Do surface hydroxyls drive the membranolytic activity of bentonite and kaolin particles?
Authors: Cananà Stefania , Chilla Giuseppe , Mino Lorenzo , Tomatis Maura , Lison Dominique , Turci Francesco , Pavan Cristina 
Published: March 2022

&lt;p&gt;Kaolin and bentonite, two clays mainly made of kaolinite and montmorillonite, respectively, are largely used in various industrial applications. However, their impact on human health has not been fully investigated and data about their mechanism of cellular toxicity are scarce. &lt;em&gt;In vivo&lt;/em&gt; and &lt;em&gt;in vitro &lt;/em&gt;studies showed that kaolin and bentonite particles can induce transient inflammation, alveolar proteinosis, and are cytotoxic to a variety of mammalian cells (Wiemann et al. 2020; Maciaszek et al. 2021). We recently demonstrated (Pavan et al. 2020) that a specific sub-population of surface silanols located at a well-defined intersilanol distance, &lt;em&gt;i.e.&lt;/em&gt;, nearly-free silanols (NFS), is responsible for the membranolytic and inflammatory activity of quartz particles. We hypothesized that a similar structure-activity relationship may exist for kaolinite and montmorillonite particles, since they exhibit tetrahedral SiO&lt;sub&gt;2&lt;/sub&gt; layers at their outer surface and hydroxyls groups, &lt;em&gt;i.e.&lt;/em&gt;, silanols and aluminols, at the crystal lattice boundaries.&lt;/p&gt;&lt;p&gt;Four bentonite (&gt; 90% montmorillonite) and kaolin (&gt; 75% kaolinite) particles were characterized for their physico-chemical properties of toxicological interest and their capacity to damage cellular membranes was assessed using red blood cells as model of membranes. All bentonite and kaolin particles resulted highly membranolytic. As clay minerals may exchange cations with suspending medium and the structural integrity of biological membranes may be compromised by significant alteration of the medium ionic strength, the membranolytic activity of kaolin and bentonite leachates was assessed. Only bentonite leachates induced membrane damage with an effect that was dependent on each sample specific cation exchange capacity (CEC). A reduction or a complete abrogation of kaolin and bentonite membranolytic activity was observed when their surface was coated with dioleoyl lecithin, indicating that surface moieties play a key role for both kaolin and bentonite interactions with membranes. Investigations by IR spectroscopy of the surface-exposed hydroxyl groups revealed the occurrence of NFS, which vibrational feature was especially well defined for kaolin. Thermal treatments carried out on kaolin modified the relative intensity of NFS and its membranolytic activity, suggesting a relationship between NFS and membrane damage.&lt;/p&gt;&lt;p&gt;In conclusion, the capacity of kaolin particles to damage membranes appears related to kaolinite specific surface hydroxylated species. On the other hand, the mechanism of interaction of montmorillonite particles with membranes is function of both mineral surface features and CEC. These findings provide a preliminary understanding of the mechanism of interaction of clay minerals with biological membranes. This interaction may represent the triggering event of kaolin and bentonite adverse cellular effects.&lt;/p&gt;&lt;p&gt;&amp;#160;&lt;/p&gt;&lt;p&gt;Bibliography&lt;/p&gt;&lt;p&gt;Maciaszek K. et al. (2022) An in vitro assessment of the toxicity of two-dimensional synthetic and natural layered silicates,Toxicol &lt;em&gt;In Vitro,&lt;/em&gt; 78:105273&lt;/p&gt;&lt;p&gt;Pavan C. et al. (2020) Nearly free surface silanols are the critical molecular moieties that initiate the toxicity of silica particles, Proc Natl Acad Sci USA, 117 (45):27836&lt;/p&gt;&lt;p&gt;Wiemann M. et al. (2020) Lung toxicity analysis of nano-sized kaolin and bentonite: missing indications for a common grouping, Nanomaterials, 10 (2):204&lt;/p&gt;