Binding of pulmonary surfactant proteins to carbon nanotubes; potential for damage to lung immune defense mechanisms
Introduction
Carbon nanotubes are widely considered to be one of the most versatile families of new materials and will be instrumental in driving forward the nanotechnology industrial revolution in the coming decade. Reported uses in fields as diverse as polymers, electronics or even health care, while promising significant improvements in quality of life, will require production of nanotubes to reach industrial levels. It is therefore essential to investigate thoroughly the effects that carbon nanotubes might have on human health. Three distinct entry routes into the human body are recognised: ingestion, penetration through the skin and inhalation. The last probably constitutes the highest risk as it is notably difficult to deal reliably with suspension in air of extremely small particles. Carbon nanotubes tend to form aggregates, many of which will not penetrate to the lung alveoli when inhaled. However, non-aggregated carbon nanotubes may, because of their small size, penetrate to the alveoli of the lungs, where they may interact with surfactant proteins and lipids.
While in vivo toxicity studies previously reported represent an important first step in highlighting the possible risks associated with exposure to carbon nanotubes, the protocols they rely upon may have produced artefacts that can be difficult to distinguish from effects produced by nanotubes themselves. For example, in instillation studies the aggregation of nanotubes produces mortality by airway obstruction in rats as reported by Warheit et al. [2] and Muller et al. [3]. Further studies are needed to elucidate how carbon nanotubes interact with host pulmonary systems at the molecular level.
The lungs are an important interface between the host and an environment that contains a plethora of potentially harmful microorganisms. The immune defense of the lung involves the pulmonary surfactant, a complex mixture of lipids, phospholipids, and proteins important for normal respiratory function [4]. Two of these surfactant proteins are SP-A and SP-D (Fig. 1), which belong to a family known as the collectins, as they contain a collagenous region and a C-type lectin domain. A major biological role of the collectins is to bind to targets (e.g., microorganisms and allergens) by recognizing patterns of carbohydrate distribution at their surface and to enhance phagocytosis/clearance of the targets. Thus, they play an important role in first-line immune defenses within the lung.
In this communication we report for the first time the selective binding of SP-A and SP-D to carbon nanotubes. These results enable us to shed new light on previously published toxicity studies and also reveal hitherto uncharacterised variability in the nanotubes. The results suggest that long-term exposure to inhalation of nanotubes has the potential to enhance susceptibility to infection and emphysema.
Section snippets
Synthesis of carbon nanotube samples
Catalytic vapor deposition double-walled nanotubes (DWNT) were made as described by Flahaut et al. [5]. After the synthesis the sample was washed with a concentrated aqueous solution of HCl (ca. 12.1 M) to dissolve all the accessible catalyst (including all MgO-based catalyst and non-protected metal particles). The sample was filtered and washed to neutrality. A final rinse was given with ethanol before drying. Subsequently, the carbon nanotube sample was dried at 80 °C in air.
Elemental analysis
Plasma atomic
Results
SDS-PAGE analysis of DWNTs after exposure to BALF (Fig. 3, track 3) shows that in the presence of ethylene diamine tetra acetic acid (EDTA) almost no protein from the BALF supernatant binds to the DWNTs. However in the presence of Ca2+ ions several proteins (6–8 bands on SDS-PAGE) bind, and are easily detectable (Fig. 3, track 6). By mass spectrometry analysis the BALF supernatant proteins attached to DWNTs were identified (Fig. 4). Transferrin, Human Serum Albumin (HSA), and IgG were
Discussion
Binding of human plasma proteins including C1q, fibrinogen and high density lipoproteins to carbon nanotubes has been characterised by SDS-PAGE, mass spectrometry and N-terminal sequence analysis [1]. These binding reactions do not require divalent metal ions. In contrast the selective binding of BALF supernatant SP-A and SP-D to carbon nanotubes is Ca2+-ion dependent. The binding of SP-A and SP-D to carbon nanotubes can be stopped by using Ca2+-ion chelators such as EDTA (Fig. 3, track 3).
Conclusion
In conclusion, this study contributes to the elucidation of a molecular mechanism involved in pulmonary toxicity of carbon nanotubes. In addition, this communication opens up new routes of research in this area, aimed at obtaining a better understanding of the role of the sequestration of SP-A and SP-D by carbon nanotubes. It also suggests that functionalisation while lowering the toxicity in some nanomaterials might also have toxicity implications of its own [32]. More studies are needed to
Acknowledgments
This work was supported partly by the Medical Research Council, UK. C. Salvador-Morales acknowledges the Mexican National Council for Science and Technology (CONACYT) for a Graduate scholarship. We thank Mr. A.C. Willis for the N-terminal sequence analysis measurements and Miss Roona Deb for providing the SP-A protein.
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