Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles

Abstract

Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.

Keywords: biomembrane; lipid extraction; lysosomal permeabilization; molecular dynamics; one-dimensional nanomaterials.

Fig. 1.

CGMD simulations of lysosomal membrane disruption induced by encapsulation of a stiff CNT. (A) The maximum noninterrupted contact time between an MWCNT and a lysosome (with a diameter D of 100 nm) at different length ratios of L/D and ΔP = 40 kPa. (Insets) Equilibrium configurations of the lysosome at L/D = 1.3 and 1.7. (B–E) Conformations of all-atom simulations in front (B and D) and top (C and E) views. (B and C) A CNT interacting with a membrane. (D and E) An MLG interacting with a membrane, representing a slice of the near-tip region of the CNT.

Fig. 2.

All-atom MD reveals a nanomechanical mechanism of lipid extraction caused by enforced mechanical contact for lysosomal permeabilization. (A–D) Representative configurations of a three-layer graphene sheet interacting with a DPPC membrane under a compressive force of 500 pN at 148 ns (A) and 264 ns (B–D). (C and D) Left and right views of B, respectively. MLG sheets are shown in yellow. Phospholipids are shown as chains of atoms colored to identify hydrophilic and hydrophobic regions (hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange). Extracted phospholipids are highlighted as chains of larger spheres. Water molecules are set to be transparent for clarity. (E) The critical damage time leading to lysosomal permeability as a function of the contact force induced by the MLG. (F) The dependence of critical damage forces on the circumference of uncapped and capped CNTs.

Fig. 3.

Cellular uptake, localization, and lysosomal interactions of long, rigid MWCNT-7 and spherical carbon black nanoparticles. (A and B) Fluorescent microscope images of hepatocytes after 24-h exposure to carbon black or MWCNT-7 combined with bright-field imaging to visualize the particles within the cells and DAPI to visualize the nucleus (blue fluorescence). (C and D) Lysosomal membrane permeabilization assessed using cathepsin B assay. Green arrow shows example of diffuse fluorescence indicating cathepsin B leakage following nanotube exposure. (E and F) Intracellular localization of nanomaterials in hepatocytes after 24 h as visualized by TEM. Carbon black colocalizes with lysosomes (yellow arrows), whereas long, rigid MWCNT-7 only partially colocalize with lysosomes. Doses are 10 μg/mL for TEM studies and 20 μg/mL for cathepsin B assay. (G) Quantification of diffuse cathepsin B release into the cytoplasm of hepatocytes after exposure to carbon nanomaterials. Diffuse cathepsin B fluorescence was determined using single-cell quantitative high content imaging (Opera Phenix). After exposure of hepatocytes to MWCNT-7, flexible MWCNTs (MWCNT-flex1 and MWCNT-flex2), carbon black and carbon nanohorns for 24 h, only MWCNT-7 induced a significant, dose-dependent release of cathepsin B into the cytoplasm. *P < 0.05.

Fig. 4.

Cellular interactions with other carbon nanoforms of diverse geometry. (A) Quantification of caspase activation 24 h after exposure to carbon nanoparticles. Caspase activation was assessed using sulforhodamine-labeled caspase after exposure of hepatocytes to 20 μg/mL carbon nanomaterials for 24 h or to 5 μM staurosporine for 3 h as positive control. MWCNT-7 significantly ($\ast P<0.05$) induced caspase activation. (B) Cell viability assessed using the dehydrogenase activity assay WST-8 24 h following exposure to carbon nanomaterials. Flexible MWCNTs, carbon black, and carbon nanohorns did not significantly decrease viability whereas long, rigid MWCNT-7 or 1-μm MWCNT-7 significantly induced toxicity ($\ast P<0.05$). (C–F) Uptake and localization of various carbon nanoforms in hepatocytes at 24 h after exposure to 10 μg/mL of various carbon nanoforms: (C) shortened MWCNT-7 sample, 0.5-μm MWCNT-7; (D) 1-μm MWCNT-7; (E) isometric carbon nanohorn aggregates; (F) thin (flexible) CNTs. Note that carbon nanohorns, flexible CNTs, and 0.5-μm-long MWCNTs are localized in cytoplasmic vesicles, whereas 1-μm-long MWCNTs are seen penetrating through the lysosomal membrane (D, yellow arrows).

Fig. 5.

Generalized classification diagram distinguishing pathogenic from biocompatible carbon nanomaterials in the mechanical pathway to lysosomal membrane damage. The classification diagram is based on the geometric criteria for unfunctionalized carbon nanomaterials to induce lysosomal permeability through mechanical stress. The nanomechanical theory of intracellular buckling (blue curve) is compared with data from the present study (square symbols) and the literature (diamonds). (See SI Appendix, Figs. S19, S27, and S29 for MWCNT-1 data.) Red (but not blue) symbols represent samples reported to activate this pathway. Data points labeled 1,2 are literature reports of statistically significant IL-1β release for THP-1 cells at 50 μg/mL MWCNT exposure (17); point 3 is statistically significant IL1-β activity in THP-1 cells above 25 μg/mL exposure to as-produced BSA-coated MWCNTs (10). Also shown are dashed lines of constant AR, which show that nanotubes with intermediate but not low or high AR, together with long length, are the most likely to induce lysosomal damage by mechanical stress.