Effects of multiwalled carbon nanotube surface modification and purification on bovine serum albumin binding and biological responses

Abstract

The potential diagnostic and therapeutic applications such as drug delivery of multi-walled carbon nanotubes (MWCNTs) are being increasingly explored due to their unique mechanical, chemical and biological properties. Carboxylation of MWCNTs has been widely used to improve the solubility in aqueous systems, and for further functionalization with biologically active moieties. Purity of carboxylated MWCNTs is of great importance in nanomedicine. An important consideration is that oxidation debris is generated during the process of carboxylation, which can be removed by base washing. We hypothesized that surface modification as well as further purification by debris removal may alter physicochemical properties of MWCNTs and their ability to bind proteins. In this study, we utilized pristine MWCNT carboxylated MWCNTs (F-MWCNTs) and base-washed carboxylated MWCNTs (BW-F-MWCNTs) to examine formation of a bovine serum albumin (BSA) protein corona and impact on biological responses. We found that carboxylation increased the capability of F-MWCNTs to bind BSA, and base washing further increased this binding by 41% implying that purification of F-MWCNTs is an important consideration in biological applications. The BSA protein corona decreased the hydrodynamic size of MWCNTs by nearly 50% because the coating improved colloidal behavior. The effect was significantly less pronounced for F-MWCNTs and BW-F-MWCNTs because they were highly dispersible to begin with. Functionalization increased cellular uptake by both rat aortic endothelial cells (RAEC) and macrophage-like murine cells (RAW264.7), while base washing showed results similar to the functionalized analog. Interestingly, BSA binding downregulated mRNA levels of interleukin-6 (IL-6) and heme oxygenase 1 (Hmox1) in RAEC cells but upregulated the expression of IL-6 and Hmox1 in RAW264.7 cells, indicating the dependence of cell types in biological responses to MWCNTs. Overall, our study demonstrated that surface modification as well as further purification impacted the interaction of MWCNTs with proteins and subsequent cellular responses. Interestingly, while the corona associated with the F-MWCNTs and BW-F-MWCNTs were significantly different, their respective cellular uptake and biological responses were similar. This implied that surface functionalization played a more important role than surface corona.

Keywords: Carbon nanotubes; biocorona; endothelial cell; macrophage; nanotoxicology.

Figure 1

Characterization of MWCNTs. SEM image of all MWCNTs including (A) pristine MWCNT, (B) functionalized MWCNT, and (C) base-washed F-MWCNT. (D) FTIR spectra of MWCNT, F-MWCNT and BW-F-MWCNT.

Figure 2

Confirmation of BSA bound to MWCNTs. (A) quantification of BSA bound to MWCNT using a Pierce BCA protein assay, (B) SDS-PAGE image of BSA binding to MWCNTs, Lanes: 1, BSA alone; 2, BSA bound to pristine MWCNT; 3, BSA bound to F-MWCNT; 4, BSA bound to BW-F-MWCNT. (C-E) UV-vis spectral analysis of MWCNTs with and without BSA coating. In (A): *, p<0.05, compared to the amount of BSA bound to MWCNT; a, p<0.05, significant difference of the amount of BSA between F-MWCNT and BW-F-MWCNT.

Figure 3

Uptake of MWCNTs with or without BSA coating by either RAEC cells or RAW264.7 cells. MWCNTs with or without BSA were internalized by RAEC cells (A) or RAW264.7 cells (B) measured by flow cytometry based on the changes in side scatter shift. The cellular uptake of MWCNTs with or without BSA coating was visualized using darkfield imaging, for RAEC cells (C) and RAW264.7 cells (D). (Magnification: 100 X) *, p<0.05, compared to the control; a, p<0.05, compared to MWCNT; b, p<0.05, comparison between MWCNTs with and without BSA coating.

Figure 4

Changes in gene expression in RAEC or RAW264.7 cells. (A) the level of IL-6 mRNA in RAEC cells, (B) the level of Hmox1 mRNA in RAEC cells, (C) the level of IL-6 mRNA in RAW264.7 cells, (D) the level of Hmox1 mRNA in RAW264.7 cells. *, p<0.05, compared to the control; a, p<0.05, compared to the BSA-treated group; b, p<0.05, comparison between MWCNTs with and without BSA coating.