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.

<p>MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity</p>

“…Adsorption of protein molecules on nanotube surfaces can increase water solubility and dispersibility of pristine MWCNTs by changing the surface tension of a pristine water-nanotube interface, 16 and the similar phenomenon of protein binding increased water solubility and dispersibility, which has been commonly observed in other carbon nanomaterials such as single-walled CNTs and graphene oxide. 17,18 In contrast to decreased size for pristine MWCNTs in BSA and IgG media by ~34% to 45%, the sizes of the MWCNTs-COOH in BSA and IgG media were slightly increased, probably due to protein adsorption. Protein corona formed on NPs are dependent on the NP-protein interaction, and NP-protein complexes lead to an increase of NP core size.…”

The Effects of Varying Degree of MWCNT Carboxylation on Bioactivity in Various In Vivo and In Vitro Exposure Models

“…Several physical changes in MWCNTs occur when the particles are carboxylated. The f-MWCNTs are more hydrophilic [ 16 ], less aggregated [ 22 , 24 ], create more unstable protein corona [ 25 ], and are more soluble [ 16 ] than the source MWCNTs. In addition, size, surface charge and zeta potential are altered, resulting in unique protein binding and protein corona upon carboxylation [ 24 , 25 ].…”

“…Further, in human alveolar A549 epithelial cells, carboxylated MWCNTs were confined within cytoplasmic vesicles, whereas pristine MWCNTs were found free in the cytoplasm as well as within vesicles [ 15 ]. Carboxylation has also been shown to increase MWCNT binding to bovine serum albumin (BSA) [ 16 ], suggesting the protein corona has an impact on the biocompatibility of functionalized MWCNTs.…”

Effect of Functionalized Carbon Nanotubes and their Citric Acid Polymerization on Mesenchymal Stem Cells In Vitro

“…A subject of great relevance is to continue the study of the interaction between PCNTs and MSCs, but there are some interesting questions to answer beforehand, for example, can the interaction between PMWNTs and MSCs induce growth factors and secretion of substances from the cells? [18,62]. Can the polymer interact with molecules found in the extracellular matrix [61][62][63][64], such as glycoproteins?…”

“…[18,62]. Can the polymer interact with molecules found in the extracellular matrix [61][62][63][64], such as glycoproteins? Will this allow binding to a range of growth receptors with high affinity [18] or simply increase the adhesive capacity of the material?…”