3.2.1. Nanoparticle Size Determination

Halloysite nanotubes are naturally formed nanoparticles with an original length between 1–1.5 μm. Recently, several studies have used shortened HNTs [11,19,20]. Rong et al. introduced a detailed procedure to produce HNTs that are uniform in length [20]. We fabricated shortened HNTs by ultra-sonication (Figure 1). Dynamic light scattering (DLS) is the most commonly used.

Technique to determine nanoparticle size, however, DLS analysis requires spherical nanoparticles instead of virgate nanotubes. Thus, we used SEM micrographs of our HNT samples NIH Image J to measure their size and plotted the data into histogram graph (Figure 4). As it showed in Figure 4, the length of commercial purchased HNTs distributes in a very wide range with the average size of 0.914 ± 0.45 μm, *n* = 102 (Figure 4A), while the size of shorten HNTs was 0.715 ± 0.25 μm, *n* = 80 (Figure 4B). Comparing the two different sources of HNTs, we observed the size distribution of shortened HNTs to be more restricted in variation, which means they are more uniform in overall size. HNTs with a uniform size will provide a more accurate means for evaluating its effect on cellular behavior.

**Figure 4.** Histogram graph of size distribution of long HNTs (**A**) and shortened HNTs (**B**).
