*2.6. Cell Culture*

HeLa cells with high folate receptor (FR) expression were used to evaluate the cellular uptake of the M-MTX/siRNA complexes. HeLa cells were cultured in DMEM (Carlsbad, CA, USA) which contained 10% fetal bovine serum (FBS) (Gemini, Woodland, CA, USA) and 1% penicillin-streptomycin (Carlsbad, CA, USA) at 37 ◦C in a humidified atmosphere of 5% CO2.

#### *2.7. Hemolytic Analysis of M-MTX and MTX-bPEI-LA on Murine Erythrocytes*

Fresh blood samples from healthy mice were collected from the orbital sinus in heparin-coated tubes. Red blood cells (RBCs) were collected by centrifuging at 3000 rpm for 5 min and washed three times with physiological saline solution. Then, the RBCs were dispersed in the physiological saline solution to obtain a 2% erythrocyte standard dispersion (*v/v*). Then, 200 μL various concentrations of MTX and MTX-bPEI-LA (equivalent to MTX-bPEI-LA at concentrations of 0, 40, 80, 100, 150, and 200 μg/mL) were incubated with 1 mL erythrocyte standard dispersion for 3 h. The suspensions were centrifuged, and the absorbance of supernatant (100 μL) was measured at 450 nm.
