Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4⁺ T-cell proliferation

Abstract

Carbon nanotubes (CNTs) are of great interest for the development of drugs and vaccines due to their unique physicochemical properties. The high surface area to volume ratio and delocalized pi-electron cloud of CNTs promote binding of proteins to the surface forming a protein corona. This unique feature of CNTs has been recognized for potential delivery of antigens for strong and long-lasting antigen-specific immune responses. Based on an earlier study that demonstrated increased protein binding, we propose that carboxylated multiwalled CNTs (MWCNTs) can function as an improved carrier to deliver antigens such as ovalbumin (OVA). To test this hypothesis, we coated carboxylated MWCNTs with OVA and measured uptake and activation of antigen-presenting cells (macrophages) and their ability to stimulate CD4⁺ T-cell proliferation. We employed two types of carboxylated MWCNTs with different surface areas and defects (MWCNT-2 and MWCNT-30). MWCNT-2 and MWCNT-30 have surface areas of ~215 m²/g and 94 m²/g, respectively. The ratios of D- to G-band areas (I_D/I_G) were 0.97 and 1.37 for MWCNT-2 and MWCNT-30, respectively, samples showing that MWCNT-30 contained more defects. The increase in defects in MWCNT-30 led to increased binding of OVA as compared to MWCNT-2 (1,066±182 μg/mL vs 582±41 μg/mL, respectively). Both types of MWCNTs, along with MWCNT–OVA complexes, showed no observable toxicity to bone-marrow-derived macrophages up to 5 days. Surprisingly, we found that MWCNT–OVA complex significantly increased the expression of major histocompatibility complex class II on macrophages and production of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin 6), while MWCNTs without OVA protein corona did not. The coculture of MWCNT–OVA-complex-treated macrophages and OVA-specific CD4⁺ T-cells isolated from OT-II mice demonstrated robust proliferation of CD4⁺ T-cells. This study provides strong evidence for a role for defects in carboxylated MWCNTs and their use in the efficient delivery of antigens for the development of next-generation vaccines.

Keywords:

• MWCNT
• protein corona
• nanoparticle
• defect
• carboxylation
• antigen presentation
• immune response

Supplementary materials

Figure S1 TEM images of MWCNTs before and after carboxylation.

Notes: (A) Pristine MWCNT-2, (B) pristine MWCNT-30, (C) carboxylated MWCNT-2, and (D) carboxylated MWCNT-30. After carboxylation, both MWCNT-2 and MWCNT-30 collapsed and became more rugged.

Abbreviations: MWCNT, multiwalled carbon nanotube; TEM, transmission electron microscopy.

Figure S2 TGA that was carried out in a Perkin Elmer Thermogravimetric Analyzer Pyris 1 TGA.

Notes: Approximately 40%–50% of the volume of the platinum pan was filled with MWCNTs (~0.1–0.2 cm³). The samples were then heated from 30°C up to 800°C at a heating rate of 20°C/min in an air atmosphere using an airflow rate of 20 mL/min. We observed a marked decrease in the weight percent for CNT–OVA complexes with increasing temperature unlike pristine CNTs confirming the presence of OVA. Based on these data, we found the amount of OVA bound to MWCNT-2 and MWCNT-30 was 150±10 μg/mL and 340±10 μg/mL, respectively. The inset shows the TGA of pristine and functionalized CNTs. While the pristine CNTs showed ~4 wt% of Fe catalyst at the end of the reaction; functionalized CNTs (viz, MWCNT-2 and MWCNT-30) had only ~0.5 wt% suggesting the removal of residual catalyst during the microwave reaction.

Abbreviations: CNTs, carbon nanotubes; MWCNT, multiwalled carbon nanotube; OVA, ovalbumin; TGA, thermogravimetric analysis.

Figure S3 Delivery of fluorescent TR-OVA by MWNCT-30 into BMDMs visualized by dark field and fluorescent microscopy.

Notes: Macrophages treated with MWCNT-30 alone showed no fluorescence, but macrophages treated with MWCNT–TR-OVA complex showed strong fluorescence (magnification: 100×).

Abbreviations: BMDMs, bone-marrow-derived macrophages; DAPI, 4′,6-diamidino-2-phenylindole; MWCNT, multiwalled carbon nanotube; OVA, ovalbumin; TR-OVA, Texas Red-conjugated OVA.

Table S1 XPS results of MWCNT-2 and MWCNT-30 samples obtained using a Kratos Axis Ultra DLD instrument and spectra were calibrated by C 1s at 284.6 eV

Acknowledgments

This study was supported by the National Institutes of Health (grant R03 ES023036).

Disclosure

The authors report no conflicts of interests in this study.