C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

Abstract

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.

Figure 1

Characterization of C60 Fullerenes and Terbium-Endohedral Fullerenes. Nanosight analysis of C60 fullerenes and Tb@C60 aggregate size: C60 fullerene aggregates were approximately 55.179 nm with a standard deviation of 34.16 nm. Tb@C60 aggregates were approximately 63.765 nm with a standard deviation of 24.46 nm.

Figure 2

No contrast transmission electron micrograph of RAW 264.7 immortalized macrophages containing terbium-endohedral C60. (A) High contrast clusters of C60 (inside with circles) are observed in membrane-bound structures and scattered “free” throughout the cytoplasm. (B) Higher power no contrast TEM shows two major clusters of Tb@C60 adjacent to the cell membrane (upper left) and not bounded by lipid membrane in the cytosol. (C) High power examination of the nuclear envelope (dotted line) reveals membrane-associated clusters that are not contained in classical phagocytotic structures.

Figure 3

Epifluorescent images of immortalized RAW 264.7 macrophages showing lack of role for endocytosis in cellular accumulation of C60. (A) Baseline uptake was established following 3 hrs exposures to C60, and a monoclonal antibody (red) was used to detect and quantify uptake. The effect of classical inhibitors of active membrane-mediated processes for cell entry was determined in the presence of (B) chlorpromazine (clathrin-mediated endocytosis), (C) cytochalasin A (F-actin polymerization) or (D) genistein (caveolin-mediated endocytosis, macropinocytosis and phagocytosis). (E) Statistical analysis of images supports diffusion rather than endo-/phagocytosis as the major route by which small clusters of C60 enter the cell membrane and cytoplasm. The apparent lack of statistically significant diminution in the uptake of C₆₀ induced by the pharmacologic inhibition of clathrin/caveolin-mediated endocytosis suggests that a significant quantity of material enters the cell through other mechanisms.

Figure 4

Free energy profile of the permeation of a C60 fullerene into a POPC bilayer as function of the distance on the membrane normal between the fullerene and the bilayer center.

Figure 5

Distribution of selected carbon atoms of the saturated chain of POPC. Shown as function of the distance on the bilayer normal. Zero corresponds to the center of the bilayer.

Figure 6

Small cluster of C60 fullerenes in a POPC lipid bilayer.

Figure 7

Free energy profile of fullerenes with various radii of gyration in a POPC bilayer. A clear well is identified around the equilibrium radius of 0.915 nm. Energy increases rapidly at smaller radii values and shows a change in slope at higher values, corresponding to detachment of a single C60.

Figure 8

Phosphorous spectra of POPC Vesicles by NMR. Prepared in the presence of fullerenes, (A) at 0 μg/mL, (B) 10 μg/mL, (C) and 100 μg/mL. Data acquired at 20 °C.

Figure 9

(A) Order parameter profile for d31-POPC prepared in the presence of fullerenes, at 0 μg/mL (circle), 10 at μg/mL (square), and 100 μg/mL C60 (triangle). (B) Perturbation in order parameter with respect to vesicles in absence of C60. Data acquired at 20 °C.

Figure 10

Temperature dependence of the first moment of deuterium NMR spectra recorded from d31-POPC in the presence of 0 μg/mL (circle), 10 μg/mL (square) and 100 μg/mL C60 (triangle).