3.2.3. Apoptosis and Necrosis

Previous cell cytotoxicity studies showed that cell viability decreased significantly when the concentration of HNTs reached 150 mg/mL [16]. To distinguish the mechanism of cell death, we examined cell death due to apoptosis or necrosis. CT26WT cells were incubated with long and short bi-HNTs; cell death was assessed using the Annexin V-FITC Apoptosis Kit. Apoptosis and necrosis were assessed by flow cytometry. The detail res as shown in Figure 5B, 20% ± 1.5% of cell death was caused by apoptosis and 10% ± 0.5% was due to necrosis when cells were cultured with long bi-HNTs. Cells cultured with shorted bi-HNTs showed apoptotic cell death at 23% ± 1.3% and 13% ± 0.6% due to necrosis. Thus, cell death was primarily due to apoptosis for long and shortened bi-HNTs. As seen in Figure 5, cells accumulated intracellularly shorter bi-HNTs as compared to long bi-HNTs and shortened bi-HNTs resulted in an overall higher cell death suggesting excessive cellular accumulation of bi-HNTs leads to apoptosis.

#### *3.3. Intracellular Location of bi-HNTs*

In order to confirm the intracellular location of bi-HNTs, we co-cultured bi-HNTs with CT26WT cells for 24 h and cells were then incubated with DiR for another 2 h. Multi-photon microscopy imaging showed cell were stained red with each reddish circular structure representing the plasma membrane; the yellow particles represent bi-HNTs (Figure 6). Multi-photon images of labeled cells showed that many bi-HNTs were present within the plasma membrane and some had accumulated in the cytoplasm. The results further confirmed the targeting capacity of bi-HNTs in vitro.

**Figure 6.** Multi-photon pictures of cells after exposure to bi-HNTs, pictures were analyzed with NIH Image J. Cell membranes were stained by DiR dye and exhibited a red color at wavelength of 850 nm. At this wavelength FITC exhibited a yellow color. The 3D pictures were captured at 36 microns in z with a 2 micron separation in each z step. (**A**) is the front view of the 3D picture for multiple cells, (**B**,**C**) are side views of the 3D picture. (**D**–**F**) are the zoom in pictures of the marked cell. (Scale bar with 50 μm).
