Not all protein-mediated single-wall carbon nanotube dispersions are equally bioactive

Abstract

Single-wall carbon nanotubes (SWCNTs) have been dispersed with proteins to increase biocompatibility and specificity, but examinations of dispersion parameters on functional cellular uptake are required for utilization of SWCNTs in biological applications. Here we correlate conditions of SWCNT dispersion with various proteins to uptake these SWCNTs in NIH-3T3 fibroblasts and J774A.1 macrophage-like cells. We varied protein types (bovine serum albumin - BSA, lysozyme - LSZ, and γ-globulins - γG), protein : SWCNT ratio and sonication time. Each protein created stable, high yield (~25%) dispersions in water while preserving intrinsic SWCNT fluorescence, but SWCNT-LSZ flocculated in media and SWCNT-γG formed clusters in both water and media, drastically altering cellular internalization. Dispersion quality and yield improved with increased protein : SWCNT - without substantial effects from depletion attraction, even at 100 : 1 protein : SWCNT - and slightly increased internalized SWCNTs for both NIH-3T3 and J774A.1 cells. Longer sonication time (12 versus 2 h) improved the dispersion yield and quality but caused minor damage to SWCNTs and altered protein structure. Cell association of SWCNT-BSA was homogenous and unaltered by sonication time. Bulk assay showed that cell association of SWCNT-LSZ and SWCNT-γG was altered with 12 versus 2 h sonication, but imaging of individual cells showed that these differences are likely from precipitation of clusters of SWCNT-LSZ and SWCNT-γG in media onto cells. Hence, the quality of SWCNT-protein dispersions in water does not necessarily correlate with bulk cellular uptake, and quantification at the level of individual cells is required to determine delivery efficacy.

Length effects on the dynamic process of cellular uptake and exocytosis of single-walled carbon nanotubes in murine macrophage cells

“…This might cause different profiles of adsorption protein on SWCNTs. It has been demonstrated that SWCNTs with different protein coating were not equally bioactive 53 . Therefore, these two types of SWCNTs might display different potent in inducing receptor-mediated internalization; (3).…”

Distribution of single wall carbon nanotubes in the Xenopus laevis embryo after microinjection

“…We have previously shown that the BSA-dispersed SWCNTs used in this study were stable upon dilution in ultrapure water (Holt et al 2011) and cell culture media (Boyer et al 2013; Holt et al 2012b). Therefore, SWCNTs should have remained well dispersed after injection into X. laevis , and indeed, Raman spectroscopy confirmed that SWCNTs remained well dispersed within X. laevis .…”

“…Centrifugation at 21,000× g for 7 min (Holt et al 2011) was performed to pellet remaining small bundles. Supernatants were decanted and the concentration of SWCNTs was determined by a known absorbance coefficient at 930 nm (Boyer et al 2013; Holt et al 2012a; Holt et al 2012b; Holt et al 2010). Dispersions were sterilized by UV lamp (UVP Black- Ray, 100 W long-wave ultraviolet lamp, Model B 100 AP/R; UVP, LLC, Upland, CA, USA) for 1 h. Previous description of ultraviolent–visible–near-infrared absorbance, fluorescence and Raman spectroscopies of the BSA-stabilized SWCNTs showing highly resolved van Hove peaks are included elsewhere (Holt et al 2011; Holt et al 2012a) and confirm that the supernatants of SWCNTs prepared in this manner contain mostly individualized SWCNTs with some very small bundles (Bachilo et al 2002; Dresselhaus et al 1996; Kataura et al 1999; O'Connell et al 2002).…”

“…The presence of SWCNT G-band in Raman spectra at ∼1591 cm -1 indicates sp 2 hybridized carbon structures (Dresselhaus et al 2005), and we have previously shown that its intensity can be used to determine SWCNT concentration (Boyer et al 2013; Holt et al 2011; Holt et al 2012a; Holt et al 2012b). The intensity ratio I D : I G between the SWCNT D-band, which characterizes the sp 3 -hybridized carbon, and the G-band indicates damage or structural defects in SWCNTs.…”

Noncovalent Protein and Peptide Functionalization of Single-Walled Carbon Nanotubes for Biodelivery and Optical Sensing Applications

“…56,73 Varying sonication times and power can alter protein secondary structure with dramatic consequences for biological applications, as conformational changes may result in the complete loss of protein activity. 63,74 …”