*2.9. Hemolysis Test*

Since it is administered by intravenous injection (iv), it is necessary to ensure that the nano-preparation will not cause hemolysis or cell aggregation. RBCs from the blood of SD rats were collected and normal saline was added to obtain a 2% (*v*/*v*) RBC suspension. The water, normal saline, RBC suspension, and RSANs were mixed into 12 tubes according to the ratio shown in Table 1. Tube 1 (RSANs) replaced with ultrapure water served as a positive control, while tube 2 (RSANs) replaced with saline as negative control. The results after incubation of 3 h and 24 h at 37 ◦C were recorded. Meanwhile, the absorbances of supernatant were determined at 3 h and 24 h through an ultraviolet spectrophotometer at the wavelength of 540 nm, and then the percentage of hemolysis was calculated according to formula:

$$\text{Percentage of Henrylysis (\%)} = (A\_{\text{sample}} - A\_{\text{tube 2}}) / (A\_{\text{tube 1}} - A\_{\text{tube 2}})$$


**Table 1.** Hemolysis test of RSANs.

### *2.10. Macrophage Uptake Study*

The immune escape ability of nanoparticles was investigated through RAW 264.7 cells. SA was labeled with 5(6)-aminofluorescein, and SANs and RSANs were prepared with labeled SA. A 12-well plate (2 × 10<sup>5</sup> cells per well) was used for 24 h incubation. Serum-free medium was used to starve cells for 1 h and then replaced by complete medium containing SANs or RSANs (SA concentration was 50 μg/mL). A complete medium without nanoparticles was used as blank group. The cells were observed with laser scanning confocal microscope (LSCM) after incubation for 2 h. Before being photographed, the cells were fixed with 4% paraformaldehyde. The excitation wavelength of 5(6)-aminofluorescein was 493 nm. Also, three parallel groups were set up to detect the uptake quantitatively. After incubation with the drugs, each group was first digested with trypsin and then washed with PBS for 3 times, then resuspended in 500 μL PBS. The detection was performed through the FITC channel of a flow cytometer.

### *2.11. In Vitro Cellular Uptake*

To investigate the uptake of nano-formulations by tumor cells and the structural integrity of NPs during this process, the test was implemented on NB4 and 7721 cells. The determination method was described in Section 2.10. For the qualitative detection, RSANs were prepared with labeled SANs and RVs (the RVs was labeled with Dil). The density of 7721 cells was 2 × 10<sup>5</sup> cells per well, and the cells were cultured for 24 h. The cells were then treated in the same way as Section 2.10. After an incubation of 2 h, Hoechst 33342 was used to stain the nuclei for 15 min, and then the uptakes were observed with LSCM. The cells were fixed with 4% paraformaldehyde before being photographed. Since NB4 cells is a kind of suspension cells, the density of inoculation was doubled to avoid loss during the experiment, and finally immobilized on a glass slide coated with polylysine. The other steps were the same as the 7721 cells. The excitation wavelengths of 5(6)-aminofluorescein, Dil and Hoechst 33342 were 493 nm, 549 nm, and 405 nm, respectively.
