*2.5. Cell Culture*

Murine colorectal cancer CT26WT, osteosarcoma cells K7M2-WT, and pre-osteoblast cells MC3T3-E1 came cryopreserved from ATCC. Cryovials were thawed and allowed to equilibrate in a water bath, all the cells were cultured in 25 cm2 tissue culture flask and incubated at 37 ◦C under humidified 5% CO2 and 95% air. CT26WT cells were cultured in RPMI 1640 medium, osteosarcoma cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) and pre-osteoblast cells were cultured in alpha-MEM (Thermo Fisher Scientific, Waltham, MA, USA), all the medium containing 10% FBS and 1% penicillin. Subconfluent cells were passaged with 0.25% trypsin, collected by centrifugation, suspended in cell culture medium and cultured at a 1:4 split into 25 cm2 tissue culture flasks. All three types of cells through passaged four times before use.

#### *2.6. MTS Assay*

Cells were seeded into 48-well plate at a concentration of 1 <sup>×</sup> 105 cells/well and cultured for 24 h. Then cells were incubated in cell media that contained different concentrations of pure HNTs or functionalized HNTs (0, 50, 100, 150, 200, 250 μg/mL), respectfully. MTS stock solution (Thermo Fisher Scientific, Waltham, MA, USA) (40 μL) were added to each well and cultured for 2 h at 37 ◦C in darkness. 200 μL of supernatant of each sample were transferred to 96-well plates and read absorbance values at 490 nm by microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.7. XTT Assay*

Cells were seeded into 48-well plate at a concentration of 1 <sup>×</sup> 105 cells/well and cultured for 24 h. 100 μL of cell suspension were added into 96-well tissue culture plates, then added with 25 μL activated XTT solution (XTT Cell Viability Assay Kit, Biotium, Fremont, CA, USA) and incubated for 2 h. Absorbance values were measured at 450 nm; background absorbance was measured at 630 nm. The final normalized absorbance values were obtained by subtracting background absorbance from signal absorbance.

## *2.8. Cell Uptake E*ffi*ciency*

CT26WT cells were seeded into 48 wells at the concentration of 1.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well and incubated for 24 h. Then, bi-HNTs were added into cell culture medium at finial concentration of 25 μg/mL and incubated for another 24 h. After three times wash with fresh RPMI 1640, cell fluorescent intensities were measured by fluorescent microplate reader at 490/525 nm (Ex/Em). The cell numbers were measured.
