Steering carbon nanotubes to scavenger receptor recognition by nanotube surface chemistry modification partially alleviates NFκB activation and reduces its immunotoxicity

Abstract

Carbon nanotubes (CNTs) cause perturbations in immune systems and limit the application of CNTs in biomedicine. Here we demonstrate that a surface chemistry modification on multiwalled CNTs (MWCNTs) reduces their immune perturbations in mice and in macrophages. The modified MWCNTs change their preferred binding pattern from mannose receptor to scavenger receptor. This switch significantly alleviates NFκB activation and reduces immunotoxicity of MWCNTs.

Figure 1

Heat map for NO generation of MWCNT 1 and a 80-member combinatorial MWCNT library and the molecular structures of selected MWCNTs for in this study. Colors from light blue to dark blue are for lower NO to higher NO generations (μmol/L, NO<7.5, ). MWCNT 1 and 2 were selected for investigation of their immune perturbations.

Figure 2

The distribution of MWCNTs in mice and the effect of MWCNT 1 and 2 on inflammatory cytokines in mice lung, liver and spleen in vivo. (A): MWCNT 1 were labeled with a specific ligand for ⁶⁴Cu. Suspension of MWCNT 1 was injected in to CD-1 female mice (4 mice per group) via tail vein. For 10 min, 60 min and 24 hrs, the accumulation of MWCNT 1 in the tissues were measured. (B and C): Suspension of MWCNT 1 and 2 in PBS solution in the presence of 0.1% Tween 80 was injected in to female BALB/c mice (5 mice per group) via tail vein with a dose of 14 mg/kg. For 24 hrs, TNF-α and IL-1β in the tissue homogenate were determined by ELISA. (*p<0.05)

Figure 3

Immune perturbations of MWCNTs in macrophages. (A) and (B): macrophages were treated with different concentrations of MWCNTs for 24 h and the nitrite concentration (A) and TNF-α (B) in the culture supernatant was analyzed by Griess reaction and ELISA separately. LPS (20 ng/mL) was used as control. (C): reactive oxygen species in macrophages was determined through increases in fluorescence intensity of dichlorofluorescin (DCF) after incubation with MWCNTs for 24 hrs. (D): The lowered mitochondrial membrane potentials elicited by MWCNTs (97.6 μg/mL for MWCNT 1 or 95.9 μg/mL for MWCNT 2, 24 hrs) were measured by JC-1 fluorescence. Mitochondria depolarization is specifically indicated by a decrease in the red (610±10 nm) to green (525±10 nm) fluorescence intensity ratio. The fluorescence was measured by confocal laser scanning microscopy.

Figure 4

TEM characterization of MWCNTs internalization and their distribution in subcellular organelles. Macrophages were incubated with MWCNT 1 (A, B, C, 97.6 μg/mL) or 2 (D, E, F, 95.9 μg/mL) for 24 hrs. A and D: cytoplasm; B and E: endosome; C and F: lysosome. Arrows point to MWCNTs. Scale bars represent 100 nm.

Figure 5

Probing the mechanisms of macrophages’ internalization of MWCNTs. Cytochalasin D, fucoidan, mannan and OxPAPC are generally classified as inhibitor of macropinicytosis/phagocytosis, scavenger receptor, mannose receptor and Toll-like receptor 4. Macrophages were first treated with various inhibitors for 30 min. treated and untreated macrophages then were incubated with MWCNT 1-FITC (48.8 μg/mL) or MWCNT 2-FITC (48.1 μg/mL) for 45 min before the amounts of internalization were quantitatively determined. The percent internalization was normalized to particle internalization in the absence of inhibitors. (Comparison between pretreated and untreated macrophages, *p<0.05. Comparison between scavenger and mannose receptor inhibitor pretreated macrophages, **p<0.05)

Figure 6

Effect of MWNCTs on NFκB activation (A), iNOS (B) and Src-family of PTK (C) expression. Degradation of IκBα and translocation of p65 protein into the nucleus are indicators for activation of NFκB signaling pathway. Macrophages were treated with 97.6 μg/mL of MWNCT 1 or 95.9 μg/mL of MWNCT 2 for 24 hrs. The cytoplasm or nuclear extracts were analyzed by western blot for IκBα, p65, iNOS and Src-family of PTK in cytoplasm and p65 in nuclear. For equal protein loading of cytoplasmic extracts, the cytoplasm extracts were analyzed with anti-β-actin antibody and for nuclear extracts with anti-PARP antibody.