**4. Conclusions**

The objective of this project was to realize sustained release of ATO and ensure the safety and efficacy of the DDS. SANs were prepared by coating ATO with SA, and then RSANs with a shell-core bilayer structure was obtained by wrapping the RBCM on the surface of the SANs. The nanoparticles were homogeneous with spherical structures and stable for 15 days with an average EE and DL of 14.31% and 4.98%, respectively. Compared to free ATO, the SANs and RSANs showed excellent sustained release in vitro for 3 days. Generally, biological characteristics of RBCM could be used to

avoid the recognition of macrophages. Even when ingested by NB4 cells and 7721 cells, RSANs still could maintain its nuclear-shell structure. At the same time, in vitro safety test showed the safety of carrier materials and without observable toxicity to 293 cells or hemolysis and aggregation of red blood cells. Free drug can kill nearly half of cells at a low concentration, while RSANs can significantly reduce the toxicity of ATO. After continuous administration of ATO formulations through IV tail injection, the results revealed that the SANs and RSANs had no significant systemic, blood, and organ toxicity. Moreover, the RSANs could maintain inhibition of NB4 cells and 7721 cells. After in vivo administration, RSANs can avoid rapid clearance and exert their sustained release properties, thereby enough drugs can be transported to the treatment site and achieve the e ffect. This may allow RSANs greater advantages in the treatment of tumors. Therefore, it could be concluded that the Nano-drug delivery system can decrease the toxicity of ATO with high safety and the potential for treating APL as well as anti-hepatocarcinoma. Meanwhile, RSANs can prolong the duration of ATO in vivo, thus reducing administration times and enhancing patient compliance. In consequence, the DDS can be developed into a safe and sustained release delivery system for ATO.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/1/21/s1, Table S1: Investigation of encapsulation e fficiency and drug loading capacity.

**Author Contributions:** Y.L. (Yumei Lian) participated in the experimental study, including the investigation, methodology, data curation, formal analysis of data, research administration, and drafting the original manuscript. X.W. participated in research administration and revising the manuscript. P.G. participated in research administration. Y.L. (Yichen Li) and F.R. participated in revising the manuscript. J.S. and M.Q., as corresponding authors, acquired the funding, participated in the study conceptualization, data analysis, reviewing and editing the manuscript, and finalized this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (No. 81573617 & 81973487), National Major Science and Technology Projects of China (2018ZX09711003-008-002), and Shanghai Association for Science and Technology (18ZR1419700).

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