## 3 Results and Discussion
### Cytotoxicity Testing of bHNTs  
Cytotoxicity of bHNTs was studied in CT-26 murine colorectal cancer cells at concentrations up to 150 \(\upmu\)g/mL. Control cells cultured with unmodified HNTs at all concentrations showed no signs of cytotoxicity based on Live/Dead analyses (Figure 4). CT-26 cells cultured with bHNTs at concentrations up to 150 \(\upmu\)g/mL also showed little evidence of cell death. When the same visual fields were observed with epifluorescence microscopy, live cell showed a green fluorescence while dead cells fluorescence a reddish orange (Figure 4). After 150 \(\upmu\)g/mL, the number of dead cells observed increased with increased bHNT concentration. There are two possible explanations for this observation. Cell death may be due to induced cytotoxicity due to exposure to increased concentrations of bHNTs. However, numerous in vitro and in vivo studies have shown that HNTs are cytocompatible and biocompatible [48; 49; 50; 51; 52]. [PERSON] et al. (2016) showed that osteosarcoma cells exposed to HNTs at a concentration of 2000 \(\upmu\)g/mL suffered no cytotoxic effect after exposure [20]. An alternative explanation is that intracellular bHNTs accumulation disrupts cellular function. The cell interior is a crowded space and once inside the cells BHNTs may accumulate leading to cell death. Nanoparticle uptake and accumulation in cells can disrupt critical intracellular transport pathways and organellar functions as detailed in [PERSON] et al. (2012) [53]. Recently, [PERSON] et al. (2017) used halloysite as a vehicle for developing an anti-cancer drug delivery system [54]. They described the synthesis of an HNT nanodelivery system modified with thiol groups, which served as a binding site for the B-cyclodextrinrin used to cap the ends of the HNTs. Disulfide bonds, which can be cleaved at both acidic and basic pH environments, were used to bind the B-cyclodextrinrin to the ends of the HNTs in order to entrap the doxorubicin (DOX) within the HNT lumen. Following this procedure, PEG conjugated with FA was deposited on the outer HNT surface. This HNT complexed DOX system accelerated the apoptosis of cancer cells as compared to non-targeted HNTs. While their system does impart a dual functionality to the HNTs, it does differ from our HNT drug delivery system. First, the HNTs were capped using cyclodextrin and our system does not include a capping system but a means to entrap the cargo and regulate its release could be can be added [54; 55; 52]. Instead, the HNT-DAS-FA/FITC includes both an imaging and targeting agent covalently attached to the surface of the HNTs. By incorporating an imaging moiety in the system, the efficacy of the treatment option can be monitored. Additional, the HNT-DAS-FITC/FA is modular  
Figure 3: Cellular Uptake Assay (**A**) Phase contrast image of CT-26 cells after exposure to bHNTs (50 \(\upmu\)g/mL). Brown intracellular accumulation indicates presence of bHNTs within the cell cytoplasm; (**B**) The same field of view (black circles in A) observed under epifluorescent light. Brown intracellular accumulation as seen in A correspond to FITC-tagged bHNTs in B showing up take by CT-26 cells. and was designed to attach bioactive molecules that use amide bonds as well as similar bonding moieties, thus allowing the system to be applied to a range of cellular targets. Secondly, the system proposed by the Hu group uses FA-conjugated to PEG deposited on the outer surface of the HNTs. Acidic and basic conditions are commonly used in the layer-by-layer surface coatings applied to devices to dissociate the bonds linking the various polyelectrolytes together [56]. Our system prevents the dissociation of FA from the surface by covalently linking it to the DAS. Furthermore, the lumen of HNTs makes them a particularly interesting nanocarrier for the loading and release of various biologically active molecules, including small molecules, enzymes, and proteins including chemotherapeutic payloads [54; 55; 56; 57; 58; 59; 60; 61].