Boron Nitride Nanosheets Can Induce Water Channels Across Lipid Bilayers Leading to Lysosomal Permeabilization

Abstract

While the interaction between 2D materials and cells is of key importance to the development of nanomedicines and safe applications of nanotechnology, still little is known about the biological interactions of many emerging 2D materials. Here, an investigation of how hexagonal boron nitride (hBN) interacts with the cell membrane is carried out by combining molecular dynamics (MD), liquid‐phase exfoliation, and in vitro imaging methods. MD simulations reveal that a sharp hBN wedge can penetrate a lipid bilayer and form a cross‐membrane water channel along its exposed polar edges, while a round hBN sheet does not exhibit this behavior. It is hypothesized that such water channels can facilitate cross‐membrane transport, with important consequences including lysosomal membrane permeabilization, an emerging mechanism of cellular toxicity that involves the release of cathepsin B and generation of radical oxygen species leading to cell apoptosis. To test this hypothesis, two types of hBN nanosheets, one with a rhomboidal, cornered morphology and one with a round morphology, are prepared, and human lung epithelial cells are exposed to both materials. The cornered hBN with lateral polar edges results in a dose‐dependent cytotoxic effect, whereas round hBN does not cause significant toxicity, thus confirming our premise.

2D Materials and Primary Human Dendritic Cells: A Comparative Cytotoxicity Study

“…2D hBN was produced by direct liquid‐phase exfoliation of pre‐milled boron nitride in the presence of bile salts dispersant using bath sonication (see Experimental Section for details). [ 35 ] After the removal of bile salts in excess via filtering, washing, and resuspension, a concentrated stock hBN dispersion (1 mg mL −1 ) was prepared, resulting in a stable suspension for weeks without formation of agglomerates and precipitates. This suspension was then characterized by using a variety of techniques.…”

“…Transmission electron microscopy (TEM) ( Figure A) and scanning electron microscopy (SEM) (Figure S2A, Supporting Information) images showed single and few‐layer sheets with an average size of 358 ± 166 nm. [ 35 ] (Figure 1D) The lateral size distribution was calculated from TEM images by measuring more than 200 sheets. Atomic force microscopy (AFM) was also used as a complementary technique to measure the lateral size and the thickness, corresponding to 225 ± 55 nm and 5.05 ± 1.35 nm, respectively (measured on 100 individual flakes) (Figure S3, Supporting Information).…”

“…Liquid‐phase exfoliation of hBN was carried out using a method already reported by the group. [ 35 ] Briefly, 3 g of boron nitride (Alfa Aesar, powder ≈325 mesh, 99,5%, Lot B21Z024) were pre‐milled with steel balls at 100 rpm for 20 h. 60 mg of the obtained material were then milled with steel balls along with 120 mg of bile salts as dispersant, containing ≈50% of sodium cholate and ≈50% of sodium deoxycholate, (Fluka, Lot 0001432751) at 100 rpm for 3 h. The powder was transferred with 40 mL of Milli‐Q water into a round flask and sonicated at 37 MHz at <35 °C for 1 h, resulting in a white dispersion. The dispersion was centrifuged at 500 g min −1 for 1 h. After centrifugation, the supernatant was recovered, filtered, and washed with water and ethanol, then dispersed by sonication in 20 mL of water.…”

Lysosomal solute and water transport

“…Notably, water may be co-transported uphill with catabolites through these water-permeable channels and transporters ( Zeuthen, 2010 ). Introducing synthetic water channels to the lysosomes dramatically increases the osmotic tension of lysosomes ( Lucherelli et al, 2021 ), suggesting the basal water permeability of lysosomes is likely low. It is likely there exist some basal water permeability in the lysosome, and osmotic stimulation may upregulate its activity.…”

Sustainable Synthesis of Highly Biocompatible 2D Boron Nitride Nanosheets

“…Self-assembled boron nitride nanosheets have been recently applied to boron neutron capture therapy (BNCT), being useful for breast cancer treatment and showing their potential in biomedicine applications [ 33 ]. Despite the interesting application of BNNs in the biomedical field, only a few studies have been conducted to understand their toxicity and some of them present conflicting results [ 15 , 34 ]. For example, sheet-like BN materials were reported to present toxicity and cause adverse effects on human hepatoma HepG2 cells, such as reducing cell viability, increasing ROS generation, or inducing damage to membrane integrity [ 35 ].…”