**5. Conclusions**

Nanospermidine, obtained by spermidine encapsulation in nanomicelles, can represent a new tool in antitumor therapy being endowed with both the pharmacokinetics characteristics of nanomedicines and the activity of polyamines catabolism modulators.

As a nanocarrier, nanospermidine can undergo accumulation at the tumor site by extravasation through the discontinuities of the tumor capillaries, thus allowing spermidine localization at the pathological site.

At the cellular level, nanospermidine can provide spermidine penetration into the cells in amounts exceeding physiological values, thus increasing ROS production over limiting values for cell tolerance and triggering cell death.

Administration of nanospermidine in combination with nanofenretinide further increases the antitumor activity of nanospermidine.

Hence, nanospermidine may represent a new approach in tumor treatment because it provides a high intracellular increase of the polyamine and tumor cell death by administration of micromolar, physiological doses of spermidine that make the treatment tolerable and biocompatible.

Finally, the proven ability of spermidine to activate macrophages and memory B cell responses makes nanospermidine worth further evaluation to assess if the antitumor effect demonstrated in this study can be further improved by inhibition of tumor immunosuppression in in vivo settings.

**Author Contributions:** P.L. and M.R. contributed equally to this work. Conceptualization, P.L., M.R., I.O., P.B. and G.F.; Methodology, P.L., M.R., P.B. and G.F.; Investigation, P.L. and M.R.; Formal Analysis, P.L., M.R., P.B., G.F. and I.O.; Writing, P.B., G.F. and I.O.; Funding acquisition, I.O., P.B. and G.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** The project work was partially funded by the "Ministero dell'Università e della Ricerca" (Targeting Hedgehog pathway: Virtual screening identification and sustainable synthesis of novel Smo and Gli inhibitors and their pharmacological drug delivery strategies for improved therapeutic effects in tumors. PRIN 2017, project n. 20175XBSX4).

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author, I.O.

**Acknowledgments:** The authors wish to acknowledge the support of the Center for Applied Biomedical Research (CRBA) of the University of Bologna.

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