**1. Introduction**

Cancer is the second leading cause of death in the United States [1]. While radiation and surgery treatments have advanced cancer treatment, chemotherapy is still one of the leading treatment modalities [2]. Unfortunately, current chemotherapeutic agents adversely affect healthy cells at the target site and elsewhere in the body [3]. Chemotherapy drugs work by impairing cell division and are effective treatments for early-stage tumors when cancer cells are rapidly multiplying. However, they also produce a range of unpleasant side effects. Systemic toxicity is an undesired consequence for most chemotherapeutic drugs [1,2]. The development of a multi-functional drug delivery system (DDS) with an ability to provide extended, controlled, and selective drug release is at the forefront of current cancer therapy research [2,3]. Targeting chemotherapeutic drugs directly at the tumor cells would increase drug effectiveness and reduce side effects.

Methotrexate (MTX) is a folic acid antagonist. It has an anti-cancer effect on lymphoblastic leukemia, lymphoma, osteosarcoma, and breast, lung, head, and neck cancers [4–6]. MTX restricts cancer cell growth by disrupting transmethylation reactions, which are essential in forming proteins, lipids, and myelin [7]. However, the high dose administration of MTX can damage cells in bone marrow, gastrointestinal mucosa, and hair follicles. Severe side effects, including renal failure, neurotoxicity, hematologic toxicity, mucocutaneous toxicity, and pulmonary toxicity, may result after high dose MTX treatments [8]. Accordingly, a targeted MTX drug delivery system designed to improve its target delivery and reduce destruction to healthy cells seems like a promising approach.

Halloysite nanotubes (HNTs) have attracted significant attention in drug delivery due to its biocompatibility, physicochemical stability, and unique structural properties. HNTs are naturally occurring nanoscale tubes composed of Al2O3·2SiO2·*n*H2O. In the process of aluminosilicate rolling, Al–OH groups are folded inside, and Si-O-Si groups are exposed to the outer surface. At neutral pH, the inner surface of HNTs is positively charged, and the outer surface is negatively charged [9]. Depending on the geological origin, the lumen and outer diameters of HNTs range between 10–15 nm and 50–80 nm, respectively [9]. While the length of HNTs averages between 0.5–2 μm. With specific surface modification, active agents can be conjugated on the surface or encapsulated in the empty HNT lumen. Curcumin [10], doxorubicin [11], irinotecan [12], and resveratrol [13] have been successfully doped into HNTs and shown to have an anti-cancer effect.

In a previous study, we demonstrated that the HNT surface could be functionalized with *N*-[3-(trimethoxysilyl)propyl) ethylenediamine (DAS) for grafting folic acid (FA) and fluorescein isothiocyanate (FITC) resulting in a bifunctionalized HNTs (bi-HNT). We used FA as a ligand for targeted drug delivery and FITC as a visual tracking agent. Surface modification using FA and FITC was further confirmed by FTIR, 13C CPMAS NMR spectrum, and UV-Vis. Folic acid directed HNTs adhered to folate receptors, which are overexpressed in numerous cancers but rarely expressed or nonexistent in most normal tissues [14–16].

In this study, we focused on identifying the cellular uptake mechanism and intracellular location of bi-HNTs after endocytosis. We further studied cellular interactions after exposure to bi-HNTs, including cell viability, proliferation, and cell uptake efficiency. When colon cancer cells (CT26WT) were co-cultured with bi-HNTs, cell viability decreased at higher doses. Further analysis showed that cell death was due to apoptosis. Also, size reduced bi-HNTs resulted in higher cell mortality. MTX loaded into bi-HNTs (MTX-bi-HNTs) was analyzed for its specific targeting ability in vitro after co-culturing with three different cell types. Our results show that bi-HNTs provided a high loading capacity for the cytotoxic cancer drug MTX and, bi-HNTs specifically targeted osteosarcoma cells and released MTX and inhibited cell proliferation. Interestingly, in the presence of MTX, non-osteosarcoma cells were unaffected.
