Toxicological Evaluation toward Refined Montmorillonite with Human Colon Associated Cells and Human Skin Associated Cells

Abstract

Montmorillonite has been refined to overcome uncertainties originating from different sources, which offers opportunities for addressing various health issues, e.g., cosmetics, wound dressings, and antidiarrheal medicines. Herein, three commercial montmorillonite samples were obtained from different sources and labeled M1, M2, and M3 for Ca-montmorillonite, magnesium-enriched Ca-montmorillonite, and silicon-enriched Na-montmorillonite, respectively. Commercial montmorillonite was refined via ultrasonic scission-differential centrifugation and labeled S, M, or L according to the particle sizes (small, medium, or large, respectively). The size distribution decreased from 2000 nm to 250 nm with increasing centrifugation rates from 3000 rpm to 12,000 rpm. Toxicological evaluations with human colon-associated cells and human skin-associated cells indicated that side effects were correlated with excess dosages and silica sand. These side effects were more obvious with human colon-associated cells. The microscopic interactions between micro/nanosized montmorillonite and human colon-associated cells or human skin-associated cells indicated that those interactions were correlated with the size distributions. The interactions of the M1 series with the human cells were attributed to size effects because montmorillonite with a broad size distribution was stored in the M1 series. The M2 series interactions with human cells did not seem to be correlated with size effects because large montmorillonite particles were retained after refining. The M3 series interactions with human cells were attributed to size effects because small montmorillonite particles were retained after refining. This illustrates that toxicological evaluations with refined montmorillonite must be performed in accordance with clinical medical practices.

Keywords: clinical medical orientation; microscopic interaction; montmorillonite; refine; toxicological evaluation.

Figure 1

Characterization and toxicological evaluation of MMT via (A) XRD, (B) FTIR, and (C) CCK-8. M1-RAW: unrefined Ca-MMT; M2-RAW: unrefined magnesium-enriched Ca-MMT; M3-RAW: unrefined silicon-enriched Na-MMT. The P value was determined by ANOVA and Student’s t tests. *** p < 0.0005.

Figure 2

Characterization of refined MMT via (A) SEM, (B) DSL, and (C) XRD. The refined MMTs with small, medium, and large particle sizes were labeled S, M, and L, respectively.

Figure 3

Toxicological effects of refined MMT on human colon-associated cells (HIEC 6 and NCM460) and human skin-associated cells (HaCaT and HUVECs). The p value was determined by ANOVA and Student’s t tests. ** p < 0.005, *** p < 0.0005.

Figure 4

SEM images of MMT–human cell interactions. Unrefined Ca-MMT was labeled M1-RAW, and refined M1-RAW with small particle sizes was labeled M1-S. Unrefined magnesium-enriched Ca-MMT was labeled M2-RAW, and refined M2-RAW with small particle sizes was labeled M2-S. Unrefined silicon-enriched Na-MMT was labeled M3-RAW, and refined M3-RAW with small particle sizes was labeled M3-S. The thumbnail showed an enlarged image featuring a white arrow indicating the presence of MMT.