*2.7. Morphological Observation*

Transmission electron microscopy (TEM, Thermo Fisher Scientific, Shanghai, China) was performed to evaluate the morphology of RSANs. 10 μL sample solutions were dropped on carbon-supported copper, air-dried and then rinses with 10 μL ultrapure water. Drip 10 μL 2% phosphotungstic acid to stain the samples, and the excess dye was removed with filter paper from the edge. After baking 30 min under an infrared baking lamp, the morphology was observed by TEM.

#### *2.8. Drug Loading Capacity and In Vitro Release Study*

The release study of ATO encapsulated in nanoparticles was performed in 1×PBS (pH 7.4). 2 mL ATO solution (final concentration of 135.6 μg/ mL), SANs, and RSANs solution were placed into dialysis bags, respectively (M w = 3500). The dialysis bags were fastened at both ends and maintained under sink conditions at 37 ◦C using 50 mL PBS, then magnetically stirred at 100 rpm. At time point of 3 h, encapsulation e fficiency (EE) and drug loading capacity (DL) of the nanoparticles were analyzed by extracting 1 mL release solution. The optimal DL and EE were investigated by adding di fferent concentrations of ATO. For the determination of in vitro release, 1 mL release sample was taken at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, and 84 h, and then replaced with 1 mL of fresh PBS. The aliquots were filtered through 0.22 μm microfiltration membrane and diluted up to 50 times. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to detect the concentration of ATO. In addition, the following formulas were used to calculated DL and EE.

> Encapsulation E fficiency (%) = (*M*1 − *M*2)/*M*1 × 100%

$$\text{Drug Loading Capacity (\%)} = (M\_1 - M\_2)(M\_1 - M\_2 + M\_3) \times 100\%$$

where *M*1 means the total amount of used ATO, *M*2 is the amount of ATO in the dialysis solution, and *M*3 is the amount of used SA.
