3.4.1. Folic Acid Coating and Drug Releasing

The chemical modification of bi-HNTs were detected by FTIR. Halloysite is an aluminosilicate clay mineral (Al2Si2O5(OH)4), the Si-O stretching vibrations and Al–OH vibrations were represented at 1000–1130 cm−1. Folic acid, FITC and MTX containing –NH and –OH bonds, their characteristic absorption is in the range of 3300–3500 cm<sup>−</sup>1, NH2 scissoring at 1550 cm−<sup>1</sup> and NH associated bending at 1652 cm−1. The deformation in 3300–3500 cm−<sup>1</sup> and 1652 cm−<sup>1</sup> indicated the successful grafting of FA, FITC, and MTX (Figure 7A) [21]. The XRD patterns also present similar results. In Figure 7B, except for the HNTs profile there is a sharp peak appeared at 2θ = 17.8◦ for other groups, which is the diffraction peaks of folic acid. The slight shift of this peak at HNTs + FA + FITC + MTX profile may due to the methotrexate loading. The loading efficiency of MTX in bi-HNTs was 34.74% ± 3.5%. Its drug release profile is presented in Figure 7D. Even through, there was a burst release during the first 20 h, this drug delivery system provided sustained drug release for more than 96 h.

**Figure 7.** (**A**) FTIR detection of pure HNTs, HNTs-FA, FA/FITC-HNTs and FA/FTIC/MTX-HNTs. (**B**) XRD patterns of pure HNTs, HNTs-FA, FA/FITC-HNTs and FA/FTIC/MTX-HNTs. (**C**) Fluorescent intensity of FITC included in cells that pretreated by chlorpromazine (CPZ) or filipin and co-cultured with bi-HNTs for different time periods. (error bar with standard deviation, *n* = 6) (**D**) Accumulated drug release profile of methotrexate in 96 h. (Error bar with standard deviation, *n* = 3).
