the DLS and UV-Vis results, W.A.S. supervised the studies and finally corrected the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Science Center, grant number UMO-2016/21/N/ST5/03360.

**Acknowledgments:** The financial support for this study was provided by the Centre of Molecular and Macromolecular Studies. K. Piorecka wishes to acknowledge the Preludium Grant UMO-2016/21/N/ST5/03360.

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

**Sample Availability:** Samples of all compounds (POSS, SAMDAU, SAMDOX, compounds **4**–**9**) are available from the authors.
